Molecular Cloning And Characterization of An Alpha-Amylase Inhibitor (Tkaai) Gene From Trichosanthes Kirilowii Maxim

Abstract Trichosanthes kirilowii Maxim., taxonomically belongs to the Cucurbitaceae and Trichosanthes genus, and whose whole fruit, fruit peel, seed and root are widely used in traditional Chinese medicines (TCM). A ribosome-inactivating protein with RNA N-glycosidase activity called Trichosanthrip was isolated and purified from the seeds of T. kirilowii in the previous research. To further explore the biological functions of Trichosanthrip, the cDNA of T. kirilowii AAI (TkAAI) was cloned through rapid-amplification of cDNA ends and its sequence was analyzed, as well as the heterologous protein was expressed in Escherichia coli and its alpha-amylase activity was further measured under optimized conditions. The full-length cDNA of TkAAI was 613 bp, the speculated open reading frame sequence encodes 141 amino acids with a molecular weight of 16.14 kDa. Phylogenetic analysis demonstrated that the AAI_SS domain sequence of TkAAI revealed significant evolutionary homology with the of 2S albumin derived from the other plants in Cucurbitaceae. In addition, TkAAI was assembled into pET28a with eGFP to generate a prokaryotic expression vector and induced to express in E. coli. The TkAAI-eGFP infusion protein was proven to exhibit alpha-amylase inhibitory activity against porcine pancreatic amylase (PPA) in a suitable reaction system. Analysis of gene expression patterns proved that the relative expression level of TkAAI in seeds is highest. Above results forecast that the TkAAI might play a crucial role during the development of T. kirilowii seeds and provide insights into the possibility of T.kirilowii-derived medicine to treat diabetes related diseases.