Positional cloning and functional verification of the epidermal protein gene KWMTBOMO04384 in silkworm, Bombyx mori

Positional cloning and functional verification of the epidermal protein gene KWMTBOMO04384 in silkworm, Bombyx mori | 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 Article Positional cloning and functional verification of the epidermal protein gene KWMTBOMO04384 in silkworm, Bombyx mori Juan Sun, Min Liu, Xin Zheng, Gui Ouyang, Heying Qian, Anli Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2558937/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A body shape mutant n08M with sunken intersegmental membrane was found during the feeding process of silkworm WT-n08. Genetic analysis showed that the mutant trait of n08M is controlled by a recessive gene located at the autosome and follows Mendelian inheritance. Results of positional cloning showed that the epidermal protein gene KWMTBOMO04384 on chromosome 26 was mutated. After KWMTBOMO04384 was knocked out by CRISPR/Cas9, the intersegmental membrane of silkworm was sunken. KWMTBOMO04384 was the target gene that caused the sunken intersegmental membrane in WT-n08 and had an important influence on the body shape of Bombyx mori . Biological sciences/Biological techniques Biological sciences/Molecular biology Bombyx mori Body shape mutant Genetic analysis Positional cloning CRISPR/Cas9 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Silkworms are important economic insects with a relatively perfect mutation system and are thus genetic model organisms second only to Drosophila 1 . After more than 5000 years of domestication and evolution, rich genetic mutation materials have accumulated. More than 600 species of silkworm mutants have been discovered and preserved in various regions of China 2 , and body color and body shape mutations are prevalent. Body color mutants include al 3 , L 4 , P S 5 , and bd 6 . Body shape mutants include mf-2 7 , tub 8 , Bo 9 , and sk 10 . Loss-of-function mutations in some genes usually result in changes in silkworm body shape. The 15 bp deletion of Bmsei led to the expression of a protein with loss of function, which resulted in the stubby body of cot mutant larvae exhibiting strong contraction, rolling, and vomiting behavior at high temperatures 11 . The nonsense mutation of BmorCPH 24 resulted in the bamboo-shaped larval body with an enlarged thoracic region and a slender but firm abdomen 9 . The specific deletion of 312 bp of Bmscarface changed the spatial structure of the protein, resulting in the tub mutant larvae body type being short and fat with a spindle shape 8 . The coding sequence mutant of BmorCPR2 led to the loss of the coding product’s ability to bind to chitin, resulting in the tight bodies of the mutant larvae, protruding internode folds, and serious defects in larval adaptability 12 . Revealing the cause of the body shape mutants of silkworm will help clarify the body shape regulation mechanism of insects. However, few mutants have been resolved at the molecular level. A new body shape mutant of silkworm was found during WT-n08 rearing. The intersegmental membrane of the mutant showed a sunken phenotype (Fig. 1 ). The mutant trait is controlled by a recessive gene located on the autosome. In this study, the mechanism of the mutant was elucidated through positional cloning and functional verification of the mutant gene. Results The n08M phenotype was controlled by a recessive gene. The body shape of the F 1 generation of n08M and P50 was normal. The self-crossing offspring of the F 1 generation showed the separation of normal and sunken individuals, and separation ratio was 3:1. Backcross populations had normal and sunken individuals, and the separation ratio was 1:1 (Table 1 ). The n08M phenotype was controlled by a recessive gene located on an autosome. Table 1 Trait segregation of each generation between the n08M and P50. Hybrid form Generation Normal number of individuals Number of mutants Total number of silkworms Separation ratio χ2 n08M×P50 F1 318 0 318 N — P50×n08M F1 394 0 394 N — (n08M·P50) × (n08M·P50) F2 279 103 382 3:1 0.78 (P50·n08M) × (P50·n08M) F2 311 116 427 3:1 1.07 (n08M·P50) × n08M BC1 F 206 185 391 1:1 1.13 (P50·n08M) × n08M BC1 F 205 219 424 1:1 0.46 n08M × (n08M·P50) BC1M 171 156 327 1:1 0.69 n08M × (P50·n08M) BC1M 173 159 332 1:1 0.59 Positional cloning. Genetic analysis showed that the mutant gene was located on the autosome. The BC 1 F population was genotyped with polymorphic SSR molecular markers of each autosome according to the nonexchange property of silkworm female chromosomes. The PCR product bands of the 14 BC 1 F individuals with sunken intersegmental membranes amplified with polymorphic SSR markers on chromosome 8 were consistent with n08M (Fig. 2 A). The PCR product bands of polymorphic molecular markers on the other 26 autosomes were distributed irregularly. Therefore, the gene controlling the sunken trait of n08M is located on the 8th linkage group of Bombyx mori . To locate and clone the mutant gene, seven SSR molecular markers were screened on the eighth linkage group. The mutant gene was located between the molecular markers S8-4-10(3) and S8-3-9(1) (Fig. 2 B, 2 C), and the distance between the markers was about 300 kb and included 12 candidate genes: KWMTBOMO04373 , KWMTBOMO04374 , KWMTBOMO04375 , KWMTBOMO04376 , KWMTBOMO04377 , KWMTBOMO04378 , KWMTBOMO04379 , KWMTBOMO04380 , KWMTBOMO04381 , KWMTBOMO04382 , KWMTBOMO04383 , and KWMTBOMO04384 ( https://kaikobase.dna.affrc.go.jp/ ). The analysis of the functions of 12 candidate genes with the silkworm website ( https://kaikobase.dna.affrc.go.jp/ ) showed that only KWMTBOMO04383 and KWMTBOMO04384 were related to the epidermis. Whether the sequences of the two genes had changed was determined by cloning their CDS sequences. The results showed that the sequence of KWMTBOMO04383 did not change, but the partial deletion of KWMTBOMO04384 exon 2 and intron 2 sequence occurred (Fig. 3 ), and a stop codon was generated in the functional region, resulting in the premature termination of gene expression. Moreover, the sequence of KWMTBOMO04384 gene had a frameshift mutation, and the lost function of the gene may have resulted in the sunken intersegmental membrane of n08M. Knockout validation. The function of KWMTBOMO04384 in silkworm epidermis was determined at the individual level, and sgRNA sites were designed on the exons 2 and 4 of KWMTBOMO04384. The phenotype of intersegmental membrane was sunken in the G2 generation after knockout (Fig. 4 ). The genomic DNA of 5 G2 generation individuals with sunken intersegmental membrane was extracted, and the sequences