黏虫HSP90/70组织蛋白基因的分子特征与表达特性

Molecular and expression characterization of an HSP90/70 organizing protein(HOP)gene in the Mythimna separata (Lepidoptera: Noctuidae)

为解析黏虫Mythimna separata响应环境胁迫的分子机制,采用RACE和RT-PCR的方法从黏虫中克隆了1个HSP90/70组织蛋白(heat shock 90/70 organizing protein, HOP)基因,并采用qRT-PCR分析了其在多种生物和非生物胁迫下的表达特性。结果表明,MsHOP具有1个1 626 bp的开放阅读框,编码541个氨基酸,预测的分子量为61.7 kDa。序列分析表明,MsHOP主要由已报道的3个保守四肽重复结构域(TPR1, TPR2A和TPR2B)和2个天冬氨酸-脯氨酸重复基序(DP)构成。基于鳞翅目昆虫HOP构建的系统进化树显示MsHOP与棉铃虫Helicoverpa armigera HOP和烟芽夜蛾Heliothis virescens HOP的亲缘关系最近。qRT-PCR分析表明,MsHOP在黏虫所有发育时期和组织均表达,分别在5龄幼虫和雌成虫中肠的表达量最高。在30℃下处理1 h后,MsHOP的表达水平较对照显著提高,而在33℃~39℃下处理1 h后MsHOP的表达水平则与对照差异不显著。饥饿24~72 h后,MsHOP的表达与对照相比无明显变化。Bt能显著诱导MsHOP的表达,其表达水平随Bt浓度的升高而上调,并在64 IU/mL浓度下的表达量最高。幼虫饲养密度对MsHOP的表达也具有显著影响,其表达水平随密度的升高而上调,在20头/瓶的饲养密度下达到最大值。这些结果表明,MsHOP可能在黏虫的生长发育、抵御杀虫剂和密度胁迫中发挥重要作用。

To analyze the responsive mechanism of Mythimna separata to environmental stress,a heat shock 90/70 organizing protein(HOP)homolog gene was identified by RACE and RT-PCR methods,and its expression characterization under multiple biotic and abiotic stressors was also examined by qRT-PCR.Results showed that the identified MsHOP had a 1626 bp ORF encoding a protein of 552 amino acids with a calculated molecular mass of approximately 61.7 kDa.Its deduced amino acids comprised of three conserved tetratricopeptide repeated domains(TPR1,TPR2A and TPR2B)and two aspartic acid-proline(DP)repeat motifs as reported in other insects.Phylogenetic tree based on Lepidoptera HOPs showed that MsHOP was closely related to Helicoverpa armigera HOP and Helicoverpa virescens HOP.qRT-PCR analysis showed that MsHOP was expressed in various developmental stages and tissues of the fourth-day-old female adult,and exhibited the highest expression level in the 5 th instar larval stage and midgut,respectively.MsHOP was significantly up-regulated after exposure to 30℃for 1 h,but its expression level showed no difference at 33℃or higher temperature as compared with that of the control(25℃).MsHOP was not changed after starvation for 24~72 h.After feeding on artificial an diet treated with Bt,MsHOP expression was significantly up-regulated and showed the highest transcription level at 64 IU/mL.The expression level of MsHOP was significantly induced with increasing larval density,and showed the highest expression at 20 larvae per jar.Taken together,the full-length cDNA of MsHOP was successfully obtained,and it might play important role in development,Bt and crowding stress response in M.separata.