{"paper_id":"13b602c4-0450-49df-9803-464f0e2d9f5e","body_text":"Molecular cloning and functional analysis of ScHAK10 gene promoter from sugarcane (Saccharum officinarum L.) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular cloning and functional analysis of ScHAK10 gene promoter from sugarcane ( Saccharum officinarum L.) Hai-Bin Luo, Cheng-Mei Huang, Hui-Qing Cao, Lin Xu, Kai-Chao Wu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3844377/v3 This work is licensed under a CC BY 4.0 License Status: Posted Version 3 posted You are reading this latest preprint version Show more versions Abstract Transcriptional regulation of the high-affinity K + ( HAK ) transporter gene is an important mechanism of plant response to low potassium stress. Cloning and analysis of the promoter of potassium transporter gene is not only helpful to its expression pattern and regulation mechanism but also important to improve the potassium absorption efficiency in sugarcane. The potassium transporter gene ScHAK10 is highly expressed and induced by low potassium stress in sugarcane，but the functions of its promoter is still unclear. In the present article, the 1918 bp promoter region of the ScHAK10 gene ( pScHAK10 ) was cloned by genomic walking technique. Computational analysis affirmed the existence of abiotic stress-responsive cis and core cis -elements, such as TATA box, CAAT box, phytohormone responsive, stress response and light response motifs. GUS histochemical staining of transgenic Arabidopsis thaliana seedlings showed that the leaf, corolla, and root tip were deeply stained, and paraffin sections of root tip tissue showed GUS staining in ductal tissue of A. thaliana shallowly stained. The 5′-terminal deletion of the promoter was cloned, and the lengths of 1918 (full), 1623 (Q1), 1332 (Q2), 957 (Q3), 576 (Q4), and 357 bp (Q5) were cloned into the GUS reporter vector for A. thaliana transient transformation. The transgenic plants generated through a single event exhibited a promising expression of the GUS reporter protein, which was treated with salt, low potassium, IAA and cold stress conditions. The results showed that the promoter activity correlates with the promoter fragment's length, and the long promoter fragment exhibits higher training. The Q5 was the least active and could not drive GUS expression. Under abiotic stress, the expression of GUS enzyme activity varies among different promoter fragments. Under low potassium and high salt stress, Q3 and Q4 showed the highest promoter activity. The Q1 and Q4 led the highest promoter activity during IAA and cold stress. These findings help to understand the molecular mechanism of ScHAK10 expression regulation and could be an excellent tool for future crop improvement. Saccharum officinarum L. ScHAK10 promoter Abiotic stress Promoter analysis cis-acting element Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Sugarcane is a high-efficiency C 4 crop with higher nutrient requirements. Therefore, the key to achieving high sugarcane yield maintaining sufficient nutrient supply. An adequate nutrient supply is crucial for high sugarcane yield [1-3]. The HAK potassium transporter gene family is an important K + transporter that plays a major role in the absorption and transport of potassium in sugarcane. The identification and functional analysis of the promoter region of the HAK potassium transporter gene family has emerged as a novel approach to enhancing potassium uptake efficiency and developing potassium-efficient sugarcane varieties. Currently, there are few studies on the promoter of HAK potassium transporter. It was found that the AP2/ERF (ethylene response factor) transcription factor RAP2.11 binds to the GCC-box region of the AtHAK5 promoter under growing conditions. Under low potassium conditions, ARF2 phosphorylated and reduced its binding ability to the promoter region of the AtHAK5 gene, resulting in the high expression of the AtHAK5 [4]. In addition, it has also been found that some transcription factors such as DDF2 (dwarf and delayed flowering 2), JLO (jagged lateral organs), and bHLH121 (basic helix-loop-helix 121) may bind to the promoter of HAK5 gene [5-7], which can respond and activate the expression of high-affinity potassium transporter ( HAK5 ). Overexpression of these transcription factors can enhance the growth of Arabidopsis thaliana roots under low potassium stress[7]. These studies demonstrated that the enhancing expression regulation of potassium transporter genes can increase the growth tolerance of plants during low potassium conditions [3]. Promoters are crucial for the regulation of gene expression, types and number of cis -acting elements affect the expression pattern and intensity of genes[8]. Therefore, it is necessary to isolate and assess its function as the promoter of genes and clarify the molecular mechanism to improving the efficiency of gene expression in sugarcane. Currently, research on the expression regulation of HAK genes mainly focuses on Arabidopsis and rice, and there have not yet to be any report on sugarcane. In our previous study, the CD sequence of the ScHAK10 was cloned, and preliminary studies on its sequence characteristics and expression properties were performed [9]. The transcript levels of ScHAK10 were highly expressed in leaf and stem at the maturation stage. Moreover, the expression of ScHAK10 induced by salinity and low potassium conditions. Transgenic A. thaliana that overexpression of ScHAK10 under CaMV35S promoter were more resistant to cold and drought conditions. Studies on the mechanism of regulating the expression of this gene have performed. This study aimed to identify the structure and function of the ScHAK10 promoter to understand the regulatory mechanism of the HAK gene and provide a theoretical basis for manipulating the expression and function of potassium transport-related genes in sugarcane. Materials And Methods Plant materials and growth conditions The sugarcane variety GuiTang 42 (GT 42) was used for cloning the ScHAK10 promoter. This variety was produced from the Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, Guangxi, China. Healthy sugarcane budsetts were planted in the greenhouse of Sugarcane Research Institute (average temperature, 28°C, soil water content 25±5%) during plant growth establishment. Leaf samples were collected at seedling stage, frozen in liquid nitrogen and stored at -80°C for DNA isolation. Arabidopsis seeds were disinfected with 75% ethanol for 1 min, then disinfected with sterile water once again, and then disinfected with 20% sodium hypochlorite disinfectant for 10 min. The seeds were placed on a clean bench to dry after thoroughly cleaning with disinfectant water six times. The disinfected seeds were vernalized for three days (4 ℃) and then grown on MS medium (18-22°C). The photoperiod in the artificial climatic chamber was 16 h light and 8 h of darkness. Arabidopsis seedlings were transplanted into pots at the four-leaf stage and grown in the atmospheric environmental conditions. After four weeks, plants were used for genetic transformation for ScHAK10 promoter analysis. DNA extraction and ScHAK10 promoter Genomic DNA was extracted from sugarcane leaves by DNA extraction kit (TIANGEN Biotech, Beijing, China). Following the instructions of the TaKaRa Genome Walking Kit (Takara, Biotech, Tokyo, Japan). Three pairs of downstream primers were designed for the 5'end of the ScHAK10 gene CDs sequence (NCBI No: MG564721), and multiplex PCR amplification was based on the sugarcane genomic DNA as a template. Based on the sequence of ScHAK10 , Primer5.0 designed downstream primers, and the primer sequence is: pScHAK10-SP1:5’-TGAGCGTGAGCGTCCAGAAGACGAA-3’; pScHAK10-SP2:5’-ACTCTGGTACGCCAGGCTCAGCGTCAT-3’； pScHAK10-SP3:5’-GACATGGGCATTCCGAGATTCAAG-3’. Perform a multiplex PCR reaction using P1, P2, P3, and P4 primers from the TaKaRa Genome Walking Kit as forward primers and pScHAK10 -SP1, pScHAK10 -SP2, and pScHAK10 -SP3 as reverse primers. Analyze by using 1% agarose gel electrophoresis. Extract the third phase PCR product from the gel and send it to TaKaRa for sequencing. Analysis of the ScHAK10 promoter sequences The promoter sequence (1918 bp) of the ScHAK10 gene was analyzed for cis-acting elements and their position using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and PlantCE (https://www.dna.affrc.go.jp/PLACE/?action=newplace) Vector construction and genetic transformation Based on the analysis of the cis -acting elements of the promoter, different lengths of forward and reverse sequences were designed for amplification (Table 1). Gene-specific primers were used to amplify the promoter sequence of pScHAK10 , which consisted of 1918 bp (Full), 1623 bp (Q1), 1332 bp (Q2), 957 bp (Q3), 576 bp (Q4), and 357 bp (Q5) fragments. The PCR procedure was as follows: 95°C for 5 min; 35 Cycle of 95℃ for 30 sec, 55℃ for 30 sec, and 72 ℃ for 30 sec; 72 ℃ for 5 min. Follow the instructions in the Seamless Clone kit (Tsingke Biotechnology, Beijing, China) and transform into competent cells of Escherichia coli DH5α. Follow the instructions of the seamless cloning kit to ligate and transform into E. coli DH5α capable cells. Observed single clones for sequencing identification. Digest the pEASY-T plasmid containing the full, Q1, Q2, Q3, Q4, and Q5 promoter regions with Hind III/ Sma I, and then ligate it to the pBI121 expression vector containing the β-glucuronidase (GUS) gene, replacing the 35S promoter upstream of GUS. Construct the recombinant vector pScHAK10 -pBI121. Table 1 : Forward and reverse primers for different lengths of pScHAK10 promoter Primer name Primer sequence (5′-3′) HAK10P-F0 gaccatgattacgccaagcttGGACTCTATAGGGCGATTGAG HAK10P-F1 gaccatgattacgccaagcttCGGAAATTTCACACATTCTTGTTTT HAK10P-F2 gaccatgattacgccaagcttAAGGGTATTTTTTTTATTAAAATCT HAK10P-F3 gaccatgattacgccaagcttCAGATTAGTGTCCATGCTCGGT HAK10P-F4 gaccatgattacgccaagcttTTAATCTATTTTTTCTTTGGATCCT HAK10P-F5 gaccatgattacgccaagcttATATTCTCTCTCCGCTCTGCAC HAK10P-R taagggactgaccacccgggCGCTGCCTCCACTGCCCGGT Identification of transgenic Arabidopsis thaliana The recombinant plasmid with the expression vector was introduced into Agrobacterium strain EHA104 by cryogenic method and transformed into Arabidopsis plants using the floral dip transformation approach [10]. Transgenic Arabidopsis was identified through baster resistance selection and PCR detection. The primer sequence for PCR detection is: F: 5'- gaccatgattacgccaagcttATATTCTCTCTCCGCTCTGCAC-3'; R: 5'-CTGATCAATTCCACAGTTTTC-3' The T 0 transgenic seeds were planted on MS medium containing hygromycin resistance for screening. Screening were designated as T 1 transgenic plants. After the T 1 plants had grown up to two leaves, transplanted into vermiculite and placed in a growth chamber (temperature 22℃ and 16 h photoperiod per day). Leaf samples was taken from the T 1 transgenic Arabidopsis plants during the seedling stage for PCR analysis, and the plants with positive PCR results were harvested for T 1 seeds at maturity. Transgenic Arabidopsis seedlings grown under abiotic stress The two-week-old transgenic A. thaliana seedlings were grown on MS medium and transferred to adverse environmental condition of MS medium supplemented with 50 μM KCl, 200 mM NaCl, low temperature (4℃), and 150 μM IAA. The seedlings were exposed to cold chamber for 3 h for low-temperature (4℃) treatment. After stress condition, qualitative and quantitative GUS analyses were performed. Histochemical GUS staining and fluorometric assay Histochemical GUS staining was performed according to