of two sgRNA sites were cloned. The results showed that a series of different sequence deletion was generated (Fig. 5 ). Quantitative reverse transcriptase PCR. The expression of KWMTBOMO04384 after knockout was detected through qRT-PCR. The expression of KWMTBOMO04384 in the knockout Nistari was significantly lower than that in the wild type (Fig. 6 ). Discussion The epidermis of insects covers the entire body surface, not only providing defense against pathogens and adverse environmental damage but also playing an essential role in shaping the body shape and maintaining normal activities during development 13 , 14 . Thus, the epidermis greatly enhances the survival and adaptability of insects, ensuring the evolution of insects into one of the most successful groups in the animal kingdom. Insect epidermis is composed of chitin and protein. The currently reported protein that can bind to chitin is mainly cuticular protein 15 , which accounts for about 1.5% of the total number of genes 16 . In the development and differentiation of the insect epidermis and the construction of important parts and organs outside the body, epidermal protein is an indispensable element and an important structural protein 17 . The silencing of the epidermal protein gene Abd-9 of Locusta migratoria led to thinning of the adult epidermis and reduction of endodermal delamination 18 . After the deletion of the epidermal protein gene TcCPAP3-B in Tribolium castaneum , the joints of the second pair of feet, the hindfoot joint, and the tibiotarsal joint became rigid and deformed. The epidermal protein gene TcCPAP3-B has been speculated to be involved in the construction of feet 19 . The silencing of the epidermal protein gene TcCPAP1 in Tribolium castaneum led to hypoplasia and shedding of the epidermis, and deletion of the epidermal genes TcCPAP3-A1 and TcCPAP3-A2 results in incomplete elytra 19 . The above findings indicate that epidermal proteins are indispensable for maintaining the integrity of insect cuticles. According to the sequence characteristics of insect epidermal proteins, epidermal proteins are divided into 12 families: CPR, CPF, CPFL, Tweedle, CPAP1, CPAP3, CPG and CPLC, etc 20 . The epidermal proteins of the CPR family, which is the largest, are the most widely distributed and the largest in number 21 . It plays an important role in the whole life activities of insects 22 . The silencing of the epidermal protein genes CPR27 and CPR18 of Tribolium castaneum resulted in structural abnormalities in the adult cuticle 23 . The silencing of the TcCPR27 gene led to disordered protocuticle structure and abnormal channel fibers, resulting in shortened and shrunken elytra. Adults died prematurely because of dehydration a week after emergence 24 . The silencing of genes for 15 CPR family epidermal protein members of Nilaparvata lugens resulted in insect death 25 . The members of the CPR family all contain Rebers & Riddiford (R&R) motif. Through the analysis of the R&R conserved motifs, the CPR family is divided into three subfamilies: RR-1 in the soft cuticle of insects, RR-2 in the hard cuticle of insects, and RR-3 in the old cuticle of insects 26 , 27 . KWMTBOMO04384 belongs to the RR-1 epidermal protein gene of the CPR family. The partial deletion of its gene sequence led to a frameshift mutation, which resulted in the sunken intersegmental membranes of the silkworm larvae. After the knocking out of the target gene with the CRISPR/Cas9 gene editing system, the mutant had a phenotype of sunken intersegmental membrane. In summary, the study demonstrated that KWMTBOMO04384 plays an important role in the formation of the silkworm epidermis and deepens understanding of the CPR family epidermal protein gene. Materials And Methods Silkworm strains. The wild-type strain P50 was preserved by Sericultural and Apicultural Research Institute, Yunnan Academy of Agricultural Sciences. The wild-type strain WT-n08 and its epidermis mutant strain (n08M) and the polyvoltine race of diapause-free Nistari and its transgenic race with Cas9 gene (Nistari-Cas9) were preserved by Sericultural Research Institute of Jiangsu University of Science and Technology. All Bombyx mori larvae were reared on fresh mulberry leaves at 25 ± 0.5°C and constant humidity of 75–80%. Genetic analysis of n08M. F 1 generation was generated by crossing P50 and n08M, and F 1 was self-crossed to produce the F 2 generation. The female moths of F 1 were backcrossed to the male moths of n08M to obtain BC 1 F, and female moths of n08M were backcrossed to the male moths of F 1 to produce BC 1 M. The phenotypes of each generation were observed, and the segregation ratio was calculated. Gene mapping. The genomic DNA of P50, n08M, F 1 , BC 1 F, and BC 1 M was extracted from the midgut of the fifth-instar larva with a cell/tissue genome DNA extraction kit (BioTeke, China). The concentration and purity of genomic DNA were determined with an ultra-micro spectrophotometer at 260/280 absorbance ratio, and the purified DNA was stored at − 20°C. Simple sequence repeat (SSR) markers for determining the linkage group of the mutant gene were obtained from a published SSR linkage map 28 , and the new SSR markers for the positional cloning were searched with SSR Hunter 1.3. The polymorphisms of SSR molecular markers were identified by agarose gel electrophoresis at a concentration of 2%. Given that the homologous chromosomes of female silkworms do not exchange, 10 BC 1 F wild-type individuals and 10 mutant BC 1 F individuals were used in determining the linkage group of the mutant gene, and 200 BC 1 M mutant individuals were used for the positional cloning of the mutant gene. The primers used for mapping are listed in Table S1 . Cloning gene. Specific primers (Table S1 ) were designed according to the gene sequence ( https://kaikobase.dna.affrc.go.jp/ ) in the location region. PCR amplification was performed using the genomic DNA of WT-n08 and n08M as template. Each 20 µL PCR reaction system consisted of 2 µL of 10× LA Taq Buffer, 3 µL of dNTP mixture, 0.2 µL of LA Taq enzyme, 1 µL of forward primer, 1 µL of reverse primer, 1 µL of DNA template, and 11.8 µL of ddH 2 O. The PCR reaction procedure was as follows: 94°C for 5 min, 31 cycles at 94°C for 30 s, 55°C for 30 s, 72°C for 2 min, and extension at 72°C for 10 min. The PCR product was detected by 1% agarose gel electrophoresis and cloned into a pMD19-T plasmid. Recombinant vectors were transformed into the competent cells of Escherichia coli and were sent for sequencing after identification by bacterial liquid PCR. Sequences were assembled and analyzed using BioEdit. Construction of target gene