钙粘蛋白氨基酸点突变介导红铃虫对Cry1Ac的抗性

Resistance of the pink bollworm Pectinophora gossypiella to Cry1Ac mediated by amino acid point mutation of cadherin

为明确室内筛选的红铃虫Pectinophora gossypiella抗性品系AQ-R对Cry1Ac的抗性机制,采用室内生物测定法明确该品系对Cry1Ac和Cry2Ab的敏感性,通过遗传杂交和基因克隆分析抗性基因的显隐性及突变位点,并进行细胞学试验分析突变蛋白的亚细胞定位。结果显示:红铃虫AQR抗性品系对Cry1Ac的抗性倍数为181.67倍,对Cry2Ab没有交互抗性;该品系携带了一种新型的隐性钙粘蛋白抗性等位基因PgCad1,其编码蛋白的钙粘蛋白重复区、前蛋白区和近膜区共发生了17个氨基酸替换。表达野生型PgCad1-s基因的Hi5细胞对Cry1Ac敏感,且钙粘蛋白定位于细胞膜;而表达抗性PgCad1-r基因的Hi5细胞则对Cry1Ac不敏感,且钙粘蛋白错误定位到内质网。表明钙粘蛋白氨基酸点突变能导致其定位错误,从而促成红铃虫AQ-R品系对Cry1Ac产生抗性。

To clarify the resistance mechanism of a laboratory-selected pink bollworm(Pectinophora gossypiella)strain AQ-R to Bt toxin Cry1Ac,the susceptibility of AQ-R to Cry1Ac and Cry2Ab was detected by bioassay;the dominance-recessiveness and mutation sites of resistant alleles were analyzed by genetic crossing and gene cloning,and subcellular localization of mutant proteins were analyzed using cytological test.The results showed that the Cry1Ac resistance of AQ-R strain was 181.67-fold,but there was no cross resistance to Cry2Ab.A novel recessive resistance allele on the cadherin locus was characterized,which encoded a mutant cadherin protein with seventeen amino acid substitutions in cadherin repeats region,proprotein region and membrane-proximal region.Hi5 cells expressing wild-type PgCad1-s gene were susceptible to Cry1Ac,and the produced cadherin protein was localized in the cell membrane;Hi5 cells expressing PgCad1-r gene were not susceptible to Cry1Ac,and the produced cadherin protein was localized in endoplasmic reticulum.These results indicated that the amino acid point mutation of cadherin might lead to mislocation,resulting in the resistance of AQ-R strain to Cry1Ac.