Chao et al [10]. Collect plant organs, such as stems and leaves from A. thaliana . Added GUS staining solution, incubated at 37°C for 24 hr, and then used 70% ethanol to decolorize until the background color completely disappeared. Finally, we observed and photographed the staining results under the dissecting microscope (AXIO Zoom.V16, Zeiss). Determination of GUS enzyme activity in transgenic plants Following the directions of the GUS enzyme activity kit manual, select transgenic A. thaliana , frozen, ground and added GUS extraction solution (1 ml). The mixture was centrifuged (15,000 rpm), and the supernatant was used for the measurement of protein content. Combined 40 µL of protein extract with 160 µL of protein reaction solution (containing P-nitrophenyl-β-D-glucuronide, PNPG) and incubated the mixture at 37℃ in the dark. Once incubation was complete, added reaction stop solution (200 µL). The GUS fluorometric assay was measured with the emission at 405 nm using an enzyme-linked immunosorbent assay. GUS activity was expressed as 4-methylumbelliferone (4-MU) produced per min per g of protein. Paraffin section of transgenic Arabidopsis thaliana roots Place the stained tissue in a container filled with fixative. A vacuum pump removes air from the container, allowing the fixative to saturate the material fully. Add alcohol (70%) for preservation. Gradually dehydrate the material using ethanol solutions of increasing concentrations, such as 85, 95, and 100%, followed by another round of 100% ethanol. The dehydrated material should be submerged in a mixture of xylene and ethanol to achieve clarity. The xylene to 100% ethanol ratio is either 1:2, 1:1, or 2:1. Shift material in pure xylene. Then, load the transparent material into wax cups for impregnation and encapsulation. Cut sections at a thickness of 50μm for microscopic observation. Results Clone of pScHAK10 promoter The genomic DNA of sugarcane variety GuiTang 42 (GT 42) was used as a template in multiplex PCR reactions, with AP1, AP2, AP3, and AP4 from TaKaRa Genome Walking Kit as forward primers and pScHAK10 -SP1, pScHAK10 -SP2, and pScHAK10 -SP3 as reverse primers. Sequencing of the PCR products revealed a 2.1 kb fragment amplified by the AP3 forward primer. This fragment contains approximately 1918bp of the promoter sequence upstream of the ScHAK10 gene in sugarcane, as assessed by the sequence alignment analysis. Bioinformatics analysis of the pScHAK10 promoter The promoter region of the ScHAK10 contains a diverse array of regulatory elements. It includes 28 TATA-boxes and 41 CAAT-boxes, crucial for gene transcription initiation. In addition to these core elements, the promoter also harbors four plant hormone-responsive elements, i.e., ABRE, CGTCA-motif, TGA-element, and TGACG-motif. Identified six light-responsive factors (Box 4, G-box, GA-motif, GT1-motif, I-box, TCT-motif), five stress-responsive elements (LTR, TC-rich repeats, WUN-motif, GT1GMSCAM4), and one transcription factor binding site (CCAAT-box). These results suggested that the expression of ScHAK10 influenced by different environmental factors such as light, phytohormones, etc. Table 2 : Analysis of cis -acting elements in the pScHAK10 Element type Name Copy number Motif sequence Function Basal element TATA-box 28 TATA/ATATAT/TTTTA Core promoter element CAAT-box 43 CAATT/CAAT/CCAAT Common cis-acting elements in promoter and enhancer regions Phytohormone ABRE 2 ACGTG AACCCGG cis-acting regulatory element involved in the abscisic acid responsiveness CGTCA-motif 2 CGTCA cis-acting regulatory element involved in the Me JA-responsiveness TGA-element 1 AACGAC Auxin-responsive element TGACG-motif 2 TGACG cis-acting regulatory element involved in the Me JA-responsiveness Light Box 4 3 ATTAAT Part of a light-responsive module G-box 1 CACGTC cis-acting regulatory element involved in light responsiveness GA-motif 1 ATAGATAA Part of a light-responsive element GT1-motif 1 GGTTAA Light responsive element I-box 1 GGATAAGGTG Part of a light-responsive element TCT-motif 1 TCTTAC Part of a light-responsive element Stress LTR 1 CCGAAA cis-acting element involved in low-temperature stress responsiveness TC-rich repeats 1 ATTCTCTAAC cis-acting element involved in defense and stress responsiveness WUN-motif 3 AAATTTCCT AAATTACT Mechanical injury response element GT1GMSCAM4 7 GAAAAA salt-induced Other CCAAT-box 2 CAACGG MYBHv1 transcription factor binding site Identification of transgenic Arabidopsis thaliana The transgenic A. thaliana with the pScHAK10 promoter was screened on a Kanamycin-resistant MS medium. Subsequently, PCR detection was performed on the seedlings of the transgenic A. thaliana . The result shows that the target band was amplified from the seedlings of the transgenic A. thaliana (Fig. 3), while no target band was amplified from the A. thaliana . It indicates that the detected transgenic A. thaliana were positive plants. GUS histochemical staining analysis of transgenic Arabidopsis thaliana GUS histochemical staining was performed on transgenic A. thaliana. The results showed that GUS driven by pScHAK10 was mainly expressed in the leaves, petals, and root tips (Fig. 4). The staining intensity of the stem leaves was higher than that of the rosette leaves. The GUS gene was primarily localized in the veins of the rosette leaves, while it was expressed in the veins and stomata of the stem leaves. The GUS gene was predominantly expressed in the corolla of the transgenic A. thaliana flowering buds, with no expression detected in the receptacle or anthers. GUS activity was mainly observed in the root tip region and root hair zone. Paraffin sectioning of the root tips of the transgenic A. thaliana revealed that the promoter predominantly drove GUS expression in the vessel tissue of the root tips (Fig. 5). The activity of GUS in transgenic Arabidopsis thaliana under abiotic stress The activity of each promoter fragment was analyzed in A. thaliana . During control conditions, GUS expression was observed in transgenic plants containing the Full (1918bp), Q1 (1623bp), Q2 (1332 bp), Q3 (957bp), Q4 (576bp) and Q5 (357bp) fragments. It was observed that Full, Q1, Q2, Q3, and Q4 could be stained, while Q5 could not be impaired. The GUS activity analysis revealed that the GUS expression level of Q1 was the highest, followed by Full and Q2, while Q5 could not drive GUS expression. This result indicated that the activity of each promoter segment driving expression in A. thaliana was Q1>Full=Q2>Q3>Q4>Q5. The promoter fragment Q5 had lost its promoter activity (Fig. 6), possibly related to the lack of enhancer elements. After transferring the active promoter fragments into Arabidopsis , their activities were detected under low-potassium, salinity, cold (4℃), and IAA treatments. The results showed that the GUS activity of Q3 and Q4 promoter fragments in transgenic A. thaliana was the highest, while the activities of Full, Q1, and Q2 decreased significantly. Under auxin (IAA) stress, the GUS activity of the Q1 promoter fragment in transgenic A. thaliana was the highest. Under low temperature, decreased significantly in the GUS activity of Full, Q1, and Q2 in transgenic A. thaliana (Fig. 6 and 7). Discussion The promoter consists of two major factors, such as the core region, which contains the core promoter element (TATA-box), and the regulatory area, which includes responsive cis-acting and enhancer elements. The type and quantity of regulatory elements directly affect the expression pattern and intensity of genes [3,8]. The GhHAK5 gene expressed under low potassium conditions in cotton plants. The promoter sequence of this gene, pGhHAK5 , contains various cis-acting elements associated with functions such as light induction, plant hormones, and stress responses. These elements suggest that the GhHAK5 gene can be influenced by multiple external factors[11]. The research findings indicated that the pScHAK10 promoter of sugarcane contains several cis-elements, including light response, plant hormone, and stress response cis-elements, in addition to the basic TATA box and CAAT box. Plant hormone and light response cis-elements are major plant growth and development regulators. Phytohormone and light signals can induce promoter expression and regulate genes[12]. The pScHAK10 promoter contains not only light response and hormone cis-elements but also various stress response elements, such as LTR (low temperature), ARE (anaerobic), and GT1 (salinity). However, the promoter may be induced by stress. Based on their expression patterns and promoters can be classified into three types, i.e., constitutive promoters, tissue-specific promoters, and inducible promoters. Constitutive promoters are expressed in all parts of the plant and are unaffected by atmospheric environmental variables. Tissue-specific promoters express only in specific tissues, while inducible promoters typically express high under specific conditions [2,13]. However, in some cases, a specific type of promoter can exhibit characteristics of other promoters. For example, the potassium transporter HAK5 is mainly expressed in the root systems. The promoters of the AtHAK5 gene in Arabidopsis and OsHAK5 gene in rice drive the GUS gene to express in the root system, which tissue specificity but also can be induced by low potassium conditions to stimulate gene expression [14-16]. Most HAK genes in plants are expressed mainly in the root system with tissue specificity, while a few members are expressed in multiple plant tissues [17]. However, recent studies have found that the EgHAK5 promoter in Eucalyptus derives GUS expression not only in the Arabidopsis root system but also in the leaf vein and embryo axis vessel tissue [16]. Present study found that the GUS driven by pScHAK10 was expressed in the root system and leaves of A. thaliana. Staining and sectioning of the root system revealed that GUS was mainly expressed in the vascular tissue of the root tip. The experimental results indicated that the pScHAK10 promoter is active and can promote potassium absorption efficiency in the plant root system. The core functional region of a promoter is typically the proximal region close to the transcription initiation site of the functional gene. Still, cis-elements located at the distal end of the promoter sequence can also affect the activation activity of the promoter [18]. The CAAT-box, position, and orientation not significantly impact their functions. By adding enhancer elements, the activation activity of the promoter can be greatly increased. Four enhancers were connected in series to increase the transcription level of the OsNAS3 gene. The resulting transcript was 30-60 times higher than the wild-type [19]. In this study, deletion analysis showed that the sequence length of the pScHAK10 promoter decreased, the promoter activity gradually reduced. As shown in Figure 6, transgenic A. thaliana of deletion mutants (Full, Q1, Q2, Q3) could be stained, while Q5 could not be impaired. The Q1 promoter fragment drove the highest expression of GUS, while the shortest Q5 promoter fragment had the lowest activity and could not drive GUS staining. The action of Q1 and Q2 promoters were higher than those of other fragments, possibly due to more CAAT-box enhancer elements in the promoters. In A. thaliana , the expression of AtHAK5 is not only affected by abiotic stresses such as low potassium, low calcium, and salt stress [20], but also can be induced by low nitrogen and low phosphorus[2,21]. The regulation of potassium transporter gene expression is influenced by