sgRNA vector and microinjection. The target gene sequence was amplified by using Nistari genomic DNA as a template. The online software CRISPRdirect ( http://crispr.dbcls.jp/ ) was used in searching two potential sgRNA targets in the ORF region of the sequenced target gene, and sgRNA primers (Table S1 ) were designed according to the sequence of sgRNA targets and transgenic vector pXL (IE1-DsRed). Each 50 µL PCR reaction system consisted of 25 µL of Primer STAR Premix (2×), 1.5 µL of forward primer (10 µmol/L), 1.5 µL of reverse primer (10 µmol/L), 2 µL of pXL (IE1-DsRed), and 20 µL of RNase-free H 2 O. The reaction procedure was as follows: 94°C for 2 min, 31 cycles at 94°C for 20 s, 56°C for 20 s, 72°C for 40 s, and extension at 72°C for 7 min. The PCR product was purified and recovered, and a homologous recombination was used in connecting the PCR product of the two sgRNA sites to the transgenic vector PXL (IE1-DsRed) linearized by SalI and NheI double digestion according to the instructions of In-Fusion Snap Assembly Master Mix kit (TaKaRa, Japan). The recombinant vector was transformed into the competent cells of Escherichia coli , and the samples were sent for sequencing after identification by bacterial liquid PCR. The recombinant plasmid was extracted by using QIAGEN Plasmid Midi Kit (QIAGEN, Germany). The recombined and helper plasmids were injected into lay 4–6 h of Nistari non-diapausing silkworm eggs by microinjection. After microinjection, the eggs (G0 generation) were incubated at a temperature of 25°C and humidity of 70–80%. Knockout validation. The G0 generation was reared under normal temperature and humidity conditions. G0 was self-crossed to produce the G1 generation. The newly-hatched larvae of the G1 generation were detected under a fluorescent stereomicroscope, and positive individuals with red fluorescence were screened. The moths of the positive individuals were crossed with the Cas9 transgenic silkworm to produce the G2 generation. The newly-hatched silkworms of the G2 generation were detected under a fluorescent stereomicroscope, and individuals that emitted red and green fluorescence were screened. Fifth instar larvae with sunken intersegmental membranes were selected, and the genome DNA was extracted by using a cell/tissue genome DNA extraction kit (BioTeke, China). The sequences of the sgRNA targets were detected with knockout detection primers (Table S1 ). Each 20 µL PCR reaction system consisted of 2 µL of 10× LA Taq buffer, 3 µL of dNTP mixture, 0.2 µL of LA Taq enzyme, 1 µL of forward primer, 1 µL of reverse primer, 1 µL of DNA template, and 11.8 µL of ddH 2 O. The PCR reaction procedure was as follows: 94°C for 5 min, 31 cycles at 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, and extension at 72°C for 10 min. The PCR products were cloned into the pMD19-T vector, and single clones were selected for sequencing. Quantitative reverse transcriptase PCR. Total RNAs were extracted from the epidermis of wild-type Nistari and G2 individuals with sunken intersegmental membranes by using RNAiso Plus (TaKaRa, Japan). RNA was reverse-transcribed using a PrimerScript RT reagent kit with gDNA eraser (TaKaRa, Japan) according to the manufacturer’s instructions. The cDNA products were diluted fivefold with ddH 2 O. Each 20 µL of the quantitative reverse transcriptase PCR (qRT-PCR) reaction system consisted of 10 µL of iTaq Universal SYBR Green Supermix (Bio-Rad, USA), 0.5 µL of each specific primer, 1 µL of cDNA template, and 8 µL of ddH 2 O, and three replicates were produced. qRT-PCR was performed in a CFX96 PCR system (Bio-Rad, USA) with a two-step reaction protocol of 40 cycles of 94°C for 5 s and 60°C for 1 min. The housekeeping B. mori actin 3 gene (GenBank ID: NM_001126254) 29 was used as a reference for eliminating bias among samples, and qRT-PCR results were converted and calculated using the 2 −ΔΔ Ct method 30 . The primers used for qRT-PCR are listed in Table S1 . Declarations Data availability The datasets generated or analysed during the current study are available in the Genbank repository, accession number: OQ547777. Acknowledgements This work was funded by China Agriculture Research System of MOF and MARA (No. CARS-18-ZJ0101), Key R & D plan of Jiangsu Province (Modern Agriculture) (No. BE2020418), the Key project of Education Department of Shaanxi Provincial Government (No. 20JS001) and the Applied Basic Research Programs of the Shaanxi Provincial Science and Technology Department (No. 2023-JC-YB-188). Author contributions J.S. and M. L. conducted all analyses, interpreted the results, and drafted the paper. A.C. designed the project. H.Q., X.Z. and G.O. contributed to interpretation of the analyses. All authors reviewed and approved the final manuscript. Competing interests The authors declare no competing interests. References Lu, C., Dai, F.Y. & Xiang, Z.H. Studies on the mutation strains of the Bombyx mori gene bank. Sci. Agric. Sin. 36, 968–975 (2003). Goldsmith, M.R., Shimada, T. & Abe, H. The genetics and genomics of the silkworm, Bombyx mori . Annu. Rev. Entomol. 50, 71–100 (2005). 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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-2558937","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":181486840,"identity":"41b2090d-e58b-4510-bd59-f987f08933c5","order_by":0,"name":"Juan Sun","email":"","orcid":"","institution":"Ankang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Sun","suffix":""},{"id":181486841,"identity":"b7ac8ab3-c2e7-45e5-b03e-fe9f6e23317f","order_by":1,"name":"Min Liu","email":"","orcid":"","institution":"The Sericultural and Apicultural Research Institute, Yunnan Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Liu","suffix":""},{"id":181486842,"identity":"2f1e9e8a-8df9-4f7e-85eb-8673896a753f","order_by":2,"name":"Xin Zheng","email":"","orcid":"","institution":"Ankang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Zheng","suffix":""},{"id":181486843,"identity":"2f163629-66b3-4ab4-8e05-10b787ff40f9","order_by":3,"name":"Gui Ouyang","email":"","orcid":"","institution":"Ankang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gui","middleName":"","lastName":"Ouyang","suffix":""},{"id":181486844,"identity":"6128b8f1-396b-485c-8463-06de544b09ca","order_by":4,"name":"Heying Qian","email":"","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Heying","middleName":"","lastName":"Qian","suffix":""},{"id":181486845,"identity":"975462cb-2a8e-4f9e-8a34-131417f0a58e","order_by":5,"name":"Anli Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIie3QoQ7CMBCA4S5LhtmYbQJhr1CCRfAoXUiYmq/sQnI1JFgE4THQ19T2AUgwzCMm52AWQXY4RD99f3J3jAXBX4o1dmq9SCYGqUnU2JPfraapl+Rk7zJw5YVvBC3IjdUu9XEFnEnWq+t4wn2p7VklNcwajA7+Pp4IVmp8+rSGOco4AkqStxoz4FXCpSAmfFgsAyHpCb+1eniyXMLwZEu6JT9uXdepV1EYYx+9IiQf8Mf5IAiC4Js37xBCweN6S2MAAAAASUVORK5CYII=","orcid":"","institution":"Ankang University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Anli","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2023-02-07 07:29:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2558937/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2558937/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":34172569,"identity":"eb11c56d-5aa5-408d-a784-85c2876e4690","added_by":"auto","created_at":"2023-03-13 14:56:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":338507,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypes of n08M and WT-n08. Intersegmental membrane of n08M mutant was sunken.