棉铃虫APN基因家族系统进化与功能分析

doi:10.16409/j.cnki.2095-039x.2020.05.001

摘要/Abstract

摘要： 氨肽酶N（APN）是锌金属蛋白酶M1家族的成员，鳞翅目昆虫中肠刷状缘膜囊泡（brush border membrane vesicles，BBMV）细胞膜上的APN不仅在蛋白消化吸收过程中起着重要的作用，而且是Bt的重要受体蛋白。本研究通过PCR技术克隆得到7条棉铃虫APN基因全长序列，HaAPN1～7（Genbank登录号为MT002819～MT002825）。经生物信息学分析，HaAPN1～7全长为2592～3099 bp，编码863～1032个氨基酸，分子量为110～115 kDa，等电点为4.7～6.4，N端信号肽为15～20 aa。系统进化分析结果表明，HaAPN1～7分属于Class 1～7类别。RT-qPCR结果表明，在敏感棉铃虫5龄幼虫中肠中APN2的表达量最高，APN7的表达量最低；Cry1Ac抗性品系棉铃虫中各类APN的表达趋势与敏感品系一致。取食Cry1Ac、Cry2Ab和Vip3Aa蛋白后棉铃虫中肠APN活性显著降低，取食Cry1Ac和Vip3Aa蛋白后中肠液和BBMV中APN活性都显著降低，而取食Cry2Ab蛋白后只有BBMV上APN活性显著降低；棉铃虫对Cry1Ac、Cry2Ab和Vip3Aa产生耐受性后，中肠液和BBMV中APN活性也都发生显著变化。因此，不同类别的棉铃虫APN在Bt的杀虫机制中可能起到不同的作用，APN活性变化可能与Bt蛋白的降解及抗性演化相关。

关键词: 氨肽酶N, 棉铃虫, 系统进化, 功能分析

Abstract: Aminopeptidase N (APN) is a member of the zinc metalloproteinase M1 family. APNs, located on the cell membrane of brush border membrane vesicles (BBMV) in Lepidoptera insects midgut, not only play an important role in the process of protein digestion and absorption, but also is an important receptor protein for Bt. In this study, seven APN genes with the full sequence of 2592-3099 bp from Helicoverpa armigera (HaAPN1-7, Genbank acc. No. MT002819-MT002825) were cloned by PCR. Bioinformatics analysis results showed that HaAPN1-7 encoded 863-1032 amino acid with a molecular weight of 110-115 kDa and an isoelectric point of 4.7-6.4, and signal peptides of 15-20 aa at the N-terminal. Phylogenetic analysis showed that HaAPN1-7 clustered into Class 1-7 families. RT-qPCR results showed that the expression level of APN2 was the highest and that of APN7 was the lowest in midgut of the fifth instar larvae from the susceptible strain. Similar APN expression pattern was also found in Cry1Ac-resistant strain. The APN activity in midgut juice and BBMV both significantly decreased when the larvae were fed Cry1Ac and Vip3Aa protein. But when the larvae were fed Cry2Ab protein, the APN activity was clearly reduced only in midgut BBMV. The APN activity in midgut juice and BBMV also significantly changed after H. armigera had tolerance to Cry1Ac, Cry2Ab, or Vip3Aa. It is suggested the different types of HaAPNs may play different roles in the insecticidal mechanism of Bt, and the change of APN activity may be related to Bt degradation and resistance evolution.

Key words: aminopeptidase N, Helicoverpa armigera, phylogenetic evolution, functional analysis

中图分类号:

S433.4

牛琳琳, ZAW Lin Naing, 张彩虹, EI Thinzar Soe, 梁革梅. 棉铃虫APN基因家族系统进化与功能分析[J]. 中国生物防治学报, 2021, 37(1): 91-101.

NIU Linlin, ZAW Lin Naing, ZHANG Caihong, EI Thinzar Soe, LIANG Gemei. Phylogenetic Evolution and Function of APN Gene Family in Helicoverpa armigera[J]. Chinese Journal of Biological Control, 2021, 37(1): 91-101.

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