麦红吸浆虫热激蛋白基因SmHsp60的分子特征及功能研究  

Molecular characteristics and functional study of heat shock protein gene SmHsp60 in Sitodiplosis mosellana (Diptera: Cecidomyidae)

作　　者：马倩 董金慧 李方向 朱克岩 成卫宁[1] MA Qian;DONG Jin-Hui;LI Fang-Xiang;ZHU Ke-Yan;CHENG Wei-Ning(Key Laboratory of Plant Protection Resources and Pest Management of Ministry of Education,College of Plant Protection,Northwest A&F University,Yangling 712100,China;Xi′an Agricultural Technology Extension Centre,Xi′an 710061,China;Department of Entomology,Texas A&M University,Texas 77843,USA)

机构地区：[1]西北农林科技大学植物保护学院,植保资源与病虫害治理教育部重点实验室,杨凌712100 [2]西安市农业技术推广中心,西安710061 [3]Department of Entomology,Texas A&M University,USA Texas 77843

出　　处：《昆虫学报》2023年第7期859-869,共11页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31371933);陕西省重点研发计划项目(2020 NY-059)。

摘　　要：【目的】麦红吸浆虫Sitodiplosis mosellana是重要的农业害虫,滞育使其度过夏季和冬季的极端环境。本研究旨在探析麦红吸浆虫滞育进程中热激蛋白60(heat shock protein 60,Hsp60)基因表达与滞育发育及温度耐受性之间的关系。【方法】采用RACE和RT-PCR技术克隆麦红吸浆虫SmHsp60 cDNA全长序列并进行生物信息学分析;采用qPCR技术检测SmHs p60在麦红吸浆虫滞育前至滞育后发育不同阶段(滞育前、滞育、滞育后静息和滞育后发育)幼虫及越夏幼虫在极端高温[35,40,45和50℃水浴中处理1 h以及35℃水浴中0(对照),15,30,60和120 min]和越冬幼虫在极端低温[0,-5,-10和-15℃处理1 h以及-5℃处理0(对照),15,30,60和120 min]胁迫下的表达量;通过原核表达和亲和柱层析技术获得SmHsp60重组蛋白,采用比色法和SDS-PAGE法测定重组蛋白SmHsp60抑制苹果酸脱氢酶(malate dehydrogenase,MDH)43℃下热聚集的能力。【结果】获得麦红吸浆虫SmHsp60(GenBank登录号:KR733065)cDNA全长序列,长2270 bp,开放阅读框长1722 bp,编码573个氨基酸,编码蛋白的相对分子量为60.7 kD。氨基酸序列分析表明,SmHsp60具有线粒体Hsp60典型的标签序列,与同为瘿蚊科(Cecidomyidae)的甘蓝瘿蚊Contarinia nasturtii Hsp60序列一致性最高、亲缘关系最近。qPCR检测结果表明,麦红吸浆虫SmHsp60在滞育阶段表达量没有显著变化,但在滞育后静息阶段逐渐升高,在滞育后静息阶段早中期即12月和翌年1月达到最高,显著高于在其他发育阶段的。与未处理的对照相比,35和40℃下1 h以及35℃下30~60 min时可显著诱导越夏幼虫SmHsp60上调表达;-5℃下1 h可显著诱导越冬幼虫SmHsp60上调表达,但高于45℃和低于-10℃的表达量没有显著变化。获得了高纯度的SmHsp60重组蛋白,可有效保护MDH免遭热聚沉,具有显著的分子伴侣功能。【结论】SmHsp60参与麦红吸浆虫滞育调控,可能与滞育终止及滞育期间的耐热和【Aim】The wheat blossom midge,Sitodiplosis mosellana,an important agricultural pest,survives under temperature extremes during summer and winter by diapause.This study aims to explore the relationship between heat shock protein 60(Hsp60)gene expression and diapause development and temperature tolerance in the diapause process of S.mosellana.【Methods】The full-length cDNA sequence of Hsp60 of S.mosellana(SmHsp60)was amplified via RACE and RT-PCR technologies,and analyzed via bioinformatics.qPCR was used to detect the expression levels of SmHsp60 in the S.mosellana larvae at different stages from pre-diapause to post-diapause development including pre-diapause,diapause,post-diapause quiescence and post-diapause development,as well as oversummering larvae under extremely high temperature stress[exposed to water bathes at 34,40,45 and 50℃for 1 h,and 35℃for 0(control),15,30,60 and 120 min]and overwintering larvae under extremely low temperature stress[exposed to 0,-5,-10 and-15℃for 1 h,and-5℃for 0(control),15,30,60 and 120 min].The recombinant SmHsp60 protein was obtained by prokaryotic expression and affinity column chromatography,and its ability to suppress the thermal aggregation of mitochondrial malate dehydrogenase(MDH)at 43℃was examined by colorimetry and SDS-PAGE.【Results】The full-length cDNA sequence of SmHsp60(GenBank accession no:KR733065)of S.mosellana obtained is 2270 bp in length,which contains an open reading frame(ORF)of 1722 bp in length encoding a protein of 573 amino acids with the relative molecular weight of 60.7 kD.Amino acid sequence analysis indicated that SmHsp60 contains the classical signature sequences of mitochondrial Hsp60,and displayed the highest amino acid identity and the closest relationship to Hsp60 from Contarinia nasturtii of Cecidomyiidae.qPCR detection result showed that the expression level of SmHsp60 did not change significantly in the diapause stage,but began to increase gradually in the post-diapause quiescence stage with a peak in early-to-mid phase of po

关 键 词：麦红吸浆虫 HSP60 滞育 温度胁迫 基因表达 分子伴侣 

分 类 号：Q966[生物学—昆虫学]