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2558937/v1/00f8780698b3be4f3be1721f.jpg"},{"id":34170077,"identity":"bbca9964-6350-47ed-8b83-4b2720fb66cd","added_by":"auto","created_at":"2023-03-13 14:40:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":399937,"visible":true,"origin":"","legend":"\u003cp\u003eMapping linkage map of mutant genes. (A) Linkage analysis of the eighth linkage group of n08M. Lane 1 is P50, lane 2 is F\u003csub\u003e1\u003c/sub\u003e, lane 3 is n08M, and lanes 4–17 are BC\u003csub\u003e1\u003c/sub\u003eF individuals with sunken intersegmental membranes. (B, C) Fine mapping narrowed the region tightly linked to the n08M phenotype between SSR markers S8-4-10 and S8-3-9. The numbers in parentheses represent the number of individuals exchanged in the BC\u003csub\u003e1\u003c/sub\u003eM population.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2558937/v1/fa0abd0b63072b3aec45befb.jpg"},{"id":34171047,"identity":"7b3fb5eb-8f13-46cb-8097-b48be83c4a0e","added_by":"auto","created_at":"2023-03-13 14:48:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":306951,"visible":true,"origin":"","legend":"\u003cp\u003eGene cloning and sequence alignment of \u003cem\u003eKWMTBOMO04384\u003c/em\u003e. A: PCR amplification map of n08M\u003cem\u003e KWMTBOMO04384\u003c/em\u003e. The CDS sequence including introns was divided into two amplifications: n08M-1 and n08M-2. B: Alignment of \u003cem\u003eKWMTBOMO04384\u003c/em\u003egene sequences between WT-n08 and n08M mutants. The partial deletion of \u003cem\u003eKWMTBOMO04384\u003c/em\u003e exon 2 and intron 2 occurred, and a stop codon was generated in the functional region, resulting in the premature termination of gene expression. Black boxes are exons. Dashed lines are deletion sequences.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2558937/v1/31e12bea2b0624ac3d067738.jpg"},{"id":34168263,"identity":"7bc0f322-ccc9-4664-90e2-ac92afcad215","added_by":"auto","created_at":"2023-03-13 14:32:17","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":328449,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotype of wild-type Nistari and knockout Nistari. The intersegmental membrane of knockout individual was sunken.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2558937/v1/83738db744a16d695b033c54.jpg"},{"id":34170075,"identity":"4b0df57f-c3ad-4fa7-b2bc-66814bd03c93","added_by":"auto","created_at":"2023-03-13 14:40:17","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":291715,"visible":true,"origin":"","legend":"\u003cp\u003eCRISPR/Cas9 induces \u003cem\u003eKWMTBOMO04384\u003c/em\u003e mutation in G2. Wild type is wild-type Nistari, the red sequence is the sgRNA sites, dashed lines indicate the knockout sequence.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2558937/v1/54d6b6a24defe09f0915a6ef.jpg"},{"id":34168267,"identity":"a1538f50-65a9-446a-a833-ee7dd3f12f02","added_by":"auto","created_at":"2023-03-13 14:32:18","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":391168,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression levels of \u003cem\u003eKWMTBOMO04384\u003c/em\u003e between wild-type Nistari and knockout Nistari. Data in the figure are presented as mean \u003cem\u003e± SE\u003c/em\u003e of three biological replicates. The expression of \u003cem\u003eKWMTBOMO04384\u003c/em\u003e in the knockout Nistari was significantly lower than that in the wild type.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2558937/v1/534d5783f08507768d9a14b4.jpg"},{"id":37814133,"identity":"8ee06569-b9a4-446f-b992-a861983966b5","added_by":"auto","created_at":"2023-06-01 06:14:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":550639,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2558937/v1/cca65983-46c4-4854-9bb7-4504a0f84c44.pdf"},{"id":34171049,"identity":"eabd5c6a-231f-4a98-8fd2-f94ce22cce93","added_by":"auto","created_at":"2023-03-13 14:48:18","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":149887,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingTable.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2558937/v1/864e39b54937af20f057460b.pdf"},{"id":34170080,"identity":"d7207e67-905e-47c2-b5b5-c7eaffe68241","added_by":"auto","created_at":"2023-03-13 14:40:18","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1390358,"visible":true,"origin":"","legend":"","description":"","filename":"TheoriginalimageofFigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-2558937/v1/94dfa5a0b7e4ce7d1aa222be.tif"},{"id":34168271,"identity":"d5b030fa-3138-47f4-a227-e741e52b58d0","added_by":"auto","created_at":"2023-03-13 14:32:18","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":2223816,"visible":true,"origin":"","legend":"","description":"","filename":"TheoriginalimageofFigure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-2558937/v1/2e349de609b03ab1be301e53.