two transcription factors, such as ARF2 and RAP2.11, and can involve multiple other transcription factors[7]. The GCC-box site is an important motif in the promoter sequence of AtHAK5 in A. thaliana that binds to the transcription factor RAP2.11[22]. The binding of transcription factor RAP2.11 to the ERE domain and GCC-box in the AtHAK5 promoter is essential for activating AtHAK5 gene expression under low potassium conditions [22]. In addition, the GhHAK5 promoter binding site (TGTCNN) involved in transcriptional regulation under low potassium conditions is also an important motif for ARF transcription factor binding [11]. The pScHAK10 promoter analyzed by bioinformatics contains multiple potential cis-acting elements related to abiotic stresses, including low potassium (ERE, ATTTCATA), high salt (GT1, GGTTAA), low temperature (LTR, CCGAAA), and phytohormones (TGA-element). These elements were also identified in the dehydration-responsive component binding protein (DREB1) promoter and reported to function in drought and cold-induced gene expression [7]. The pScHAK10 promoter sequence of sugarcane does not contain GCC box sites and (TGTCNN) sites but contains an ERE structure, indicating that the expression of RAP2.11 may regulate in sugarcane. In this study, the activity of the full, Q1, and Q2 under low potassium stress significantly downregulated. At the same time, Q3 and Q4 significantly increased, indicating that there may be negative regulatory factors upstream of the promoter. Under saline condition, high-affinity K+ transport proteins promote K + uptake in plants, balance K + /Na + ratio and improving salt tolerance capacity[23-25]. The promoter elements responsive to high salt stress that have been reported as GT1GMSCAM4 and GT1CONSENSUS. The GT1GMSCAM4 element has been reported to regulate salt stress expression [26]. The GAAAAA (GT-1 cis-acting element) sequence has been identified in the SCAM-4 promoter as a core cis-acting element responsive to pathogens and salt induction[27-28]. This sequence significantly enhances salt tolerance in soybean and Arabidopsis. This study found that the pScHAK10 promoter contains the GT1GMSCAM4 element (GAAAAA). The full-length pScHAK10 promoter and Q2 contain GT-1 cis-acting elements. However, under saline stress (200 mM), the GUS enzyme activity of the full-length pScHAK10 promoter and Q2 significantly reduced, indicating that the GAAAAA sequence in the full-length promoter and Q2 regions not enhance salt tolerance in transgenic Arabidopsis plants . Other unknown core sequences in the promoter may affect its salt response-ability. The significant loss in expression activity under salt stress suggests that this region may contain inhibitory cis-acting elements. Q1, Q3, and Q4 promoter fragments contain GT1GMSCAM4 elements; their GUS activities significantly increased under salt stress, indicating that the -961bp to -1918bp fragment may contain enhancer elements and other unknown cis-acting elements that respond to salt induction and enhance gene expression. Auxin is a vital hormone for proper plant growth and development, associated in various processes, including fruit formation and abscission [29-31]. However, it is currently unknown whether pScHAK10 can be activated by auxin. In this study, a potential auxin response element TGA (-1381bp) was identified in the promoter of pScHAK10 through cis-acting element prediction. It was found that auxin can significantly enhance the gene expression of Q1 and Q4 segments of the pScHAK10 promoter, indicating that the auxin response element present in the promoter or auxin indirectly activates the promoter expression via various mechanisms[32]. The cis-acting LTR element is widely present in the promoters of genes related to the low-temperature stress response[33]. It plays a significant role in the plant's response to abiotic stresses. Cucumber GR-RBP3 was induced by low-temperature stress, and an LTR element (-565bp) identified in its promoter region[34]. This study found that the activities of Full, Q1, and Q2 were significantly reduced, while the activity of the Q4 segment was increased under low temperatures. This result indicates that the low-temperature response element LTR in this promoter plays a negative regulatory role in the expression regulation of its gene during low temperatures. It can be functional elements related to cold stress between -576bp and -357bp in the promoter. Conclusion In this study, the upstream promoter of the ScHAK10 gene ( pScHAK10 ) was cloned and functionally analyzed from the sugarcane variety GT42. The results of bioinformatics prediction analysis indicated that the pScHAK10 promoter sequence includes TATA-box core elements, light-responsive elements, phytohormone-responsive elements, stress-response elements, and some specific protein-binding cis-acting elements. A fusion expression vector ( pScHAK10 -GUS) was constructed and successfully transformed into A. thaliana. By analyzing the expression and tissue-specific localization of GUS in transgenic A. thaliana , it was found that pScHAK10 could drive the expression of the GUS gene in A. thaliana root, corolla, and leaf tissues, indicating that the cloned pScHAK10 promoter is an active tissue-specific promoter. By transferring different promoter fragments into A. thaliana and subjecting them to stress, the analysis of GUS activity in transgenic A. thaliana suggests that the region between 296bp and 1561bp may be a key region for plant response to low potassium, salt, cold, and phytohormone stresses. The research findings provide theoretical basis for further elucidating the key regulatory elements of the pScHAK10 promoter and its response mechanism to atmospheric environmental stresses. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests Author's Contributions Conceptualization, Luo HB and Huang CM; Methodology, Luo HB and Wei YW; Software, Xu L and Wu XJ; Supervision, Deng ZN. Wu KC. Wei YW; Data Curation, Luo HB and Ye LP; Writing Original Draft Preparation, Luo HB; Writing Review and Editing, Luo HB and Yi XP; Project Administration, Luo HB; Funding Acquisition, Luo HB and Huang CM. Funding This work was supported by Natural Science Foundation of Guangxi Province (2022GXNSFAA035444; 2023GXNSFAA026482), Basic Scientific Research Projects of Guangxi Academy of Agricultural Sciences (Guinong ke2020YM107; Guinongke 2024YP087) and Fundamental Research Fund of Guangxi Academy of Agriculture Sciences (2021YT118). References [1] Li Q-W, Lu Y-L, Zhou W-L, Chen D-W, Ao J-H, Jiang Y(2011)Effects of Low Potassium Stress on Growth and Photosynthetic Characteristics of Different Sugarcane Lines. Sugarcane and Canesugar (6):1-5. [2] Herrera WFB, Arruda B, de Carvalho HWP, Pavinato PS(2022) Improving potassium use efficiency of sugarcane through the use of polyhalite. CABI Agric Biosci 3. 2022, 55. https://doi.org/10.1186/s43170-022-00124-4 [3] Castro SG, Coelho AP, Castro SA, Souza Chiachia TR, Castro RA, Lemos LB(2023) Fertilizer source and application method influence sugarcane production and nutritional status. 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Plant Cell 9:2281-2289. https://doi.org/10.1105/tpc.9.12.2281 [15] Ahn SJ, Shin R, Schachtman DP(2004) Expression of KT/KUP genes in Arabidopsis and the role of root hairs in K + uptake. Plant Physiology 134(3): 1135-1145. https://doi.org/10.1104/pp.103.034660 [16] Costa CS, Bravo JP, Ribeiro CL, Soprano AS, Sassaki FT, Maia IG(2017) Vascular expression is driven by the promoter of a gene encoding a high-affinity potassium transporter HAK5 from Eucalyptus grandis . Plant Cell, Tissue and Organ Culture 131:213-222. https:/doi.org/10.1007/s11240-017-1276-6 [17] Bañuelos MA, Garciadeblas B, Cubero B, Rodríguez-Navarro A(2002) Inventory and functional characterization of the HAK potassium transporters of rice. Plant Physiology 130(2):784-795. https://doi.org/10.1104/pp.007781 [18] Blankvoort S, Descamps L, Kentros C(2020) Enhancer-driven gene expression (EDGE) enables the generation of cell type specific tools for the analysis of neural circuits. 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Plant 152:558-570. https://doi.org/10.1111/ppl.12205 [22] Kim MJ, Ruzicka D, Shin R, Schachtman DP(2012) The Arabidopsis AP2/ERF transcription factor RAP2.11 modulates plant response to low-potassium conditions. Molecular Plant 5(5):1042-1057. https://doi:%2010.1093/mp/sss003 [23] Zhu M-M, Zhang F-Y, Lv Z-Y, Shen Q, Zhang L, Lu X, Jiang W-M, Fu X-Q, Yan T-X, Chen L-X, Wang G-F, Tang K-X(2014) Characterization of the promoter of Artemisia annua Amorpha-4,11-dime Synthase (ADS) gene using homologous and heterologous expression as well as deletion analysis. Plant Mol. Biol. Rep 32:406-418. https://doi.org/10.1007/s11105-013-0656-2. [24] Chinnusamy V, Jagendorf A, Zhu J-K(2005) Understanding and improving salt tolerance in plants. Crop Sci 45:437-448. https://doi.org/10.2135/cropsci2005.0437 [25] Feng S-Y(2007) Cloning and characterization of a high affinity K + transporter gene from Aeluropus littoralis . Masteral dissertation. Dalian, Dalian University of Technology, pp 61-76. [26] Liang M-H, Lu Y, Chen H-H, Jiang J-G(2017) The salt-regulated element in the promoter of lycopene β-cyclase gene confers a salt regulatory pattern in carotenogenesis of Dunaliella bardawil . Environ Microbiol 19:982-989. https://doi.org/10.1111/1462-2920.13539 . [27] Park HC, Kim ML, Kang YH, Jeon JM, Yoo JH, Kim MC, Park CY, Jeong JC, Moon BC, Lee JH, Yoon HW, Lee SH, Chung WS, Lim CO, Lee SY, Hong JC, Cho MJ(2004) Pathogen- and NaCl-induced expression of the SCaM-4 promoter is mediated in part by a GT-1 box that interacts with a GT-1-like transcription factor. Plant Physiol 135(4):2150-2161. https://doi.org/10.1104/pp.104.041442 [28] Chow CN, Chiang-Hsieh YF, Chien CH, Zheng HQ, Lee TY, Wu NY, Tseng KC, Hou PF, Chang WC(2018) Delineation of condition specific Cis - and Trans -acting elements in plant promoters under various Endo- and exogenous stimuli. BMC Genomics 19(Suppl 2),85. https://doi.org/10.1186/s12864-018-4469-4 [29] Ni Y-W, Lin K-H, Chen K-H, Wu C-W, Chang Y-S(2020) Flavonoid compounds and photosynthesis in passiflora plant leaves under varying light intensities. Plants 9(5):633-651. https://doi.org/10.3390/plants9050633 [30] Li J-Y, Tao X-Y, Li L, Mao L-C, Luo Z-S, Khan Z-U, Ying T-J(2016) Comprehensive RNA-Seq analysis on the regulation of tomato ripening by exogenous auxin. PLoS One 11:5. https://doi.org/10.1371/journal.pone.0156453 [31] Olatunji D, Geelen D, Verstraeten I(2017) Control of Endogenous Auxin Levels in Plant Root Development. Int. J. Mol. Sci 18(12):2587. https://doi.org/10.3390/ijms18122587 [32] Grzybkowska D, Nowak K, Gaj MD(2020) Hypermethylation of auxin-responsive motifs in the promoters of the transcription factor genes accompanies the somatic embryogenesis induction in arabidopsis. Int. J. Mol. Sci 21(18):6849-6871. https://doi.org/10.3390/ijms21186849 [33] Dunn MA, White AJ, Vural S, Hughes MA(1998) Identification of promoter elements in a low-temperature-responsive gene ( blt4.9 ) from barley ( Hordeum vulgare L.). Plant Mol Biol 38(4):551-564. https://doi.org/10.1023/A:1006098132352 [34] Wang B, Huang Y-Y, Yi J-Y,Yuan Y(2022) Molecular cloning of cucumber GR-BP3 promoter and induction of low temperature on its activity. Shandong Agric. Sci 54(7):15-23. Supplementary Files QQ20240128174951.png Fig 6 Cite Share Download PDF Status: Posted Version 3 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-3844377\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":269927281,\"identity\":\"fb3dd478-e269-4a4e-b881-774278b10b12\",\"order_by\":0,\"name\":\"Hai-Bin 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03:16:27\",\"currentVersionCode\":3,\"declarations\":{\"humanSubjects\":false,\"vertebrateSubjects\":false,\"conflictsOfInterestStatement\":false,\"humanSubjectEthicalGuidelines\":false,\"humanSubjectConsent\":false,\"humanSubjectClinicalTrial\":false,\"humanSubjectCaseReport\":false,\"vertebrateSubjectEthicalGuidelines\":false,\"coiExplicitlySet\":false},\"doi\":\"10.21203/rs.3.rs-3844377/v3\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-3844377/v3\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":51566973,\"identity\":\"9140d136-4c65-4a79-8513-dffd2e56e9d9\",\"added_by\":\"auto\",\"created_at\":\"2024-02-23 19:35:16\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":10158,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePCR products of \\u003cem\\u003eScHAK10 \\u003c/em\\u003epromoter. 