梁林 吕志创 武强 刘怀 刘万学 万方浩

摘要 ：瞬時感受器离子通道（transient receptor potential， TRP）是影响昆虫温度感知系统的关键组成。为探讨苹果蠹蛾温度感知和温度适应的机理，本研究以苹果蠹蛾Cydia pomonella为研究对象，通过分子克隆获得TRPA家族中的CpPainless和CpWater_witch基因，进行生物信息学分析，并利用实时荧光定量PCR技术分析靶基因在高低温胁迫后的表达。结果表明，CpPainless基因编码938个氨基酸，N端有8个锚蛋白重复序列。CpWater_witch基因编码980个氨基酸，N端有10个锚蛋白重复序列，二者编码产物均有6个跨膜结构，具瞬时感受器离子通道家族成员结构典型特征。表达分析结果显示，和对照26℃相比，高低温胁迫1 h后，CpPainless在5龄幼虫雌虫体内的表达量显著下调，而5龄幼虫雄虫经低温胁迫1 h后CpPainless表达量显著上调，但高温胁迫1 h后表达差异不显著;CpWater_witch在雌雄虫体内均没有显著变化。研究结果为研究瞬时感受器离子通道在苹果蠹蛾温度感知中的作用奠定基础。

关键词 ：苹果蠹蛾; 瞬时感受器离子通道; 温度感知; 温度胁迫; 表达量

中图分类号： S 436.611.29

文献标识码： A

DOI： 10.16688/j.zwbh.2019564

Cloning and expression analysis of transient receptor potential gene in Cydia pomonella under temperature stress

LIANG Lin1，2， L Zhichuang2， WU Qiang2， LIU Huai1， LIU Wanxue2， WAN Fanghao2*

（1. College of Plant Protection， Southwest University， Chongqing 400715， China; 2. State Key

Laboratory for Biology of Plant Diseases and Insect Pests， Institute of Plant Protection，

Chinese Academy of Agricultural Sciences， Beijing 100193， China）

Abstract ：TRP （transient receptor potential） is a key component of the temperature sensing system in insects. To understand the mechanism of temperature perception and temperature adaptation in Cydia pomonella， CpPainless and CpWater_witch， which belong to TRPA subfamily， were cloned by RTPCR （reverse transcription PCR）. After conducting bioinformatics analysis， quantitative realtime PCR was applied to characterize the relative expression levels of two genes at different temperature stresses. CpPainless and CpWater_witch were successfully predicted and cloned， which encoded 938 amino acids and 980 amino acids， respectively. Protein structure analysis indicated that CpPainless and CpWater_witch had 8 ankyrin repeats and 10 ankyrin repeats， respectively. Both of the genes had 6 transmembrane domains， which is the structural signature of TRP. The RTPCR analysis showed that the expression level of CpPainless was significantly decreased in female 5thinstar larvae 1 h after temperature stress， but significantly increased in male 5thinstar larvae 1 h after lowtemperature stress， whereas there was no significant difference in male larvae 1 h after hightemperature stress; besides， CpWater_witch did not show significant difference in both male and female 5thinstar larvae after temperature stress. The results laid a foundation for further understanding of the role of transient receptor channels in temperature perception of C.pomonella.

Key words ：Cydia pomonella; TRP channel; temperature sensing; temperature stress; expression

苹果蠹蛾Cydia pomonella （L.）属鳞翅目Lepidoptera小卷蛾科Olethreutidae，是最重要的世界性检疫性害虫之一，其以幼虫蛀食果实为害，严重降低果实品质[1]，并导致果实成熟前腐烂和脱落[2]，给世界各地苹果、沙果、梨、桃、杏、石榴等水果的生产带来了毁灭性危害[3]。苹果蠹蛾原产于欧亚大陆南部，现已广泛分布于除南极洲外其他各大洲的各大苹果产区[4]。在我国，苹果蠹蛾也具有广泛的适生区域，其中中高度适生区主要包括黑……