甜菜夜蛾Secrt基因克隆及其对甜菜夜蛾核型多角体病毒DNA复制的影响

Cloning of Secrt Gene from Spodoptera exigua and Its Effect on DNA Replication of Spodoptera exigua Multiple Nucleopolyhedrovirus

钙网蛋白(calreticulin,CRT)是一类真核生物中高度保守的Ca^(2+)结合蛋白,在病毒复制中起重要作用。为研究甜菜夜蛾(Spodoptera exigua)Secrt基因及其在甜菜夜蛾核型多角体病毒(Spodoptera exigua multiple nucleopolyhedrovirus,SeMNPV)感染细胞中的作用,本研究以宿主Se301细胞cDNA为模板,通过无缝克隆技术获得Secrt基因的完整开放阅读框1200 bp,其编码399个氨基酸。SeCRT蛋白的相对分子量为45.86 kDa,生物信息学分析发现其含有信号肽、无跨膜结构域,含有2个O-糖基化位点及28个磷酸化位点,C结构域末端具ER驻留信号HDEL,是定位于内质网上的亲水性蛋白质。以最大似然法(Maximum likelihood method)构建系统进化树,聚类结果表明SeCRT与其他鳞翅目(Lepidoptera)昆虫CRT聚为一类,其中与斜纹夜蛾(Spodoptera litura)CRT的相似性高达98.25%。实时荧光定量PCR(real-time fluorescent quantitative PCR,RT-qPCR)检测表明,SeMNPV感染Se301细胞后,其Secrt基因表达在mRNA水平上呈现先降低后升高最终趋于平稳的趋势,表明Secrt基因响应病毒的感染。随后通过RNAi干扰技术敲低Secrt基因,发现SeMNPV病毒DNA复制减少43.4%,表明Secrt基因影响病毒DNA复制。研究结果为进一步阐明宿主基因Secrt在SeMNPV感染中的分子机理及病毒和宿主间相互作用机制奠定基础。

Calreticulin(CRT)is a highly conserved Ca^(2+)binding protein in eukaryotes and plays an important role in viral replication.To study the Secrt gene of Spodoptera exigua and its role in cells infected by Spodoptera exigua multiple nucleopolyhedrovirus(SeMNPV),the complete open reading frame of 1200 bp of Secrt gene encoding 399 amino acids was obtained by seamless cloning with the cDNA of host Se301 cell as the template.The relative molecular weight of SeCRT protein was 45.86 kDa,bioinformatic analysis showed that it had a signal peptide,no transmembrane domain,2 O-glycosylation sites,28 phosphorylation sites,and the terminal of the C domain had an ER retention signal HDEL,which was a hydrophilic protein located in the endoplasmic reticulum.The phylogenetic tree was constructed by the maximum likelihood method.The clustering results showed that the SeCRT was clustered with CRT of other Lepidoptera insects,and the similarity with the CRT of Spodoptera litura was as high as 98.25%.Real-time fluorescent quantitative PCR(RT-qPCR)showed that Secrt gene mRNA level first decreased,then increased,and finally stabilized when SeMNPV infected Se301 cells,indicating that Secrt gene responded to virus infection.Subsequently knockdown of Secrt expre-ssion in cells by RNAi interference resulted in a 43.4%reduction in SeMNPV virus DNA replication,suggesting that Secrt affects viral DNA replication.These results lay a foundation for further elucidating the molecular mechanism of host gene Secrt in SeMNPV infection and the mechanism of virus-host interaction.