烟芽夜蛾囊泡病毒3h株3h-38基因原核表达及转录、表达时相分析  

Prokaryotic expression and transcription analysis of Heliothis virescens ascovirus 3h encoded 3h-38 gene

作　　者：曹生凯 何磊 黎妮 王星[1,2] 于欢[1,2] CAO Sheng-Kai;HE Lei;LI Ni;WANG Xing;YU Huan(College of Plant Protection,Hunan Agricultural University,Changsha 410128,China;Hunan Provincial Key Laboratory for Biology and Control of Plant Diseases and Insect Pests,Hunan Agricultural University,Changsha 410128,China)

机构地区：[1]湖南农业大学植物保护学院,长沙410128 [2]湖南农业大学植物病虫害生物学与防治湖南省重点实验室,长沙410128

出　　处：《环境昆虫学报》2021年第5期1113-1121,共9页Journal of Environmental Entomology

基　　金：国家自然科学基金(32070168)。

摘　　要：烟芽夜蛾囊泡病毒3h株(Heliothis virescens ascovirus 3h,HvAV-3h)作为一种极具生防潜力的环状双链DNA昆虫病毒,自被分离后,对其基因的特性和相关蛋白功能研究从未间断。本研究通过生物信息学预测3H-38蛋白序列发现其131~150氨基酸位置有一段由胞外向胞内的跨膜区序列,N端23~121氨基酸位置有一个BRO家族结构域,和其同源蛋白的序列对比发现3H-38与烟芽夜蛾囊泡病毒3i株(Heliothis virescens ascovirus 3i,HvAV-3i)和烟芽夜蛾囊泡病毒3j株(Heliothis virescens ascovirus 3j,HvAV-3j)编码的同源蛋白3I-40和3J-43相似性高达85%以上。进一步通过RT-PCR克隆、构建pET-28a-38原核表达载体、IPTG诱导表达、Ni^(2+)-NTA亲和层析柱纯化蛋白等方法获得了His-tag融合的3H-38重组蛋白,并制备了该蛋白的兔多克隆抗体。通过检测3h-38基因在HvAV-3h感染的甜菜夜蛾Spodoptera exigua(Hübner)幼虫中的转录和表达时相,本研究发现3h-38从感染后3 h开始转录,从感染后36 h开始表达,即3h-38是一个早期转录、晚期表达的基因。3h-38基因的生物学信息分析和转录表达时相检测为进一步研究该蛋白的功能和特性奠定了基础。As a large double stranded DNA insect virus,Heliothis virescens ascovirus 3h(HvAV-3h)has high potential to develop into biological control agent,and the studies on the gene characteristics and related protein functions of HvAV-3h has been carried on since it was isolated.In this study,the putative protein sequence of 3H-38 was predicted to contain a transmembrane region and a bro family domain(23~121 amino acid in the N-terminal).Compared with the homologous proteins in other ascovirus isolates,a high identity above 85%was found between 3H-38 and 3I-40(encoded by HvAV-3i)and 3J-43(encoded by HvAV-3j).Besides,RT-PCR,construction of pET-28a-38 vector,induction by IPTG,and purification of Ni^(2+)-NTA affinity chromatography column was employed to obtain the His-tag fused 3H-38,followed by rabbit polyclonal antibody preparation.The transcription and expression phase detection of 3h-38 gene in Spodoptera exigua(Hübner)larvae infected by HvAV-3h were analyzed,and the results showed that 3h-38 was started to transcribe from 3 hpi(hours post infection),but started to express from 36 hpi.The results indicated that 3h-38 was an early transcriptional gene but a late expressional protein.The analysis of the biological information and the detection of transcription and expression phase of 3h-38 gene laid a foundation for further study on the function and characteristics of the protein.

关 键 词：HvAV-3h 原核表达 多克隆抗体 转录时相 表达时相 

分 类 号：Q963[生物学—昆虫学] S476[农业科学—农业昆虫与害虫防治]