Studies on the expression and biological activity of a bruchid resistance-related cDNA clone from mung bean

• Main Authors: Kuan, Cheng-Chun, 官振群
• Other Authors: Ching-San Chen
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/62398376889682173451

碩士 === 國立臺灣大學 === 農業化學系 === 86 === Vigna radiata L. VC1973A and VC6089A are isogenic lines provided by Asian Vegetable Research and Development Center (AVRDC). Seeds of the former are susceptible to a mung bean insect bruchid Callosobruchus chinesis, whereas the latter was showed to resis bruchid. VC6089A was bred by crossing VC1973A and a wild mung bean TC1966. Eighteen cDNA clones were previously isolated from VC6089A and showed positive Northern blotting, indicating possible correlation with bruchid resistance. one of the clones SSH60 is a full-length cDNA with deduced amino acid sequence similar to a protease inhibitor (41.9%) and an insect alpha-amylase inhibitor (42.6%). Expression of SSH60 cDNA in E.coli and analysis of the expressed protein for bruchid resistance activity are described in this thesis. E. coli M15 transformed with pQE30 harboring full-length SSH60 failed to overexpress SSH60 protein. The transformed lysed after IPTG induction. This was improved by expressing signal peptide deleted SSH60 (SSH60/sp). The SSH60/sp protein was purified with a Ni-NTA agarose column and assayed for resistant activity. Artificial seeds containing 0.25% SSH60/sp protein were shown to resist the attack of bruchid. Larvae that hatched on the artificial seeds with addition of 0.25% SSH60/sp were not able to survive, while those hatched on the artificial seeds without SSH60/sp shown normal adult emergence. The results suggest that SSH60 protein is one of the antibiosis factors that confers resistance against mung bean bruchid.