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来自对苏云金芽孢杆菌毒素具有不同敏感性的印度谷螟菌株的氨肽酶样蛋白基因的cDNA

cDNAs of aminopeptidase-like protein genes from Plodia interpunctella strains with different susceptibilities to Bacillus thuringiensis toxins.

作者信息

Zhu Y C, Kramer K J, Oppert B, Dowdy A K

机构信息

Grain Marketing and Production Research Center, ARS-USDA, Manhattan, Kansas 66502-2736, USA.

出版信息

Insect Biochem Mol Biol. 2000 Mar;30(3):215-24. doi: 10.1016/s0965-1748(99)00118-6.

Abstract

Aminopeptidase N has been reported to be a Bacillus thuringiensis (Bt) Cry1A toxin-binding protein in several lepidopteran insects. cDNAs of aminopeptidase-like proteins from both Bt-susceptible RC688s and Bt-resistant HD198r strains of the Indianmeal moth, Plodia interpunctella, were cloned and sequenced. They contain 3345 and 3358 nucleotides, respectively, and each has a 3048 bp open reading frame that encodes 1016 amino acids. Putative protein sequences include 10 potential glycosylation sites and a zinc metal binding site motif of HEXXH, which is typical of the active site of zinc-dependent metallopeptidases. Sequence analysis indicated that the deduced protein sequences are most similar to an aminopeptidase from Heliothis virescens with 62% sequence identity and highly similar to three other lepidopteran aminopeptidases from Plutella xylostella, Manduca sexta, Bombyx mori with sequence identities of 51-52%. Four nucleotide differences were observed in the open reading frames that translated into two amino acid differences in the putative protein sequences. Polymerase chain reaction (PCR) confirmed an aminopeptidase gene coding difference between RC688s and HD198r strains of P. interpunctella in the PCR amplification of a specific allele (PASA) using preferential primers designed from a single base substitution. The gene mutation for Asp185-->Glu185 was also confirmed in two additional Bt-resistant P. interpunctella strains. This mutation is located within a region homologous to the conserved Cry1Aa toxin binding regions from Bombyx mori and Plutella xylostella. The aminopeptidase-like mRNA expression levels in the Bt-resistant strain were slightly higher than those in the Bt-susceptible strain. The sequences reported in this paper have been deposited in the GenBank database (accession numbers AF034483 for susceptible strain RC688s and AF034484 for resistant strain HD198r).

摘要

据报道,氨肽酶N是几种鳞翅目昆虫体内的一种苏云金芽孢杆菌(Bt)Cry1A毒素结合蛋白。已克隆并测序了印度谷螟敏感品系RC688s和抗性品系HD198r中类氨肽酶蛋白的cDNA。它们分别含有3345和3358个核苷酸,每个都有一个3048 bp的开放阅读框,编码1016个氨基酸。推测的蛋白质序列包括10个潜在的糖基化位点和一个HEXXH锌金属结合位点基序,这是锌依赖性金属肽酶活性位点的典型特征。序列分析表明,推导的蛋白质序列与棉铃虫的一种氨肽酶最相似,序列同一性为62%,与小菜蛾、烟草天蛾、家蚕的其他三种鳞翅目氨肽酶高度相似,序列同一性为51 - 52%。在开放阅读框中观察到四个核苷酸差异,这些差异在推测的蛋白质序列中转化为两个氨基酸差异。聚合酶链反应(PCR)使用从单个碱基替换设计的优先引物,在特定等位基因PCR扩增(PASA)中证实了印度谷螟RC688s和HD198r品系之间氨肽酶基因编码的差异。在另外两个Bt抗性印度谷螟品系中也证实了Asp185→Glu185的基因突变。该突变位于与家蚕和小菜蛾保守的Cry1Aa毒素结合区域同源的区域内。Bt抗性品系中类氨肽酶mRNA的表达水平略高于Bt敏感品系。本文报道的序列已存入GenBank数据库(敏感品系RC688s的登录号为AF034483,抗性品系HD198r的登录号为AF034484)。

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