草地贪夜蛾热激蛋白基因SfHsp90的克隆及在高低温和UV-A胁迫下的表达分析

Cloning of heat shock protein gene SfHsp 90 and its expression under high and low temperature and UV-A stresses in Spodoptera frugiperda(Lepidoptera:Noctuidae)

【目的】本研究旨在探索草地贪夜蛾Spodoptera frugiperda响应高低温和UV-A胁迫的分子机制。【方法】通过RT-PCR技术克隆草地贪夜蛾热激蛋白基因Hsp90,利用生物信息学方法分析其序列特征;采用RT-qPCR技术检测草地贪夜蛾Hsp90基因在不同发育阶段(卵、1-6龄幼虫、蛹和成虫)、成虫不同组织(去除触角和复眼的头、胸、腹、触角、复眼、足、翅、中肠、精巢和卵巢)及在不同时长(0, 30, 60, 90, 120和150 min)高温(36℃)、低温(4℃)和UV-A胁迫下成虫中的相对表达量。【结果】克隆获得草地贪夜蛾Hsp90基因,命名为SfHsp90(GenBank登录号:MN832694),该基因开放阅读框(ORF)长2 154 bp,编码717个氨基酸,编码蛋白质相对分子量为82.52 kD,等电点(pI)为5.01,C末端序列含保守基序EEVD,说明该蛋白为胞质型热激蛋白。系统进化分析结果表明,昆虫Hsp90高度保守。发育表达模式显示SfHsp90在草地贪夜蛾1龄幼虫中表达量最高;组织表达模式显示,SfHsp90在草地贪夜蛾雌雄成虫的触角、复眼和去除触角和复眼的头中的表达量显著高于在其他组织中的。高低温胁迫对草地贪夜蛾SfHsp90表达具有明显的诱导作用,36℃胁迫下,SfHsp90表达量显著高于对照,且随着处理时间的延长,在雄成虫中表达量先上升后下降,在60 min时达到最高值,在雌成虫中表达量随处理时间延长逐渐升高;4℃胁迫下,在雄成虫中表达量随着处理时间的延长先上升后下降,在30 min时表达量达到峰值,在雌成虫中表达量随着处理时间延长逐渐上升;随着UV-A照射时间的延长,在雌雄成虫中表达量先上升后下降,90 min时在雄成虫中表达量达到最高值,60 min时在雌成虫中表达量达到最高值。【结论】草地贪夜蛾SfHsp90基因在高低温和UV-A胁迫下的差异表达说明该基因在草地贪夜蛾响应环境胁迫的分子机制中发挥重要作用。

【Aim】This study aims to explore the molecular mechanism of Spodoptera frugiperda in response to high and low temperature and UV-A stresses.【Methods】The heat shock protein Hsp90 gene of S.frugiperda was cloned by RT-PCR technique,and its sequence characteristics were analyzed using bioinformatics methods.The relative expression levels of Hsp90 in different developmental stages(egg,1st-6th instar larva,pupa and adult),different adult tissues(head with antennae and compound eyes removed,thorax,abdomen,antenna,compound eye,leg,wing,midgut,testis and ovary)and adults exposed to high temperature of 36℃,low temperature of 4℃and UV-A radiation for different time(0,30,60,90,120 and 150 min)were detected by RT-qPCR.【Results】The Hsp90 gene was cloned from S.frugiperda and named SfHsp90(GenBank accession no.:MN832694).Its open reading frame(ORF)is 2154 bp in length,encoding 717 amino acids,with the relative molecular weight of 82.52 kD and the isoelectric point(pI)of 5.01.The C-terminal sequence contains the conserved motif EEVD,suggesting that the protein is a cytoplasmic heat shock protein.Phylogenetic analysis showed that insect Hsp90 proteins are highly conserved.Developmental expression profile showed that the expression level of SfHsp90 was the highest in the 1st instar larva,and the tissue expression profile revealed that the expression levels of SfHsp90 were significantly higher in the antenna,compound eyes and head with antennae and compound eyes removed of male and female adults than in other tissues.High and low temperature stresses induced the expression of SfHsp90 obviously.The expression level of SfHsp 90 in adults exposed to high temperature of 36℃was significantly higher than that of the control group.After the adults were exposed to high temperature of 36℃,the expression level of SfHsp 90 increased at first and then decreased with the increase of exposure time,and reached the maximum at 60 min after exposure in male adults,while its expression level in female adults increased gradually with the prolonging of exposure time.After the adults were exposed to low temperature of 4℃,the expression level of SfHsp90 increased firstly and then decreased in male adults with the prolonging of exposure time,and reached the maximum at 30 min after exposure,while its expression level in female adults increased gradually with the prolonging of exposure time.The expression of SfHsp90 in female and male adults was induced by UV-A stress.After the adults were exposed to UV-A radiation,the expression level of SfHsp90 increased firstly and then decreased with the increase of exposure time,and reached the highest at 90 min in male adults and at 60 min in female adults,respectively.【Conclusion】The differential expression of SfHsp90 in S.frugiperda adults under high and low temperature and UV-A stresses suggests that this gene plays an important role in the molecular mechanism in S.frugiperda in response to environmental stresses.