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Positional cloning and functional verification of the epidermal protein gene KWMTBOMO04384 in silkworm, Bombyx mori","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSilkworms are important economic insects with a relatively perfect mutation system and are thus genetic model organisms second only to \u003cem\u003eDrosophila\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. After more than 5000 years of domestication and evolution, rich genetic mutation materials have accumulated. More than 600 species of silkworm mutants have been discovered and preserved in various regions of China\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, and body color and body shape mutations are prevalent. Body color mutants include \u003cem\u003eal\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eL\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eP\u003c/em\u003e\u003csup\u003e\u003cem\u003eS\u003c/em\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003ebd\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Body shape mutants include \u003cem\u003emf-2\u003c/em\u003e\u003csup\u003e7\u003c/sup\u003e, \u003cem\u003etub\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eBo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003esk\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLoss-of-function mutations in some genes usually result in changes in silkworm body shape. The 15 bp deletion of \u003cem\u003eBmsei\u003c/em\u003e led to the expression of a protein with loss of function, which resulted in the stubby body of \u003cem\u003ecot\u003c/em\u003e mutant larvae exhibiting strong contraction, rolling, and vomiting behavior at high temperatures\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The nonsense mutation of \u003cem\u003eBmorCPH\u003c/em\u003e24 resulted in the bamboo-shaped larval body with an enlarged thoracic region and a slender but firm abdomen\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The specific deletion of 312 bp of \u003cem\u003eBmscarface\u003c/em\u003e changed the spatial structure of the protein, resulting in the \u003cem\u003etub\u003c/em\u003e mutant larvae body type being short and fat with a spindle shape\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The coding sequence mutant of \u003cem\u003eBmorCPR2\u003c/em\u003e led to the loss of the coding product\u0026rsquo;s ability to bind to chitin, resulting in the tight bodies of the mutant larvae, protruding internode folds, and serious defects in larval adaptability\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Revealing the cause of the body shape mutants of silkworm will help clarify the body shape regulation mechanism of insects. However, few mutants have been resolved at the molecular level.\u003c/p\u003e \u003cp\u003eA new body shape mutant of silkworm was found during WT-n08 rearing. The intersegmental membrane of the mutant showed a sunken phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The mutant trait is controlled by a recessive gene located on the autosome. In this study, the mechanism of the mutant was elucidated through positional cloning and functional verification of the mutant gene.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eThe n08M phenotype was controlled by a recessive gene.\u003c/strong\u003e The body shape of the F\u003csub\u003e1\u003c/sub\u003e generation of n08M and P50 was normal. The self-crossing offspring of the F\u003csub\u003e1\u003c/sub\u003e generation showed the separation of normal and sunken individuals, and separation ratio was 3:1. Backcross populations had normal and sunken individuals, and the separation ratio was 1:1 (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The n08M phenotype was controlled by a recessive gene located on an autosome.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eTrait segregation of each generation between the n08M and P50.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHybrid form\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGeneration\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNormal number of individuals\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber of mutants\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal number of silkworms\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSeparation ratio\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026chi;2\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en08M\u0026times;P50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e318\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e318\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP50\u0026times;n08M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e394\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e394\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(n08M\u0026middot;P50) \u0026times; (n08M\u0026middot;P50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e279\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e103\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e382\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(P50\u0026middot;n08M) \u0026times; (P50\u0026middot;n08M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e311\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e427\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(n08M\u0026middot;P50) \u0026times; n08M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBC1 F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e206\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e185\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e391\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(P50\u0026middot;n08M) \u0026times; n08M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBC1 F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e205\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e219\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e424\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en08M \u0026times; (n08M\u0026middot;P50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBC1M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e171\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e327\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en08M \u0026times; (P50\u0026middot;n08M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBC1M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e173\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e159\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e332\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003ePositional cloning.\u003c/strong\u003e Genetic analysis showed that the mutant gene was located on the autosome. The BC\u003csub\u003e1\u003c/sub\u003eF population was genotyped with polymorphic SSR molecular markers of each autosome according to the nonexchange property of silkworm female chromosomes. The PCR product bands of the 14 BC\u003csub\u003e1\u003c/sub\u003eF individuals with sunken intersegmental membranes amplified with polymorphic SSR markers on chromosome 8 were consistent with n08M (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). The PCR product bands of polymorphic molecular markers on the other 26 autosomes were distributed irregularly. Therefore, the gene controlling the sunken trait of n08M is located on the 8th linkage group of \u003cem\u003eBombyx mori\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTo locate and clone the mutant gene, seven SSR molecular markers were screened on the eighth linkage group. The mutant gene was located between the molecular markers S8-4-10(3) and