1-3：PCR products and M： DNA marker (DL 2000)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3844377/v3/a54e13ddf47f3b5bcfd307c3.jpg\"},{\"id\":51567192,\"identity\":\"d6e7b019-c765-4290-b075-d6bc307476a4\",\"added_by\":\"auto\",\"created_at\":\"2024-02-23 19:43:15\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":86576,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSchematic of the \\u003cem\\u003epScHAK10\\u003c/em\\u003e promoter deletion-GUS construct. Fragments of the promoter of varying sizes were inserted into the pBI121 vector containing the GUS reporter gene.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3844377/v3/22221c7e2ba3dfc4e460ea67.jpg\"},{\"id\":51566974,\"identity\":\"5838cae3-4154-4c36-9c78-c4702a74331f\",\"added_by\":\"auto\",\"created_at\":\"2024-02-23 19:35:17\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":29641,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEnzyme restriction sites PCR products of promoter fragment. 1-2: Q1 fragments with transgenic \\u003cem\\u003eA. thaliana\\u003c/em\\u003e, 3-4: Q2 fragments with transgenic \\u003cem\\u003eA. thaliana, \\u003c/em\\u003e5-6: Q3 fragments with transgenic \\u003cem\\u003eA. thaliana, \\u003c/em\\u003e7-8: Q4 fragments with transgenic \\u003cem\\u003eA. thaliana, \\u003c/em\\u003e9-10: Q5 fragments with transgenic \\u003cem\\u003eA. thaliana, \\u003c/em\\u003e11: DL 2000 DNA marker, 12-13: \\u003cem\\u003epScHAK10\\u003c/em\\u003ewith transgenic \\u003cem\\u003eA. thaliana \\u003c/em\\u003eand 14-15: Non-transgenic control\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3844377/v3/7bb1aca86293c72525c59d33.jpg\"},{\"id\":51566971,\"identity\":\"0ef22b62-6a0c-4272-a36e-0974573f5587\",\"added_by\":\"auto\",\"created_at\":\"2024-02-23 19:35:16\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":574936,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eHistochemical staining of GUS expression in transgenic plants containing \\u003cem\\u003epScHAK10\\u003c/em\\u003e promoters. A: \\u003cem\\u003epScHAK10 \\u003c/em\\u003etransgenic \\u003cem\\u003eA. thaliana, \\u003c/em\\u003eA1: enlarged view of transgenic \\u003cem\\u003eA. thaliana\\u003c/em\\u003e rosette leaf, A2: enlarged view of transgenic \\u003cem\\u003eA. thaliana\\u003c/em\\u003e stem leaf, A3: enlarged view of transgenic \\u003cem\\u003eA. thaliana \\u003c/em\\u003ecorolla, A4: enlarged view of transgenic \\u003cem\\u003eA. thaliana \\u003c/em\\u003eanther, B: \\u003cem\\u003epScHAK10 \\u003c/em\\u003etransgenic \\u003cem\\u003eA. thaliana \\u003c/em\\u003eroot and B1: enlarged view of transgenic \\u003cem\\u003eA. thaliana \\u003c/em\\u003eroot tips\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3844377/v3/29cc136ab01dbec6943b6d3b.png\"},{\"id\":51566970,\"identity\":\"bfa8580d-4c14-48f3-8648-7ddf4fcd1725\",\"added_by\":\"auto\",\"created_at\":\"2024-02-23 19:35:16\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":25854,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eParaffin sections of transgenic \\u003cem\\u003eArabidopsis \\u003c/em\\u003eroot tips.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3844377/v3/1812a380b05e227f86af4d7d.jpg\"},{\"id\":51566972,\"identity\":\"113f5837-736f-44f2-9b08-3b522be22486\",\"added_by\":\"auto\",\"created_at\":\"2024-02-23 19:35:16\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1127903,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eHistochemical assay for GUS expression in transgenic \\u003cem\\u003eA. thaliana\\u003c/em\\u003e treated with low potassium, salinity, IAA and cold (4℃) stress conditions. The treated transgenic plants were incubated overnight in GUS staining solution at 37 ℃. Later, treated with ethyl alcohol for 4-h to remove chlorophyll and taken photographed.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3844377/v3/ef738d80e0682f5781d81581.png\"},{\"id\":51566967,\"identity\":\"8fb6db06-31c8-4d77-a549-456562bf2234\",\"added_by\":\"auto\",\"created_at\":\"2024-02-23 19:35:15\",\"extension\":\"jpg\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":45764,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAnalysis of GUS activity for \\u003cem\\u003epScHAK10\\u003c/em\\u003e deletions in transformed \\u003cem\\u003eA. thaliana\\u003c/em\\u003e under different stress conditions. The low potassium stress condition was simulated by 50 nmol K\\u003csup\\u003e+\\u003c/sup\\u003e in MS medium. Salt stress (200 mmol L\\u003csup\\u003e−1\\u003c/sup\\u003e) was affected by the MS medium. 4 °C simulated the cold stress for 3 hr. The IAA\\u0026nbsp; (150 μmol L\\u003csup\\u003e−1\\u003c/sup\\u003e) stress was simulated in MS medium. The GUS activity was expressed as nmol 4-methylumbelliferone/min/g protein. Bars represent the mean, and error bars (±) represent SD from three independent experiments. Different letters represented significant differences among treatments at \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure7.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3844377/v3/0ce954f249b5ce52ebe7e3be.jpg\"},{\"id\":51567459,\"identity\":\"dc72a9f4-5c6c-4556-a97a-a1b8144155e1\",\"added_by\":\"auto\",\"created_at\":\"2024-02-23 19:51:15\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2222277,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3844377/v3/45160b51-6268-44ef-8da8-d13b00c7f02f.pdf\"},{\"id\":51566968,\"identity\":\"08cf195a-2038-4376-9015-1025898aaaa6\",\"added_by\":\"auto\",\"created_at\":\"2024-02-23 19:35:15\",\"extension\":\"png\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1402679,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eFig 6\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"QQ20240128174951.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3844377/v3/a700e3edf640f8b110240e60.png\"}],\"financialInterests\":\"\",\"formattedTitle\":\"\\u003cp\\u003e\\u003cstrong\\u003eMolecular cloning and functional analysis of \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eScHAK10 \\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003egene promoter from sugarcane (\\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eSaccharum officinarum \\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003eL.)\\u003c/strong\\u003e\\u003c/p\\u003e\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eSugarcane is a high-efficiency C\\u003csub\\u003e4\\u003c/sub\\u003e crop with higher nutrient requirements. Therefore, the key to achieving high sugarcane yield maintaining sufficient nutrient supply. An adequate nutrient supply is crucial for high sugarcane yield [1-3]. The \\u003cem\\u003eHAK\\u003c/em\\u003e potassium transporter gene family is an important K\\u003csup\\u003e+\\u003c/sup\\u003e transporter that plays a major role in the absorption and transport of potassium in sugarcane. The identification and functional analysis of the promoter region of \\u003cem\\u003ethe HAK\\u003c/em\\u003e potassium transporter gene family has emerged as a novel approach to enhancing potassium uptake efficiency and developing potassium-efficient sugarcane varieties. Currently, there are few studies on the promoter of \\u003cem\\u003eHAK\\u003c/em\\u003e potassium transporter. It was found that the \\u003cem\\u003eAP2/ERF\\u003c/em\\u003e (ethylene response factor) transcription factor \\u003cem\\u003eRAP2.11\\u003c/em\\u003e binds to the GCC-box region of the \\u003cem\\u003eAtHAK5\\u003c/em\\u003e promoter under growing conditions. Under low potassium conditions, \\u003cem\\u003eARF2\\u003c/em\\u003e phosphorylated and reduced its binding ability to the promoter region of the \\u003cem\\u003eAtHAK5\\u003c/em\\u003e gene, resulting in the high expression of the \\u003cem\\u003eAtHAK5\\u0026nbsp;\\u003c/em\\u003e[4]. In addition, it has also been found that some transcription factors such as \\u003cem\\u003eDDF2\\u003c/em\\u003e (dwarf and delayed flowering 2), \\u003cem\\u003eJLO\\u003c/em\\u003e (jagged lateral organs), and \\u003cem\\u003ebHLH121\\u003c/em\\u003e (basic helix-loop-helix 121) may bind to the promoter of \\u003cem\\u003eHAK5\\u0026nbsp;\\u003c/em\\u003egene [5-7], which can respond and activate the expression of high-affinity potassium transporter (\\u003cem\\u003eHAK5\\u003c/em\\u003e). Overexpression of these transcription factors can enhance the growth of \\u003cem\\u003eArabidopsis thaliana\\u003c/em\\u003e roots under low potassium stress[7]. These studies demonstrated that the enhancing expression regulation of potassium transporter genes can increase the growth tolerance of plants during low potassium conditions [3].\\u003c/p\\u003e\\n\\u003cp\\u003ePromoters are crucial for the regulation of gene expression, types and number of \\u003cem\\u003ecis\\u003c/em\\u003e-acting elements affect the expression pattern and intensity of genes[8].\\u0026nbsp;Therefore, it is necessary to isolate and assess its function as the promoter of genes\\u0026nbsp;and clarify the molecular mechanism to improving the efficiency of gene expression in sugarcane. Currently, research on the expression regulation of \\u003cem\\u003eHAK\\u003c/em\\u003e genes mainly focuses on Arabidopsis and rice, and there have not yet to be any report on sugarcane. In\\u0026nbsp;our previous study,\\u0026nbsp;the CD sequence of the \\u003cem\\u003eScHAK10\\u003c/em\\u003e was cloned, and preliminary studies on its sequence characteristics and expression properties were performed\\u0026nbsp;[9]. The transcript levels of \\u003cem\\u003eScHAK10\\u003c/em\\u003e were\\u0026nbsp;highly expressed in leaf and stem at the maturation stage. Moreover, the expression of\\u0026nbsp;\\u003cem\\u003eScHAK10\\u003c/em\\u003e induced by salinity and low potassium conditions. Transgenic\\u003cem\\u003e\\u0026nbsp;A. thaliana\\u003c/em\\u003e that overexpression of\\u0026nbsp;\\u003cem\\u003eScHAK10\\u003c/em\\u003e under\\u0026nbsp;\\u003cem\\u003eCaMV35S\\u003c/em\\u003e promoter were more resistant to cold and drought conditions.\\u0026nbsp;Studies on the mechanism of regulating the expression of this gene have performed.\\u0026nbsp;This study aimed to identify the structure and function of the\\u0026nbsp;\\u003cem\\u003eScHAK10\\u003c/em\\u003e promoter\\u0026nbsp;to understand the regulatory mechanism of \\u003cem\\u003ethe HAK\\u003c/em\\u003e gene and provide a theoretical basis for manipulating the expression and function of potassium transport-related genes in sugarcane.