Abstract
Trichosanthes kirilowii Maxim taxonomically belongs to the Cucurbitaceae family and Trichosanthes genus. Its whole fruit, fruit peel, seed and root are widely used in traditional Chinese medicines. A ribosome-inactivating protein with RNA N-glycosidase activity called Trichosanthrip was isolated and purified from the seeds of T. kirilowii in our recent previous research. To further explore the biological functions of Trichosanthrip, the cDNA of T. kirilowii alpha-amylase inhibitor (TkAAI) was cloned through rapid-amplification of cDNA ends and its sequence was analyzed. Also, 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 encoded 141 amino acids with a molecular weight of 16.14 kDa. Phylogenetic analysis demonstrated that the Alpha-Amylase Inhibitors Seed Storage domain sequence of TkAAI revealed significant evolutionary homology with the 2S albumin derived from the other plants in the Cucurbitaceae group. In addition, TkAAI was assembled into pET28a with eGFP to generate a prokaryotic expression vector and was induced to express in E. coli. The TkAAI-eGFP infusion protein was proven to exhibit alpha-amylase inhibitory activity against porcine pancreatic amylase in a suitable reaction system. Analysis of gene expression patterns proved that the relative expression level of TkAAI in seeds is highest. The results presented here forecasted that the TkAAI might play a crucial role during the development of T. kirilowii seeds and provided fundamental insights into the possibility of T. kirilowii derived medicine to treat diabetes related diseases.
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The datasets generated during the current study are available from the corresponding author on reasonable request.
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The study was supported by the Fundamental Research Funds for the Central Universities (Grant Numbers 2011QC085).
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All authors contributed to the study conception and design. ZYP participated in design of the study, performed the clone, and identify of gene, analyzed the data and drafted manuscript; HQY helped to RNA extraction; WKY contributed to enzymatic reaction. SSH helped designed the research and revised the manuscript. All authors read and approved the final manuscript.
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Zhang, Y., Wang, K., Huang, Q. et al. Molecular cloning and characterization of an alpha-amylase inhibitor (TkAAI) gene from Trichosanthes kirilowii Maxim.. Biotechnol Lett 44, 1127–1138 (2022). https://doi.org/10.1007/s10529-022-03277-4
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DOI: https://doi.org/10.1007/s10529-022-03277-4