S8-3-9(1) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC), and the distance between the markers was about 300 kb and included 12 candidate genes: \u003cem\u003eKWMTBOMO04373\u003c/em\u003e, \u003cem\u003eKWMTBOMO04374\u003c/em\u003e, \u003cem\u003eKWMTBOMO04375\u003c/em\u003e, \u003cem\u003eKWMTBOMO04376\u003c/em\u003e, \u003cem\u003eKWMTBOMO04377\u003c/em\u003e, \u003cem\u003eKWMTBOMO04378\u003c/em\u003e, \u003cem\u003eKWMTBOMO04379\u003c/em\u003e, \u003cem\u003eKWMTBOMO04380\u003c/em\u003e, \u003cem\u003eKWMTBOMO04381\u003c/em\u003e, \u003cem\u003eKWMTBOMO04382\u003c/em\u003e, \u003cem\u003eKWMTBOMO04383\u003c/em\u003e, and \u003cem\u003eKWMTBOMO04384\u003c/em\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://kaikobase.dna.affrc.go.jp/\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe analysis of the functions of 12 candidate genes with the silkworm website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://kaikobase.dna.affrc.go.jp/\u003c/span\u003e\u003c/span\u003e) showed that only \u003cem\u003eKWMTBOMO04383\u003c/em\u003e and \u003cem\u003eKWMTBOMO04384\u003c/em\u003e were related to the epidermis. Whether the sequences of the two genes had changed was determined by cloning their CDS sequences. The results showed that the sequence of \u003cem\u003eKWMTBOMO04383\u003c/em\u003e did not change, but the partial deletion of \u003cem\u003eKWMTBOMO04384\u003c/em\u003e exon 2 and intron 2 sequence occurred (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e), and a stop codon was generated in the functional region, resulting in the premature termination of gene expression. Moreover, the sequence of \u003cem\u003eKWMTBOMO04384\u003c/em\u003e gene had a frameshift mutation, and the lost function of the gene may have resulted in the sunken intersegmental membrane of n08M.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKnockout validation.\u003c/strong\u003e The function of \u003cem\u003eKWMTBOMO04384\u003c/em\u003e in silkworm epidermis was determined at the individual level, and sgRNA sites were designed on the exons 2 and 4 of \u003cem\u003eKWMTBOMO04384.\u003c/em\u003e The phenotype of intersegmental membrane was sunken in the G2 generation after knockout (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The genomic DNA of 5 G2 generation individuals with sunken intersegmental membrane was extracted, and the sequences of two sgRNA sites were cloned. The results showed that a series of different sequence deletion was generated (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative reverse transcriptase PCR.\u003c/strong\u003e The expression of \u003cem\u003eKWMTBOMO04384\u003c/em\u003e after knockout was detected through qRT-PCR. The expression of \u003cem\u003eKWMTBOMO04384\u003c/em\u003e in the knockout Nistari was significantly lower than that in the wild type (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe epidermis of insects covers the entire body surface, not only providing defense against pathogens and adverse environmental damage but also playing an essential role in shaping the body shape and maintaining normal activities during development\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Thus, the epidermis greatly enhances the survival and adaptability of insects, ensuring the evolution of insects into one of the most successful groups in the animal kingdom. Insect epidermis is composed of chitin and protein. The currently reported protein that can bind to chitin is mainly cuticular protein\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, which accounts for about 1.5% of the total number of genes\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In the development and differentiation of the insect epidermis and the construction of important parts and organs outside the body, epidermal protein is an indispensable element and an important structural protein\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The silencing of the epidermal protein gene \u003cem\u003eAbd-9\u003c/em\u003e of \u003cem\u003eLocusta migratoria\u003c/em\u003e led to thinning of the adult epidermis and reduction of endodermal delamination\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. After the deletion of the epidermal protein gene \u003cem\u003eTcCPAP3-B\u003c/em\u003e in \u003cem\u003eTribolium castaneum\u003c/em\u003e, the joints of the second pair of feet, the hindfoot joint, and the tibiotarsal joint became rigid and deformed. The epidermal protein gene \u003cem\u003eTcCPAP3-B\u003c/em\u003e has been speculated to be involved in the construction of feet\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The silencing of the epidermal protein gene \u003cem\u003eTcCPAP1\u003c/em\u003e in \u003cem\u003eTribolium castaneum\u003c/em\u003e led to hypoplasia and shedding of the epidermis, and deletion of the epidermal genes \u003cem\u003eTcCPAP3-A1\u003c/em\u003e and \u003cem\u003eTcCPAP3-A2\u003c/em\u003e results in incomplete elytra\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The above findings indicate that epidermal proteins are indispensable for maintaining the integrity of insect cuticles.\u003c/p\u003e \u003cp\u003eAccording to the sequence characteristics of insect epidermal proteins, epidermal proteins are divided into 12 families: CPR, CPF, CPFL, Tweedle, CPAP1, CPAP3, CPG and CPLC, etc\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The epidermal proteins of the CPR family, which is the largest, are the most widely distributed and the largest in number\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. It plays an important role in the whole life activities of insects\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The silencing of the epidermal protein genes \u003cem\u003eCPR27\u003c/em\u003e and \u003cem\u003eCPR18\u003c/em\u003e of \u003cem\u003eTribolium castaneum\u003c/em\u003e resulted in structural abnormalities in the adult cuticle\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The silencing of the \u003cem\u003eTcCPR27\u003c/em\u003e gene led to disordered protocuticle structure and abnormal channel fibers, resulting in shortened and shrunken elytra. Adults died prematurely because of dehydration a week after emergence\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The silencing of genes for 15 CPR family epidermal protein members of \u003cem\u003eNilaparvata lugens\u003c/em\u003e resulted in insect death\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The members of the CPR family all contain Rebers \u0026amp; Riddiford (R\u0026amp;R) motif. Through the analysis of the R\u0026amp;R conserved motifs, the CPR family is divided into three subfamilies: RR-1 in the soft cuticle of insects, RR-2 in the hard cuticle of insects, and RR-3 in the old cuticle of insects\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eKWMTBOMO04384\u003c/em\u003e belongs to the RR-1 epidermal protein gene of the CPR family. The partial deletion of its gene sequence led to a frameshift mutation, which resulted in the sunken intersegmental membranes of the silkworm larvae. After the knocking out of the target gene with the CRISPR/Cas9 gene editing system, the mutant had a phenotype of sunken intersegmental membrane.