\\u003c/p\\u003e\"},{\"header\":\"Materials And Methods\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003ePlant materials and growth conditions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe sugarcane variety GuiTang 42 (GT 42) was used for cloning the \\u003cem\\u003eScHAK10\\u003c/em\\u003e promoter. This variety was produced from the Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, Guangxi, China. Healthy sugarcane budsetts were planted in the greenhouse of Sugarcane Research Institute\\u0026nbsp;(average temperature, 28\\u0026deg;C,\\u0026nbsp;soil water content 25\\u0026plusmn;5%) during plant growth establishment.\\u0026nbsp;Leaf samples were collected at seedling stage, frozen in liquid nitrogen and\\u0026nbsp;stored at -80\\u0026deg;C for DNA isolation. Arabidopsis seeds were disinfected with 75% ethanol for 1 min, then disinfected with sterile water once again, and then disinfected with 20% sodium hypochlorite disinfectant for 10 min. The seeds were placed on a clean bench to dry after thoroughly cleaning with disinfectant water six times. The disinfected seeds were vernalized for three days (4 ℃) and then grown on MS medium (18-22\\u0026deg;C). The photoperiod in the artificial climatic chamber was 16 h light and 8 h of darkness. Arabidopsis seedlings were transplanted into pots at the four-leaf stage and grown in the atmospheric environmental conditions. After four weeks, plants were used for genetic transformation for\\u0026nbsp;\\u003cem\\u003eScHAK10\\u003c/em\\u003e promoter analysis.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDNA extraction and \\u003cem\\u003eScHAK10\\u003c/em\\u003e promoter\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eGenomic DNA was extracted from sugarcane leaves by DNA extraction kit (TIANGEN Biotech, Beijing, China). Following the instructions of the TaKaRa Genome Walking Kit (Takara, Biotech, Tokyo, Japan). Three pairs of downstream primers were designed for the 5\\u0026apos;end of the \\u003cem\\u003eScHAK10\\u003c/em\\u003e gene CDs sequence (NCBI No: MG564721), and multiplex PCR amplification was based on the sugarcane genomic DNA as a template. Based on the sequence of \\u003cem\\u003eScHAK10\\u003c/em\\u003e, Primer5.0 designed downstream primers, and the primer sequence is:\\u003c/p\\u003e\\n\\u003cp\\u003epScHAK10-SP1:5\\u0026rsquo;-TGAGCGTGAGCGTCCAGAAGACGAA-3\\u0026rsquo;;\\u003c/p\\u003e\\n\\u003cp\\u003epScHAK10-SP2:5\\u0026rsquo;-ACTCTGGTACGCCAGGCTCAGCGTCAT-3\\u0026rsquo;；\\u003c/p\\u003e\\n\\u003cp\\u003epScHAK10-SP3:5\\u0026rsquo;-GACATGGGCATTCCGAGATTCAAG-3\\u0026rsquo;.\\u003c/p\\u003e\\n\\u003cp\\u003ePerform a multiplex PCR reaction using P1, P2, P3, and P4 primers from the TaKaRa Genome Walking Kit as forward primers and \\u003cem\\u003epScHAK10\\u003c/em\\u003e-SP1, \\u003cem\\u003epScHAK10\\u003c/em\\u003e-SP2, and \\u003cem\\u003epScHAK10\\u003c/em\\u003e-SP3 as reverse primers. Analyze by using 1% agarose gel electrophoresis. Extract the third phase PCR product from the gel and send it to TaKaRa for sequencing.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAnalysis of the \\u003cem\\u003eScHAK10\\u003c/em\\u003e promoter sequences\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe promoter sequence (1918 bp) of the \\u003cem\\u003eScHAK10\\u003c/em\\u003e gene was analyzed for cis-acting elements and their position using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and PlantCE (https://www.dna.affrc.go.jp/PLACE/?action=newplace)\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eVector construction and genetic transformation\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eBased on the analysis of the \\u003cem\\u003ecis\\u003c/em\\u003e-acting elements of the promoter, different lengths of forward and reverse sequences were designed for amplification (Table 1). Gene-specific primers were used to amplify the promoter sequence of \\u003cem\\u003epScHAK10\\u003c/em\\u003e, which consisted of 1918 bp (Full), 1623 bp (Q1), 1332 bp (Q2), 957 bp (Q3), 576 bp (Q4), and 357 bp (Q5) fragments. The PCR procedure was as follows: 95\\u0026deg;C for 5 min; 35 Cycle of 95℃ for 30 sec, 55℃ for 30 sec, and 72 ℃ for 30 sec; 72 ℃ for 5 min. Follow the instructions in the Seamless Clone kit (Tsingke Biotechnology, Beijing, China) and transform into competent cells of \\u003cem\\u003eEscherichia coli\\u0026nbsp;\\u003c/em\\u003eDH5\\u0026alpha;. Follow the instructions of the seamless cloning kit to ligate and transform into \\u003cem\\u003eE. coli\\u003c/em\\u003e DH5\\u0026alpha; capable cells. Observed single clones for sequencing identification. Digest the pEASY-T plasmid containing the full, Q1, Q2, Q3, Q4, and Q5 promoter regions with \\u003cem\\u003eHind\\u003c/em\\u003eIII/ \\u003cem\\u003eSma\\u003c/em\\u003eI, and then ligate it to the pBI121 expression vector containing the \\u0026beta;-glucuronidase (GUS) gene, replacing the 35S promoter upstream of GUS. Construct the recombinant vector \\u003cem\\u003epScHAK10\\u003c/em\\u003e-pBI121.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\\n\\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;line-height:150%;'\\u003e\\u003cspan style=\\\"line-height:150%;color:black;\\\"\\u003eTable\\u0026nbsp;\\u003c/span\\u003e\\u003cspan style=\\\"line-height:150%;color:black;\\\"\\u003e1\\u003c/span\\u003e\\u003cspan style=\\\"line-height:150%;color:black;\\\"\\u003e: Forward and reverse primers for different lengths of pScHAK10 promoter\\u003c/span\\u003e\\u003c/p\\u003e\\n\\u003ctable style=\\\"border-collapse:collapse;border:none;\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 138.25pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003ePrimer name\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 276.55pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003ePrimer sequence (5\\u0026prime;-3\\u0026prime;)\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 138.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eHAK10P-F0\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 276.55pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003egaccatgattacgccaagcttGGACTCTATAGGGCGATTGAG\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 138.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eHAK10P-F1\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 276.55pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003egaccatgattacgccaagcttCGGAAATTTCACACATTCTTGTTTT\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 138.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eHAK10P-F2\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 276.55pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003egaccatgattacgccaagcttAAGGGTATTTTTTTTATTAAAATCT\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 138.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eHAK10P-F3\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 276.55pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;line-height:115%;'\\u003e\\u003cspan style=\\\"line-height:115%;color:black;\\\"\\u003egaccatgattacgccaagcttCAGATTAGTGTCCATGCTCGGT\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 138.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eHAK10P-F4\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 276.55pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003egaccatgattacgccaagcttTTAATCTATTTTTTCTTTGGATCCT\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 138.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eHAK10P-F5\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 276.55pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;line-height:115%;'\\u003e\\u003cspan style=\\\"line-height:115%;color:black;\\\"\\u003egaccatgattacgccaagcttATATTCTCTCTCCGCTCTGCAC\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 138.25pt;border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003eHAK10P-R\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 276.55pt;border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;line-height:115%;'\\u003e\\u003cspan style=\\\"line-height:115%;color:black;\\\"\\u003etaagggactgaccacccgggCGCTGCCTCCACTGCCCGGT\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eIdentification of transgenic \\u003cem\\u003eArabidopsis thaliana\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe recombinant plasmid with the expression vector was introduced into Agrobacterium strain \\u003cem\\u003eEHA104\\u003c/em\\u003e by cryogenic method and transformed into \\u003cem\\u003eArabidopsis\\u003c/em\\u003e plants using\\u0026nbsp;the floral dip transformation approach [10]. Transgenic \\u003cem\\u003eArabidopsis\\u003c/em\\u003e was identified through baster resistance selection and PCR detection. The primer sequence for PCR detection is: F: 5\\u0026apos;-\\u0026nbsp;gaccatgattacgccaagcttATATTCTCTCTCCGCTCTGCAC-3\\u0026apos;;\\u0026nbsp;R: 5\\u0026apos;-CTGATCAATTCCACAGTTTTC-3\\u0026apos;\\u003c/p\\u003e\\n\\u003cp\\u003eThe T\\u003csub\\u003e0\\u003c/sub\\u003e transgenic seeds were planted on MS medium containing hygromycin resistance for screening. Screening were designated as T\\u003csub\\u003e1\\u003c/sub\\u003e transgenic plants. After the T\\u003csub\\u003e1\\u003c/sub\\u003e plants had grown up to two leaves, transplanted into vermiculite and placed in a growth chamber (temperature 22℃ and 16 h photoperiod per day). Leaf samples was taken from the T\\u003csub\\u003e1\\u003c/sub\\u003e transgenic \\u003cem\\u003eArabidopsis\\u003c/em\\u003e plants during the seedling stage for PCR analysis, and the plants with positive PCR results were harvested for T\\u003csub\\u003e1\\u003c/sub\\u003e seeds at maturity.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTransgenic \\u003cem\\u003eArabidopsis\\u003c/em\\u003e seedlings grown under abiotic stress\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe two-week-old transgenic \\u003cem\\u003eA. thaliana\\u003c/em\\u003e seedlings were grown on MS medium and transferred to adverse environmental condition of MS medium supplemented with 50 \\u0026mu;M KCl, 200 mM NaCl, low temperature (4℃), and 150\\u0026nbsp;\\u0026mu;M IAA. The seedlings were exposed to cold chamber for 3 h for low-temperature (4℃) treatment. After stress condition, qualitative and quantitative GUS analyses were performed.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eHistochemical GUS staining and fluorometric assay\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eHistochemical GUS staining was performed according to Chao et al [10]. Collect plant organs, such as stems and leaves from \\u003cem\\u003eA. thaliana\\u003c/em\\u003e. Added GUS staining solution, incubated at 37\\u0026deg;C for 24 hr, and then used 70% ethanol to decolorize until the background color completely disappeared. Finally, we observed and photographed the staining results under the dissecting microscope (AXIO Zoom.V16, Zeiss).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDetermination of GUS enzyme activity in transgenic plants\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFollowing the directions of the GUS enzyme activity kit manual, select transgenic \\u003cem\\u003eA. thaliana\\u003c/em\\u003e, frozen, ground and added GUS extraction solution (1 ml). The mixture was centrifuged (15,000 rpm), and the supernatant was used for the measurement of protein content. Combined 40 \\u0026micro;L of protein extract with 160 \\u0026micro;L of protein reaction solution (containing P-nitrophenyl-\\u0026beta;-D-glucuronide, PNPG) and incubated the mixture at 37℃ in the dark. Once incubation was complete, added reaction stop solution (200 \\u0026micro;L). The GUS fluorometric assay was measured with the emission at 405 nm using an enzyme-linked immunosorbent assay. GUS activity was expressed as 4-methylumbelliferone (4-MU) produced per min per g of protein.