昆虫学报 ›› 2021, Vol. 64 ›› Issue (7): 790-799.doi: 10.16380/j.kcxb.2021.07.003

• 研究论文 • 上一篇    下一篇

美国白蛾热激蛋白70基因的鉴定及功能分析

乔恒1,2, 李慧1,2, 耿薏舒1,2, 赵旭东1,2, 于晓航1,2, 郝德君1,2,*    

  1.   (1. 南京林业大学南方现代林业协同创新中心, 南京 210037; 2. 南京林业大学林学院, 南京 210037)
  • 出版日期:2021-07-20 发布日期:2021-08-02

Identification and functional analysis of heat shock protein 70 gene of Hyphantria cunea (Lepidoptera: Arctiidae)

 QIAO Heng1,2, LI Hui1,2, GENG Yi-Shu1,2, ZHAO Xu-Dong1,2, YU Xiao-Hang1,2, HAO De-Jun1,2,*    

  1.   (1. Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China; 2. College of Forestry, Nanjing Forestry University, Nanjing 210037, China)
  • Online:2021-07-20 Published:2021-08-02

摘要:

 【目的】探究热激蛋白70(heat shock protein 70, HSP70)基因在美国白蛾Hyphantria cunea抵御高温胁迫过程中的作用,为揭示美国白蛾的扩散机制以及预测潜在分布区提供理论支撑。【方法】利用PCR技术克隆美国白蛾HSP70基因,并进行生物信息学分析;利用qPCR技术检测新蜕皮的美国白蛾4龄第2天幼虫在25, 30, 35和40℃处理下HSP70基因的表达特性;构建美国白蛾HSP70的原核表达载体,并在大肠杆菌Escherichia coli BL21感受态细胞中诱导表达该蛋白,利用Ni2+-His柱纯化蛋白,并通过Western blot验证该蛋白;利用体外实验测定原核表达获得的重组蛋白在高温胁迫下的ATP酶活性。【结果】克隆获得美国白蛾2条HSP70基因HcHSP70(GenBank登录号: MT995848)和HcHSC70(GenBank登录号: MT261583),其ORF长度分别为1 917和2 061 bp,分别编码637和687个氨基酸,分子质量分别约为69.66和74.96 kD,等电点分别为5.90和5.96;氨基酸序列结构均含有3个高度保守的区域GIDLGTTYS, IFDLGGGTFDVSIL和VGGSTRIPKVQ,符合热激蛋白70家族的特征;蛋白三维结构均由N端ATPase功能域和C端底物结合功能域所组成。系统进化树显示HcHSP70与鳞翅目HSP70家族其他成员聚为一支,而HcHSC70与鳞翅目HSC70家族的其他成员聚为另一支。qPCR结果表明,新蜕皮的美国白蛾4龄第2天幼虫在热胁迫下HcHSP70的相对表达量显著上调,且在35℃处理2 h下达到峰值,而热胁迫下HcHSC70的表达响应较微弱。成功构建了HcHSP70的原核表达载体并在体外诱导表达。纯化后的重组蛋白HcHSP70具备ATPase活性,且在高温胁迫下酶活性稳定。【结论】本研究克隆获得美国白蛾2条HSP70基因HcHSP70和HcHSC70,明确了两条基因在高温处理下的表达特性;成功对美国白蛾HcHSP70进行原核表达及纯化,并证明重组蛋白HcHSP70在高温下具有稳定的ATPase活性,推测其在美国白蛾应对高温胁迫中发挥着重要作用。

关键词: 美国白蛾, 热激蛋白70, 高温胁迫, 原核表达, 分子伴侣

Abstract: 【Aim】 This study aims to explore the role of heat shock protein 70 (HSP70) genes in the process of resisting high temperature stress and to provide a theoretical basis for revealing the expansion mechanism of Hyphantria cunea and predicting its potential distribution area. 【Methods】 HSP70 genes of H. cunea were cloned by PCR and subjected to bioinformatical analysis. The expression characteristics of HSP70 genes in the day-2 4th instar newly molted larvae of H. cunea under 25, 30, 35 and 40℃ were detected by qPCR. The prokaryotic expression vector of HSP70 of H. cunea was constructed and induced to express in Escherichia coli BL21. The protein was purified by Ni2+-His column and verified by Western blot. Then, the ATPase activity of the recombinant protein obtained by prokaryotic expression was determined by in vitro experiments. 【Results】 The two HSP70 genes of H. cunea including HcHSP70 (GenBank accession no.: MT995848) and HcHSC70 (GenBank accession no.: MT261583) were cloned and sequenced. Their ORFs are 1 917 and 2 061 bp in length, encoding 637 and 687 amino acids with the predicted molecular weights of about 69.66 and 74.96 kD, and the isoelectric points of 5.90 and 5.96, respectively. The structure prediction conformed to the characteristics of the heat shock protein 70 family, which contains three highly conserved regions GIDLGTTYS, IFDLGGGTFDVSIL, and VGGSTRIPKVQ. The 3D structure of the two HSP70 proteins is composed of the N-terminal ATPase functional domain and C-terminal substrate binding domain. The phylogenetic tree showed that HcHSP70 and other members of the HSP70 family of Lepidoptera were clustered into one branch, while HcHSC70 and other members of the HSC70 family of Lepidoptera were clustered into another branch. The qPCR results showed that the expression of HcHSP70 in the day-2 4th instar newly molted larvae of H. cunea was significantly upregulated under heat stress and reached the peak under 35℃ for 2 h, while HcHSC70 had a weak expression response under heat stress. The prokaryotic expression vector of HcHSP70 was successfully constructed, and HcHSP70 was expressed in vitro. The purified recombinant protein HcHSP70 had ATPase activity, which was stable under high temperature stress. 【Conclusion】 In this study, HcHSP70 and HcHSC70 of H. cunea were cloned, and their expression characteristics under high temperature were confirmed. The prokaryotic expression and purification of HcHSP70 were successfully performed. The recombinant HcHSP70 has stable ATPase activity under high temperature, suggesting that it may play an important role in the response of H. cunea to high temperature stress.

Key words: Hyphantria cunea, heat shock protein 70, heat stress, prokaryotic expression, molecular chaperone