\u003c/p\u003e \u003cp\u003eIn summary, the study demonstrated that \u003cem\u003eKWMTBOMO04384\u003c/em\u003e plays an important role in the formation of the silkworm epidermis and deepens understanding of the CPR family epidermal protein gene.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e \u003cb\u003eSilkworm strains.\u003c/b\u003e The wild-type strain P50 was preserved by Sericultural and Apicultural Research Institute, Yunnan Academy of Agricultural Sciences. The wild-type strain WT-n08 and its epidermis mutant strain (n08M) and the polyvoltine race of diapause-free Nistari and its transgenic race with Cas9 gene (Nistari-Cas9) were preserved by Sericultural Research Institute of Jiangsu University of Science and Technology. All \u003cem\u003eBombyx mori\u003c/em\u003e larvae were reared on fresh mulberry leaves at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C and constant humidity of 75\u0026ndash;80%.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic analysis of n08M.\u003c/b\u003e F\u003csub\u003e1\u003c/sub\u003e generation was generated by crossing P50 and n08M, and F\u003csub\u003e1\u003c/sub\u003e was self-crossed to produce the F\u003csub\u003e2\u003c/sub\u003e generation. The female moths of F\u003csub\u003e1\u003c/sub\u003e were backcrossed to the male moths of n08M to obtain BC\u003csub\u003e1\u003c/sub\u003eF, and female moths of n08M were backcrossed to the male moths of F\u003csub\u003e1\u003c/sub\u003e to produce BC\u003csub\u003e1\u003c/sub\u003eM. The phenotypes of each generation were observed, and the segregation ratio was calculated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGene mapping.\u003c/b\u003e The genomic DNA of P50, n08M, F\u003csub\u003e1\u003c/sub\u003e, BC\u003csub\u003e1\u003c/sub\u003eF, and BC\u003csub\u003e1\u003c/sub\u003eM was extracted from the midgut of the fifth-instar larva with a cell/tissue genome DNA extraction kit (BioTeke, China). The concentration and purity of genomic DNA were determined with an ultra-micro spectrophotometer at 260/280 absorbance ratio, and the purified DNA was stored at \u0026minus;\u0026thinsp;20\u0026deg;C. Simple sequence repeat (SSR) markers for determining the linkage group of the mutant gene were obtained from a published SSR linkage map\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, and the new SSR markers for the positional cloning were searched with SSR Hunter 1.3. The polymorphisms of SSR molecular markers were identified by agarose gel electrophoresis at a concentration of 2%. Given that the homologous chromosomes of female silkworms do not exchange, 10 BC\u003csub\u003e1\u003c/sub\u003eF wild-type individuals and 10 mutant BC\u003csub\u003e1\u003c/sub\u003eF individuals were used in determining the linkage group of the mutant gene, and 200 BC\u003csub\u003e1\u003c/sub\u003eM mutant individuals were used for the positional cloning of the mutant gene. The primers used for mapping are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCloning gene.\u003c/b\u003e Specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were designed according to the gene sequence (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://kaikobase.dna.affrc.go.jp/\u003c/span\u003e\u003cspan address=\"https://kaikobase.dna.affrc.go.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) in the location region. PCR amplification was performed using the genomic DNA of WT-n08 and n08M as template. Each 20 \u0026micro;L PCR reaction system consisted of 2 \u0026micro;L of 10\u0026times; LA Taq Buffer, 3 \u0026micro;L of dNTP mixture, 0.2 \u0026micro;L of LA Taq enzyme, 1 \u0026micro;L of forward primer, 1 \u0026micro;L of reverse primer, 1 \u0026micro;L of DNA template, and 11.8 \u0026micro;L of ddH\u003csub\u003e2\u003c/sub\u003eO. The PCR reaction procedure was as follows: 94\u0026deg;C for 5 min, 31 cycles at 94\u0026deg;C for 30 s, 55\u0026deg;C for 30 s, 72\u0026deg;C for 2 min, and extension at 72\u0026deg;C for 10 min. The PCR product was detected by 1% agarose gel electrophoresis and cloned into a pMD19-T plasmid. Recombinant vectors were transformed into the competent cells of \u003cem\u003eEscherichia coli\u003c/em\u003e and were sent for sequencing after identification by bacterial liquid PCR. Sequences were assembled and analyzed using BioEdit.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConstruction of target gene sgRNA vector and microinjection.\u003c/b\u003e The target gene sequence was amplified by using Nistari genomic DNA as a template. The online software CRISPRdirect (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispr.dbcls.jp/\u003c/span\u003e\u003cspan address=\"http://crispr.dbcls.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used in searching two potential sgRNA targets in the ORF region of the sequenced target gene, and sgRNA primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were designed according to the sequence of sgRNA targets and transgenic vector pXL (IE1-DsRed). Each 50 \u0026micro;L PCR reaction system consisted of 25 \u0026micro;L of Primer STAR Premix (2\u0026times;), 1.5 \u0026micro;L of forward primer (10 \u0026micro;mol/L), 1.5 \u0026micro;L of reverse primer (10 \u0026micro;mol/L), 2 \u0026micro;L of pXL (IE1-DsRed), and 20 \u0026micro;L of RNase-free H\u003csub\u003e2\u003c/sub\u003eO. The reaction procedure was as follows: 94\u0026deg;C for 2 min, 31 cycles at 94\u0026deg;C for 20 s, 56\u0026deg;C for 20 s, 72\u0026deg;C for 40 s, and extension at 72\u0026deg;C for 7 min. The PCR product was purified and recovered, and a homologous recombination was used in connecting the PCR product of the two sgRNA sites to the transgenic vector PXL (IE1-DsRed) linearized by SalI and NheI double digestion according to the instructions of In-Fusion Snap Assembly Master Mix kit (TaKaRa, Japan).\u003c/p\u003e \u003cp\u003eThe recombinant vector was transformed into the competent cells of \u003cem\u003eEscherichia coli\u003c/em\u003e, and the samples were sent for sequencing after identification by bacterial liquid PCR. The recombinant plasmid was extracted by using QIAGEN Plasmid Midi Kit (QIAGEN, Germany). The recombined and helper plasmids were injected into lay 4\u0026ndash;6 h of Nistari non-diapausing silkworm eggs by microinjection. After microinjection, the eggs (G0 generation) were incubated at a temperature of 25\u0026deg;C and humidity of 70\u0026ndash;80%.