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eParaffin section of transgenic \\u003cem\\u003eArabidopsis thaliana\\u003c/em\\u003e roots\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ePlace the stained tissue in a container filled with fixative. A vacuum pump removes air from the container, allowing the fixative to saturate the material fully. Add alcohol (70%) for preservation. Gradually dehydrate the material using ethanol solutions of increasing concentrations, such as 85, 95, and 100%, followed by another round of 100% ethanol. The dehydrated material should be submerged in a mixture of xylene and ethanol to achieve clarity. The xylene to 100% ethanol ratio is either 1:2, 1:1, or 2:1. Shift material in pure xylene. Then, load the transparent material into wax cups for impregnation and encapsulation. Cut sections at a thickness of 50\\u0026mu;m for microscopic observation.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eClone of \\u003cem\\u003epScHAK10\\u003c/em\\u003e promoter\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe genomic DNA of sugarcane variety GuiTang 42 (GT 42) was used as a template in multiplex PCR reactions, with AP1, AP2, AP3, and AP4 from TaKaRa Genome Walking Kit as forward primers and \\u003cem\\u003epScHAK10\\u003c/em\\u003e-SP1, \\u003cem\\u003epScHAK10\\u003c/em\\u003e-SP2, and \\u003cem\\u003epScHAK10\\u003c/em\\u003e-SP3 as reverse primers. Sequencing of the PCR products revealed a 2.1 kb fragment amplified by the AP3 forward primer. This fragment contains approximately 1918bp of the promoter sequence upstream of the \\u003cem\\u003eScHAK10\\u003c/em\\u003e gene in sugarcane, as assessed by the sequence alignment analysis.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eBioinformatics analysis of the \\u003cem\\u003epScHAK10\\u003c/em\\u003e promoter\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe promoter region of the \\u003cem\\u003eScHAK10\\u003c/em\\u003e contains a diverse array of regulatory elements. It includes 28 TATA-boxes and 41 CAAT-boxes, crucial for gene transcription initiation. In addition to these core elements, the promoter also harbors four plant hormone-responsive elements, i.e., ABRE, CGTCA-motif, TGA-element, and TGACG-motif. Identified six light-responsive factors (Box 4, G-box, GA-motif, GT1-motif, I-box, TCT-motif), five stress-responsive elements (LTR, TC-rich repeats, WUN-motif, GT1GMSCAM4), and one transcription factor binding site (CCAAT-box). These results suggested that the expression of \\u003cem\\u003eScHAK10\\u003c/em\\u003e influenced by different environmental factors such as light, phytohormones, etc.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\\n\\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;line-height:150%;'\\u003e\\u003cspan style=\\\"line-height:150%;color:black;\\\"\\u003eTable\\u0026nbsp;\\u003c/span\\u003e\\u003cspan style=\\\"line-height:150%;color:black;\\\"\\u003e2\\u003c/span\\u003e\\u003cspan style=\\\"line-height:150%;color:black;\\\"\\u003e: Analysis of \\u003cem\\u003ecis\\u003c/em\\u003e-acting elements in the \\u003cem\\u003epScHAK10\\u003c/em\\u003e\\u003c/span\\u003e\\u003c/p\\u003e\\n\\u003ctable style=\\\"width:432.1pt;border-collapse:collapse;border:none;\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eElement type\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 62.7pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eName\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 42.3pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eCopy number\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 100.05pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eMotif sequence\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 153.25pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eFunction\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eBasal \\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eelement\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 62.7pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eTATA-box\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 42.3pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e28\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 100.05pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eTATA/ATATAT/TTTTA\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 153.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eCore promoter element\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 62.7pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eCAAT-box\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 42.3pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e43\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 100.05pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eCAATT/CAAT/CCAAT\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 153.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eCommon cis-acting elements in promoter and enhancer regions\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 62.7pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 42.3pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 100.05pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 153.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003ePhytohormone\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 62.7pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eABRE\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 42.3pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e2\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 100.05pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eACGTG\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eAACCCGG\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 153.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003ecis-acting regulatory element involved in the abscisic acid responsiveness\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 62.7pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eCGTCA-motif\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 42.3pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e2\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 100.05pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eCGTCA\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 153.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003ecis-acting regulatory element involved in the Me JA-responsiveness\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 62.7pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eTGA-element\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 42.3pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e1\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 100.05pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eAACGAC\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 153.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eAuxin-responsive element\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 62.7pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eTGACG-motif\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 42.3pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp 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style=\\\"font-size:13px;color:black;\\\"\\u003ecis-acting regulatory element involved in the Me JA-responsiveness\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eLight\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 62.7pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eBox 4\\u003c/span\\u003e\\u003c/p\\u003e\\n 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style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003ePart of a light-responsive module\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 62.7pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New 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153.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003ecis-acting regulatory element involved in light responsiveness\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 62.7pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eGA-motif\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 42.3pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e1\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 100.05pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eATAGATAA\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 153.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003ePart of a light-responsive element\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n 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style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eGGTTAA\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 153.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eLight responsive element\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New 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100.05pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eGGATAAGGTG\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 153.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003ePart of a light-responsive element\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 62.7pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eTCT-motif\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 42.3pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e1\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 100.05pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eTCTTAC\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 153.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003ePart of a light-responsive element\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eStress\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 62.7pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eLTR\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 42.3pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e1\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 100.05pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eCCGAAA\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 153.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003ecis-acting element involved in low-temperature stress responsiveness\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 62.7pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eTC-rich repeats\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 42.3pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e1\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 100.05pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eATTCTCTAAC\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 153.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003ecis-acting element involved in defense and stress responsiveness\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 62.7pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e\\u0026nbsp;WUN-motif\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 42.3pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e3\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 100.05pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eAAATTTCCT\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eAAATTACT\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 153.