Engineering of multiple trypsin/chymotrypsin sites in Cry3A to enhance its activity against Monochamus alternatus Hope larvae

文献类型： 外文期刊

第一作者： Guo, Yajie

作者： Guo, Yajie;Liang, Guanghong;Hu, Xia;Wang, Rong;Zhang, Feiping;Wu, Songqing;Guo, Yajie;Liang, Guanghong;Hu, Xia;Wang, Rong;Zhang, Feiping;Wu, Songqing;Guo, Yajie;Wang, Yafang;Guan, Xiong;Wu, Songqing;Wang, Yafang;O'Donoghue, Anthony J.;Jiang, Zhenze;Carballar-Lejarazu, Rebeca;Xu, Lei

作者机构：

关键词： Monochamus alternatus Hope; Cry3Aa toxin; proteolytic activation; molecular modification

期刊名称：PEST MANAGEMENT SCIENCE （ 影响因子：4.845； 五年影响因子：4.674 ）

ISSN： 1526-498X

年卷期： 2020 年 76 卷 9 期

页码：

收录情况： SCI

摘要： BACKGROUND Bacillus thuringiensis Cry3 toxins exhibit specific toxicity against several coleopteran larvae. However, owing to its low toxicity to Monochamus alternatus, Cry3A toxin is not useful for managing M. alternatus larvae. Here we assessed the proteolytic activation of Cry3Aa toxin in M. alternatus larval midgut and increased its toxicity by molecular modification. RESULTS Our results indicated that insufficient processing of Cry3Aa protoxin and non-specific enzymatic digestion of Cry3Aa toxin in the midgut of M. alternatus larvae led to low toxicity. The results of transcriptome analysis, enzymatic assay with fluorogenic substrates, and multiplex substrate profiling by mass spectrometry showed that the main digestive enzymes in M. alternatus larval midgut were trypsin-like proteases that preferentially cleaved peptides with arginine and lysine residues. Consequently, trypsin recognition sites were introduced into the Domain I of Cry3Aa protoxin in the loop regions between alpha-helix 3 and alpha-helix 4 to facilitate proteolytic activation. Multiple potential trypsin cleavage sites away from the helix sheet and functional regions in Cry3Aa proteins were also mutated to alanine to prevent non-specific enzymatic digestion. Bioassays indicated that a modified Cry3Aa-T toxin (K65A, K70A, K231A, K468A, and K596A) showed a 9.5-fold (LC50 = 12.3 mu g/mL) increase in toxicity to M. alternatus larvae when compared to native Cry3Aa toxin. CONCLUSION This study highlights an effective way to increase the toxicity of Cry3Aa toxin to M. alternatus, which may be suitable for managing the resistance of transgenic plants to other pests, including some of the most important pests in agriculture.

分类号：

  • 相关文献
作者其他论文 更多>>

微信小程序