环链棒束孢IcHsp104与IcHsp78的克隆及在不同胁迫条件下的表达检测

Molecular cloning, expression detection of IcHsp104 and IcHsp78 genes under different stress conditions in Isaria cateinannulata

摘要:

【背景】环链棒束孢(Isaria cateinannulata)是一种重要的虫生真菌，许多环境因子的胁迫作用影响了该菌在田间的生防效果。【目的】热休克蛋白酪蛋白溶解蛋白酶(Heat Shock Protein Casein Lytic Proteinase，Hsp100/Clp)是一类ATP依赖型Hsp100家族蛋白，克隆该菌株Hsp100/ClpB家族的2个关键基因IcHsp104与IcHsp78，探索该对基因在应对不同温度及盐浓度胁迫下的作用。【方法】通过前期获得的转录组数据库，采用本地Blast方法对环链棒束孢Hsp100家族基因进行分析筛选。通过RT-PCR技术，对环链棒束孢Hsp100基因的编码区(Open Reading Frame，ORF)进行碱基验证。通过分子生物信息学分析软件，对环链棒束孢Hsp100基因的氨基酸结构、进化树、功能结构域和三级结构进行分析。通过不同温度及盐浓度处理菌株，采用荧光定量PCR (Real-Time Quantitative PCR，RT-qPCR)技术，对获得的基因进行表达检测。【结果】共筛选出2个环链棒束孢Hsp100/ClpB基因Hsp104与Hsp78，将其命名为IcHsp104与IcHsp78，2个基因分别编码923个和805个氨基酸，分子量分别为103.199 kD和88.805 kD；2个基因均与棒束孢属、白僵菌属和虫草属3个物种的亲缘关系最近，但2个基因之间的同源性较低；2个基因编码的蛋白均为经典的AAA+-ATPase家族蛋白，三级结构以α螺旋为主。另外，经高低温处理菌株后，2个基因的表达均会上调，并随处理时间的延长上升越显著，而且高温胁迫组显著强于低温组。经不同浓度氯化钠处理后，低浓度组2个基因的表达量均上调，高浓度组2个基因的表达量均受到抑制。【结论】环链棒束孢IcHsp104与IcHsp78基因在抵抗外界温度及盐胁迫过程中起到重要的作用，为后续环链棒束孢应对环境胁迫的机理研究提供了理论基础。

Abstract:

[Background] Isaria cateinannularis is an important entomogenous fungus, and its biocontrol efficiency in the field is affected by the stress of many environmental factors. [Objective] Hsp100/ClpB is a class of ATP-dependent Hsp100 family protein, to clone two crucial Hsp100/ClpB (heat shock protein, casein lytic proteinase B) family genes IcHsp104 and IcHsp78 from Isaria cateinannulata, and to explore its role in response to different temperatures and concentrations salt stress. [Methods] The Hsp100 family genes were analyzed and screened by BLAST method using transcriptomic database obtained in early stage. RT-PCR technologies were used to verify the open reading frame (ORF) sequence of Isaria cateinannulata Hsp100 genes. The amino acid structure, phylogenetic tree, functional domain and tertiary structure of Isaria cateinannulata Hsp100 genes were analyzed by molecular bioinformatics analysis software. The expression of Isaria cateinannulata Hsp100 genes were detected by RT-qPCR technology after the fungus were treated with different temperature and salt concentration. [Results] A pair of Hsp100/ClpB genes Hsp104 and Hsp78 were screened and named IcHsp104 and IcHsp78, which encoded 923 and 805 amino acids, and their molecular weights were 103.199 kD and 88.805 kD, respectively. The relationship of both genes were close to Isaria spp., Beauveria spp. and Cordyceps spp., but it showed lower similarity between the two genes. The proteins encoded by the two genes were classic AAA+-ATPase family proteins, and the tertiary structure was mainly by alpha helix. In addition, after the treatment of high and low temperature, the expression levels of both genes were up-regulated, and increased more significantly with the extension of treatment time. Additionally, the effect of high temperature stress was significantly stronger than the low temperature stress. Then, in terms of the treatment of different concentrations of sodium chloride, the expression levels of both genes were up-regulated in the low concentration stress, while they were inhibited in the high concentration stress. [Conclusion] The IcHsp104 and IcHsp78 genes from Isaria cateinannulata play an important role in the response to external temperature and salt stress, those findings provide a theoretical basis for the further mechanism studies on Isaria cateinannulata responding to environmental stress.