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eMechanical injury response element\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 62.7pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eGT1GMSCAM4\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 42.3pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e7\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 100.05pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eGAAAAA\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 153.25pt;border: medium;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003esalt-induced\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 73.8pt;border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eOther\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 62.7pt;border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eCCAAT-box\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 42.3pt;border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003e2\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 100.05pt;border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eCAACGG\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 153.25pt;border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5.4pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"font-size:13px;color:black;\\\"\\u003eMYBHv1 transcription factor binding site\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp style='margin-top:6.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;font-size:16px;font-family:\\\"Times New Roman\\\",serif;text-align:justify;'\\u003e\\u003cspan style=\\\"color:black;\\\"\\u003e\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eIdentification of transgenic \\u003cem\\u003eArabidopsis thaliana\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe transgenic \\u003cem\\u003eA. thaliana\\u003c/em\\u003e with the\\u003cem\\u003e\\u0026nbsp;pScHAK10\\u003c/em\\u003e promoter was screened on a Kanamycin-resistant MS medium. Subsequently, PCR detection was performed on the seedlings of the transgenic \\u003cem\\u003eA. thaliana\\u003c/em\\u003e. The result shows that the target band was amplified from the seedlings of the transgenic \\u003cem\\u003eA. thaliana\\u0026nbsp;\\u003c/em\\u003e(Fig. 3), while no target band was amplified from the \\u003cem\\u003eA. thaliana\\u003c/em\\u003e. It indicates that the detected transgenic \\u003cem\\u003eA. thaliana\\u003c/em\\u003e were positive plants.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eGUS histochemical staining analysis of transgenic \\u003cem\\u003eArabidopsis thaliana\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eGUS histochemical staining was performed on transgenic \\u003cem\\u003eA. thaliana.\\u003c/em\\u003e The results showed that GUS driven by \\u003cem\\u003epScHAK10\\u003c/em\\u003e was mainly expressed in the leaves, petals, and root tips (Fig. 4). The staining intensity of the stem leaves was higher than that of the rosette leaves. The GUS gene was primarily localized in the veins of the rosette leaves, while it was expressed in the veins and stomata of the stem leaves. The GUS gene was predominantly expressed in the corolla of the transgenic \\u003cem\\u003eA. thaliana\\u003c/em\\u003e flowering buds, with no expression detected in the receptacle or anthers. GUS activity was mainly observed in the root tip region and root hair zone. Paraffin sectioning of the root tips of the transgenic \\u003cem\\u003eA. thaliana\\u003c/em\\u003e revealed that the promoter predominantly drove GUS expression in the vessel tissue of the root tips (Fig. 5).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eThe activity of GUS in transgenic \\u003cem\\u003eArabidopsis thaliana\\u0026nbsp;\\u003c/em\\u003eunder abiotic stress\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe activity of each promoter fragment was analyzed in \\u003cem\\u003eA. thaliana\\u003c/em\\u003e. During control conditions, GUS expression was observed in transgenic plants containing the Full (1918bp), Q1 (1623bp), Q2 (1332 bp), Q3 (957bp), Q4 (576bp) and Q5 (357bp) fragments. It was observed that Full, Q1, Q2, Q3, and Q4 could be stained, while Q5 could not be impaired. The GUS activity analysis revealed that the GUS expression level of Q1 was the highest, followed by Full and Q2, while Q5 could not drive GUS expression. This result indicated that the activity of each promoter segment driving expression in \\u003cem\\u003eA. thaliana\\u003c/em\\u003e was Q1\\u0026gt;Full=Q2\\u0026gt;Q3\\u0026gt;Q4\\u0026gt;Q5. The promoter fragment Q5 had lost its promoter activity (Fig. 6), possibly related to the lack of enhancer elements.\\u003c/p\\u003e\\n\\u003cp\\u003eAfter transferring the active promoter fragments into \\u003cem\\u003eArabidopsis\\u003c/em\\u003e, their activities were detected under low-potassium, salinity, cold (4℃), and IAA treatments. The results showed that the GUS activity of Q3 and Q4 promoter fragments in transgenic \\u003cem\\u003eA. thaliana\\u003c/em\\u003e was the highest, while the activities of Full, Q1, and Q2 decreased significantly. Under auxin (IAA) stress, the GUS activity of the Q1 promoter fragment in transgenic \\u003cem\\u003eA. thaliana\\u003c/em\\u003e was the highest. Under low temperature, decreased significantly in the GUS activity of Full, Q1, and Q2 in transgenic \\u003cem\\u003eA. thaliana\\u003c/em\\u003e (Fig. 6 and 7).\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eThe promoter consists of two major factors, such as the core region, which contains the core promoter element (TATA-box), and the regulatory area, which includes responsive cis-acting and enhancer elements. The type and quantity of regulatory elements directly affect the expression pattern and intensity of genes\\u0026nbsp;[3,8]. The \\u003cem\\u003eGhHAK5\\u0026nbsp;\\u003c/em\\u003egene expressed under low potassium conditions in cotton plants. The promoter sequence of this gene, \\u003cem\\u003epGhHAK5\\u003c/em\\u003e, contains various cis-acting elements associated with functions such as light induction, plant hormones, and stress responses. These elements suggest that the \\u003cem\\u003eGhHAK5\\u0026nbsp;\\u003c/em\\u003egene can be influenced by multiple external factors[11]. The research findings indicated that the \\u003cem\\u003epScHAK10\\u003c/em\\u003e promoter of sugarcane contains several cis-elements, including light response, plant hormone, and stress response cis-elements, in addition to the basic TATA box and CAAT box. Plant hormone and light response cis-elements are major plant growth and development regulators. Phytohormone and light signals can induce promoter expression and regulate genes[12]. The \\u003cem\\u003epScHAK10\\u003c/em\\u003e promoter contains not only light response and hormone cis-elements but also various stress response elements, such as LTR (low temperature), ARE (anaerobic), and GT1 (salinity). However, the promoter may be induced by stress.\\u003c/p\\u003e\\n\\u003cp\\u003eBased on their expression patterns and promoters can be classified into three types, i.e., constitutive promoters, tissue-specific promoters, and inducible promoters. Constitutive promoters are expressed in all parts of the plant and are unaffected by atmospheric environmental variables. Tissue-specific promoters express only in specific tissues, while inducible promoters typically express high under specific conditions\\u0026nbsp;[2,13]. However, in some cases, a specific type of promoter can exhibit characteristics of other promoters. For example, the potassium transporter \\u003cem\\u003eHAK5\\u003c/em\\u003e is mainly expressed in the root systems. The promoters of the \\u003cem\\u003eAtHAK5\\u003c/em\\u003e gene in Arabidopsis and \\u003cem\\u003eOsHAK5\\u003c/em\\u003e gene in rice drive the GUS gene to express in the root system, which tissue specificity but also can be induced by low potassium conditions to stimulate gene expression [14-16]. Most \\u003cem\\u003eHAK\\u003c/em\\u003e genes in plants are expressed mainly in the root system with tissue specificity, while a few members are expressed in multiple plant tissues\\u0026nbsp;[17]. However, recent studies have found that the \\u003cem\\u003eEgHAK5\\u003c/em\\u003e promoter in \\u003cem\\u003eEucalyptus\\u003c/em\\u003e derives GUS expression not only in the Arabidopsis root system but also in the leaf vein and embryo axis vessel tissue\\u0026nbsp;[16]. Present study found that the GUS driven by \\u003cem\\u003epScHAK10\\u003c/em\\u003e was expressed in the root system and leaves of \\u003cem\\u003eA. thaliana.\\u003c/em\\u003e Staining and sectioning of the root system revealed that GUS was mainly expressed in the vascular tissue of the root tip. The experimental results indicated that the \\u003cem\\u003epScHAK10\\u003c/em\\u003e promoter is active and can promote potassium absorption efficiency in the plant root system.\\u003c/p\\u003e\\n\\u003cp\\u003eThe core functional region of a promoter is typically the proximal region close to the transcription initiation site of the functional gene. Still, cis-elements located at the distal end of the promoter sequence can also affect the activation activity of the promoter [18]. The\\u0026nbsp;CAAT-box, position, and orientation not significantly impact their functions. By adding enhancer elements, the activation activity of the promoter can be greatly increased. Four enhancers were connected in series to increase the transcription level of the \\u003cem\\u003eOsNAS3\\u003c/em\\u003e gene. The resulting transcript was 30-60 times higher than the wild-type\\u0026nbsp;[19]. In this study, deletion analysis showed that the sequence length of the \\u003cem\\u003epScHAK10\\u003c/em\\u003e promoter decreased, the promoter activity gradually reduced. As shown in Figure 6, transgenic \\u003cem\\u003eA. thaliana\\u003c/em\\u003e of deletion mutants (Full, Q1, Q2, Q3) could be stained, while Q5 could not be impaired. The Q1 promoter fragment drove the highest expression of GUS, while the shortest Q5 promoter fragment had the lowest activity and could not drive GUS staining. The action of Q1 and Q2 promoters were higher than those of other fragments, possibly due to more CAAT-box enhancer elements in the promoters.\\u003c/p\\u003e\\n\\u003cp\\u003eIn \\u003cem\\u003eA. thaliana\\u003c/em\\u003e, the expression of \\u003cem\\u003eAtHAK5\\u003c/em\\u003e is not only affected by abiotic stresses such as low potassium, low calcium, and salt stress [20], but also can be induced by low nitrogen and low phosphorus[2,21]. The regulation of potassium transporter gene expression is influenced by two transcription factors, such as ARF2 and RAP2.11, and can involve multiple other transcription factors[7]. The GCC-box site is an important motif in the promoter sequence of \\u003cem\\u003eAtHAK5\\u003c/em\\u003e in \\u003cem\\u003eA. thaliana\\u003c/em\\u003e that binds to the transcription factor RAP2.11[22]. The binding of transcription factor RAP2.11 to the ERE domain and GCC-box in the \\u003cem\\u003eAtHAK5\\u003c/em\\u003e promoter is essential for activating \\u003cem\\u003eAtHAK5\\u003c/em\\u003e gene expression under low potassium conditions\\u0026nbsp;[22]. In addition, the \\u003cem\\u003eGhHAK5\\u003c/em\\u003e promoter binding site (TGTCNN) involved in transcriptional regulation under low potassium conditions is also an important motif for ARF transcription factor binding\\u0026nbsp;[11]. The \\u003cem\\u003epScHAK10\\u003c/em\\u003e promoter analyzed by bioinformatics contains multiple potential cis-acting elements related to abiotic stresses, including low potassium (ERE, ATTTCATA), high salt (GT1, GGTTAA), low temperature (LTR, CCGAAA), and phytohormones (TGA-element).