麦红吸浆虫热激蛋白基因 SmHsp 40的克隆及其在滞育进程中和极端温度胁迫下的表达  

Cloning of heat shock protein gene SmHsp40 and its expression during diapause and under extreme temperature stress in Sitodiplosis mosellana (Diptera: Cecidomyiidae)

作　　者：赵佳佳[1] 李雪娇[1] 刘珍珠 成卫宁[1] 朱克岩 ZHAO Jia-Jia;LI Xue-Jiao;LIU Zhen-Zhu;CHENG Wei-Ning;ZHU Ke-Yan(Key Laboratory of Integrated Pest Management on Crops in Northwestern Loess Plateau,Ministry of Agriculture,Northwest A＆F University,Yangling,Shaanxi 712100,China;Library,Northwest A＆F University,Yangling,Shaanxi 712100,China;Department of Entomology,Texas A＆M University,Texas 77843,USA)

机构地区：[1]西北农林科技大学、农业部西北黄土高原作物有害生物综合治理重点实验室,陕西杨凌712100 [2]西北农林科技大学图书馆,陕西杨凌712100 [3]Department of Entomology,Texas A Muniversity,Texas 77843,USA

出　　处：《昆虫学报》2018年第11期1253-1262,共10页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31371933); 国家重点研发计划(2018YFD020040302); 杨凌示范区科技计划项目(2018NY-07)

摘　　要：【目的】麦红吸浆虫Sitodiplosis mosellana是一种典型的专性滞育昆虫,滞育过程中经历酷暑和严寒。本研究旨在探讨麦红吸浆虫热激蛋白基因Hsp40在滞育中的作用。【方法】以麦红吸浆虫滞育前幼虫为材料,利用RT-PCR和RACE技术克隆Hsp40全长cDNA序列;利用生物信息学软件分析该基因及其编码蛋白的特性;采用实时荧光定量PCR技术分析其在滞育不同阶段幼虫及越夏滞育幼虫在短期(≤120 min)极端高温(35～50℃)和越冬滞育幼虫在短期极端低温(0～-15℃)胁迫下的表达模式。【结果】克隆获得麦红吸浆虫Hsp40基因,命名为SmHsp40(GenBank登录号:MH001167),其cDNA全长1 553 bp,开放阅读框长为1 074 bp,编码357个氨基酸,相对分子量为40.87 kD,理论等电点为5.31。推导的氨基酸序列中含有Hsp40蛋白家族典型的J结构域和HPD基序。蛋白序列相似性和系统进化关系分析表明,SmHsp40蛋白与冈比亚按蚊Anopheles gambiae等长角亚目(Nematocera)昆虫Hsp40的相似性最高、亲缘关系最近。麦红吸浆虫滞育不同时期幼虫SmHsp40表达量存在显著差异,进入滞育后表达量上调;滞育进程中,夏季7-8月和冬季12月至翌年1月表达量显著高于其他季节。与未处理的对照相比,40℃处理越夏滞育幼虫和-10℃处理越冬滞育幼虫1 h明显诱导SmHsp40的表达,但超过45℃或低于-15℃极端温度处理诱导效果均不明显。【结论】SmHsp40参与了麦红吸浆虫滞育进程,可能在滞育状态的维持及滞育期间耐热和耐寒能力的调控方面起重要作用。【Aim】 The orange wheat blossom midge, Sitodiplosis mosellana , escapes temperature extremes during hot summer and cold winter by undergoing obligatory larval diapause. This study aims to explore the potential roles of heat shock protein 40 gene （ Hsp 40） during diapause of S. mosellana . 【Methods】 The full-length cDNA of Hsp 40 was cloned from S. mosellana larvae at pre-diapause stage by using RT-PCR and RACE technologies, and its nucleotide and amino acid sequences were subjected to bioinformatic analysis. The mRNA expression level of Hsp 40 in pre-diapause, diapause and post-diapause larvae of S. mosellana , over-summering diapausing larvae exposed to short-term heat stress （35-50℃, ≤120 min） and over-wintering diapausing larvae exposed to short-term cold stress （0--15℃, ≤120 min） were detected with real-time quantitative PCR. 【Results】 The full-length cDNA sequence of Hsp 40 was obtained from S. mosellana , and named SmHsp40 （GenBank accession number： MH001167）, which is 1 553 bp in length and contains a 1 074 bp ORF encoding a protein of 357 amino acids with a predicted molecular weight of 40.87 kD and a theoretical pI of 5.31. The deduced amino acid sequence contains the conserved J-domain and HPD motif of Hsp40 protein family. Multiple sequence alignment and phylogenetic analysis indicated that SmHsp40 was most similar and closely related to Hsp40s from the Nematocera including Anopheles gambiae . The expression of SmHsp 40 differed significantly among different diapause stages, and was up-regulated during the diapause period, with significantly higher expression level during summer （July to August） and winter （December to January of the following year）. The expression of SmHsp 40 was induced in over-summering diapausing larvae exposed to heat stress （40℃ for 1 h） or over-wintering diapausing larvae exposed to cold stress （-10℃ for 1 h） as compared to the untreated control, but not induced significantly under extreme temperatures higher than 45℃ or

关 键 词：麦红吸浆虫 Hsp40 基因克隆 滞育 温度胁迫 实时定量PCR 

分 类 号：Q966[生物学—昆虫学]