\u003c/p\u003e \u003cp\u003e \u003cb\u003eKnockout validation.\u003c/b\u003e The G0 generation was reared under normal temperature and humidity conditions. G0 was self-crossed to produce the G1 generation. The newly-hatched larvae of the G1 generation were detected under a fluorescent stereomicroscope, and positive individuals with red fluorescence were screened. The moths of the positive individuals were crossed with the Cas9 transgenic silkworm to produce the G2 generation. The newly-hatched silkworms of the G2 generation were detected under a fluorescent stereomicroscope, and individuals that emitted red and green fluorescence were screened. Fifth instar larvae with sunken intersegmental membranes were selected, and the genome DNA was extracted by using a cell/tissue genome DNA extraction kit (BioTeke, China). The sequences of the sgRNA targets were detected with knockout detection primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Each 20 \u0026micro;L PCR reaction system consisted of 2 \u0026micro;L of 10\u0026times; LA Taq buffer, 3 \u0026micro;L of dNTP mixture, 0.2 \u0026micro;L of LA Taq enzyme, 1 \u0026micro;L of forward primer, 1 \u0026micro;L of reverse primer, 1 \u0026micro;L of DNA template, and 11.8 \u0026micro;L of ddH\u003csub\u003e2\u003c/sub\u003eO. The PCR reaction procedure was as follows: 94\u0026deg;C for 5 min, 31 cycles at 94\u0026deg;C for 30 s, 55\u0026deg;C for 30 s, 72\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 10 min. The PCR products were cloned into the pMD19-T vector, and single clones were selected for sequencing.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantitative reverse transcriptase PCR.\u003c/b\u003e Total RNAs were extracted from the epidermis of wild-type Nistari and G2 individuals with sunken intersegmental membranes by using RNAiso Plus (TaKaRa, Japan). RNA was reverse-transcribed using a PrimerScript RT reagent kit with gDNA eraser (TaKaRa, Japan) according to the manufacturer\u0026rsquo;s instructions. The cDNA products were diluted fivefold with ddH\u003csub\u003e2\u003c/sub\u003eO. Each 20 \u0026micro;L of the quantitative reverse transcriptase PCR (qRT-PCR) reaction system consisted of 10 \u0026micro;L of iTaq Universal SYBR Green Supermix (Bio-Rad, USA), 0.5 \u0026micro;L of each specific primer, 1 \u0026micro;L of cDNA template, and 8 \u0026micro;L of ddH\u003csub\u003e2\u003c/sub\u003eO, and three replicates were produced. qRT-PCR was performed in a CFX96 PCR system (Bio-Rad, USA) with a two-step reaction protocol of 40 cycles of 94\u0026deg;C for 5 s and 60\u0026deg;C for 1 min. The housekeeping \u003cem\u003eB. mori\u003c/em\u003e actin 3 gene (GenBank ID: NM_001126254)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e was used as a reference for eliminating bias among samples, and qRT-PCR results were converted and calculated using the 2\u003csup\u003e\u0026minus;ΔΔ\u003cem\u003eCt\u003c/em\u003e\u003c/sup\u003e method\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The primers used for qRT-PCR are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated or analysed during the current study are available in the Genbank repository, accession number: OQ547777.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by China Agriculture Research System of MOF and MARA (No. CARS-18-ZJ0101), Key R \u0026amp; D plan of Jiangsu Province (Modern Agriculture) (No. BE2020418), the Key project of Education Department of Shaanxi Provincial Government (No. 20JS001) and the Applied Basic Research Programs of the Shaanxi Provincial Science and Technology Department (No. 2023-JC-YB-188).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.S.\u0026nbsp;and M. L. conducted all analyses, interpreted the results, and drafted the paper. A.C. designed the project. H.Q., X.Z. and G.O. contributed to interpretation of the analyses. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLu, C., Dai, F.Y. \u0026amp; Xiang, Z.H. Studies on the mutation strains of the \u003cem\u003eBombyx mori\u003c/em\u003e gene bank. Sci. Agric. Sin. 36, 968\u0026ndash;975 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoldsmith, M.R., Shimada, T. \u0026amp; Abe, H. The genetics and genomics of the silkworm, \u003cem\u003eBombyx mori\u003c/em\u003e. Annu. Rev. 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Methods 25, 402\u0026ndash;408 (2001).\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"[email protected]","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":"Bombyx mori, Body shape mutant, Genetic analysis, Positional cloning, CRISPR/Cas9","lastPublishedDoi":"10.21203/rs.3.rs-2558937/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2558937/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA body shape mutant n08M with sunken intersegmental membrane was found during the feeding process of silkworm WT-n08. Genetic analysis showed that the mutant trait of n08M is controlled by a recessive gene located at the autosome and follows Mendelian inheritance. Results of positional cloning showed that the epidermal protein gene \u003cem\u003eKWMTBOMO04384\u003c/em\u003e on chromosome 26 was mutated. After \u003cem\u003eKWMTBOMO04384\u003c/em\u003e was knocked out by CRISPR/Cas9, the intersegmental membrane of silkworm was sunken. \u003cem\u003eKWMTBOMO04384\u003c/em\u003e was the target gene that caused the sunken intersegmental membrane in WT-n08 and had an important influence on the body shape of \u003cem\u003eBombyx mori\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Positional cloning and functional verification of the epidermal protein gene KWMTBOMO04384 in silkworm, Bombyx mori","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-13 14:32:11","doi":"10.21203/rs.3.rs-2558937/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","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":"79f7b414-d402-44f7-abda-bfdb6319b8cb","owner":[],"postedDate":"March 13th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":19727959,"name":"Biological sciences/Biological techniques"},{"id":19727960,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2023-06-01T06:14:38+00:00","versionOfRecord":[],"versionCreatedAt":"2023-03-13 14:32:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2558937","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2558937","identity":"rs-2558937","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}