\\u0026nbsp;These elements were also identified in the dehydration-responsive component binding protein (DREB1) promoter and reported to function in drought and cold-induced gene expression\\u0026nbsp;[7]. The\\u0026nbsp;\\u003cem\\u003epScHAK10\\u003c/em\\u003e promoter sequence of sugarcane does not contain GCC box sites and (TGTCNN) sites but contains an ERE structure, indicating that the expression of RAP2.11 may regulate in sugarcane. In this study, the activity of the full, Q1, and Q2 under low potassium stress significantly downregulated. At the same time, Q3 and Q4 significantly increased, indicating that there may be negative regulatory factors upstream of the promoter.\\u003c/p\\u003e\\n\\u003cp\\u003eUnder saline condition, high-affinity K+ transport proteins promote K\\u003csup\\u003e+\\u003c/sup\\u003e uptake in plants, balance K\\u003csup\\u003e+\\u003c/sup\\u003e/Na\\u003csup\\u003e+\\u003c/sup\\u003e ratio and improving salt tolerance capacity[23-25].\\u0026nbsp;The promoter elements responsive to high salt stress that have been reported as GT1GMSCAM4 and GT1CONSENSUS. The GT1GMSCAM4 element has been reported to regulate salt stress expression [26]. The GAAAAA (GT-1 cis-acting element) sequence has been identified in the \\u003cem\\u003eSCAM-4\\u003c/em\\u003e promoter as a core cis-acting element responsive to pathogens and salt induction[27-28]. This sequence significantly enhances salt tolerance in soybean and Arabidopsis. This study found that the \\u003cem\\u003epScHAK10\\u0026nbsp;\\u003c/em\\u003epromoter contains the GT1GMSCAM4 element (GAAAAA). The full-length \\u003cem\\u003epScHAK10\\u003c/em\\u003e promoter and Q2 contain GT-1 cis-acting elements. However, under saline stress (200 mM), the GUS enzyme activity of the full-length \\u003cem\\u003epScHAK10\\u003c/em\\u003e promoter and Q2 significantly reduced, indicating that the GAAAAA sequence in the full-length promoter and Q2 regions not enhance salt tolerance in transgenic \\u003cem\\u003eArabidopsis\\u0026nbsp;\\u003c/em\\u003eplants\\u003cem\\u003e.\\u003c/em\\u003e Other unknown core sequences in the promoter may affect its salt response-ability. The significant loss in expression activity under salt stress suggests that this region may contain inhibitory cis-acting elements. Q1, Q3, and Q4 promoter fragments contain GT1GMSCAM4 elements; their GUS activities significantly increased under salt stress, indicating that the -961bp to -1918bp fragment may contain enhancer elements and other unknown cis-acting elements that respond to salt induction and enhance gene expression.\\u003c/p\\u003e\\n\\u003cp\\u003eAuxin is a vital hormone for proper plant growth and development, associated in various processes, including fruit formation and abscission\\u0026nbsp;[29-31]. However, it is currently unknown whether \\u003cem\\u003epScHAK10\\u003c/em\\u003e can be activated by auxin. In this study, a potential auxin response element TGA (-1381bp) was identified in the promoter of \\u003cem\\u003epScHAK10\\u003c/em\\u003e through cis-acting element prediction. It was found that auxin can significantly enhance the gene expression of Q1 and Q4 segments of the \\u003cem\\u003epScHAK10\\u003c/em\\u003e promoter, indicating that the auxin response element present in the promoter or auxin indirectly activates the promoter expression via various mechanisms[32].\\u003c/p\\u003e\\n\\u003cp\\u003eThe cis-acting LTR element is widely present in the promoters of genes related to the low-temperature stress response[33]. It plays a significant role in the plant\\u0026apos;s response to abiotic stresses. Cucumber \\u003cem\\u003eGR-RBP3\\u0026nbsp;\\u003c/em\\u003ewas induced by low-temperature stress, and an LTR element (-565bp) identified in its promoter region[34]. This study found that the activities of Full, Q1, and Q2 were significantly reduced, while the activity of the Q4 segment was increased under low temperatures. This result indicates that the low-temperature response element LTR in this promoter plays a negative regulatory role in the expression regulation of its gene during low temperatures. It can be functional elements related to cold stress between -576bp and -357bp in the promoter.\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eIn this study, the upstream promoter of the \\u003cem\\u003eScHAK10\\u003c/em\\u003e gene (\\u003cem\\u003epScHAK10\\u003c/em\\u003e) was cloned and functionally analyzed\\u0026nbsp;from the sugarcane variety GT42.\\u0026nbsp;The results of bioinformatics prediction analysis indicated that the \\u003cem\\u003epScHAK10\\u003c/em\\u003e promoter sequence includes TATA-box core elements, light-responsive elements, phytohormone-responsive elements, stress-response elements, and some specific protein-binding cis-acting elements.\\u0026nbsp;A fusion expression vector (\\u003cem\\u003epScHAK10\\u003c/em\\u003e-GUS) was constructed and successfully transformed into \\u003cem\\u003eA. thaliana.\\u003c/em\\u003e By analyzing the expression and tissue-specific localization of GUS in transgenic \\u003cem\\u003eA. thaliana\\u003c/em\\u003e, it was found that \\u003cem\\u003epScHAK10\\u003c/em\\u003e could drive the expression of the GUS gene in \\u003cem\\u003eA. thaliana\\u003c/em\\u003e root, corolla, and leaf tissues, indicating that the cloned \\u003cem\\u003epScHAK10\\u003c/em\\u003e promoter is an active tissue-specific promoter.\\u0026nbsp;By transferring different promoter fragments into \\u003cem\\u003eA. thaliana\\u003c/em\\u003e and subjecting them to stress, the analysis of GUS activity in transgenic \\u003cem\\u003eA. thaliana\\u003c/em\\u003e suggests that the region between 296bp and 1561bp may be a key region for plant response to low potassium, salt, cold, and phytohormone stresses. The research findings provide theoretical basis for further elucidating the key regulatory elements of the \\u003cem\\u003epScHAK10\\u003c/em\\u003e promoter and its response mechanism to atmospheric environmental stresses.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and material\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no competing interests\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor\\u0026apos;s Contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eConceptualization, Luo HB and Huang CM; Methodology, Luo HB and Wei YW; Software, Xu L and Wu XJ; Supervision, Deng ZN. Wu KC. Wei YW; Data Curation, Luo HB and Ye LP; Writing Original Draft Preparation, Luo HB; Writing Review and Editing, Luo HB and Yi XP; Project Administration, Luo HB; Funding Acquisition, Luo HB and Huang CM.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by Natural Science Foundation of Guangxi Province (2022GXNSFAA035444; 2023GXNSFAA026482), Basic Scientific Research Projects of Guangxi Academy of Agricultural Sciences (Guinong ke2020YM107; Guinongke 2024YP087) and Fundamental Research Fund of Guangxi Academy of Agriculture Sciences (2021YT118).\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003cp\\u003e[1] Li Q-W, Lu Y-L, Zhou W-L, Chen D-W, Ao J-H, \\u0026nbsp;Jiang Y(2011)Effects of Low Potassium Stress on Growth and Photosynthetic Characteristics of Different Sugarcane Lines. 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Plant Mol Biol \\u0026nbsp;38(4):551-564.\\u0026nbsp;\\u003ca href=\\\"https://doi.org/10.1023/A:1006098132352\\\"\\u003ehttps://doi.org/10.1023/A:1006098132352\\u003c/a\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e[34]\\u0026nbsp;Wang B, Huang Y-Y, Yi J-Y,Yuan Y(2022) Molecular cloning of cucumber \\u003cem\\u003eGR-BP3\\u003c/em\\u003e promoter and induction of low temperature on its activity. Shandong Agric. Sci 54(7):15-23.\\u003c/p\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Saccharum officinarum L., ScHAK10 promoter, Abiotic stress, Promoter analysis, cis-acting element\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-3844377/v3\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-3844377/v3\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eTranscriptional regulation of the high-affinity K\\u003csup\\u003e+ \\u003c/sup\\u003e(\\u003cem\\u003eHAK\\u003c/em\\u003e) transporter gene is an important mechanism of plant response to low potassium stress. Cloning and analysis of the promoter of potassium transporter gene is not only helpful to its expression pattern and regulation mechanism but also important to improve the potassium absorption efficiency in sugarcane.\\u003cem\\u003e \\u003c/em\\u003eThe potassium transporter gene \\u003cem\\u003eScHAK10 \\u003c/em\\u003eis highly expressed and induced by low potassium stress in sugarcane，but the functions of its promoter is still unclear. In the present article, the 1918 bp promoter region of the \\u003cem\\u003eScHAK10 \\u003c/em\\u003egene (\\u003cem\\u003epScHAK10\\u003c/em\\u003e) was cloned by genomic walking technique. Computational analysis affirmed the existence of abiotic stress-responsive \\u003cem\\u003ecis \\u003c/em\\u003eand core \\u003cem\\u003ecis\\u003c/em\\u003e-elements, such as TATA box, CAAT box, phytohormone responsive, stress response and light response motifs. GUS histochemical staining of transgenic \\u003cem\\u003eArabidopsis thaliana\\u003c/em\\u003e seedlings showed that the leaf, corolla, and root tip were deeply stained, and paraffin sections of root tip tissue showed GUS staining in ductal tissue of \\u003cem\\u003eA. thaliana \\u003c/em\\u003eshallowly stained. The 5′-terminal deletion of the promoter was cloned, and the lengths of 1918 (full), 1623 (Q1), 1332 (Q2), 957 (Q3), 576 (Q4), and 357 bp (Q5) were cloned into the GUS reporter vector for \\u003cem\\u003eA. thaliana\\u003c/em\\u003e transient transformation. The transgenic plants generated through a single event exhibited a promising expression of the GUS reporter protein, which was treated with salt, low potassium, IAA and cold stress conditions. The results showed that the promoter activity correlates with the promoter fragment's length, and the long promoter fragment exhibits higher training. The Q5 was the least active and could not drive GUS expression. Under abiotic stress, the expression of GUS enzyme activity varies among different promoter fragments. Under low potassium and high salt stress, Q3 and Q4 showed the highest promoter activity. The Q1 and Q4 led the highest promoter activity during IAA and cold stress. These findings help to understand the molecular mechanism of \\u003cem\\u003eScHAK10\\u003c/em\\u003e expression regulation and could be an excellent tool for future crop improvement.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Molecular cloning and functional analysis of ScHAK10 gene promoter from sugarcane (Saccharum officinarum L.)\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":3,\"date\":\"2024-02-23 19:35:09\",\"doi\":\"10.21203/rs.3.rs-3844377/v3\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}},{\"code\":2,\"date\":\"2024-01-29 17:44:10\",\"doi\":\"10.21203/rs.3.rs-3844377/v2\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}},{\"code\":1,\"date\":\"2024-01-10 08:31:49\",\"doi\":\"10.21203/rs.3.rs-3844377/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"6d9fb6d2-f756-4fcc-94b1-0ea3f44e28d2\",\"owner\":[],\"postedDate\":\"February 23rd, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-01-24T09:29:13+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-02-23 19:35:09\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v3\",\"identity\":\"rs-3844377\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-3844377\",\"identity\":\"rs-3844377\",\"version\":[\"v3\"]},\"buildId\":\"WrCJVZZCHTDjtuVLN7oU0\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}