桑螟胞外铜锌超氧化物歧化酶基因的克隆与融合表达及重组蛋白酶活性检测

Cloning,Fusion Expression and Enzyme Activity Analysis of Extracellular Copper Zinc Superoxide Dismutase in Mulberry Pyralid,Glyphodes pyloalis

桑螟（Glyphodes pyloalis）是蚕区桑园的主要害虫之一,鉴定其抵御各种环境胁迫相关的基因,是制定防治新策略的重要理论依据。对桑螟幼虫转录组数据的分析中,发现桑螟胞外铜锌超氧化物歧化酶（ec Cu/Zn SOD）基因在应对高温胁迫时的表达量发生显著变化。为了明确ec Cu/Zn SOD在桑螟应对不良环境中发挥的作用,克隆和鉴定了其编码基因Gpec Cu/Zn SOD（Gen Bank登录号：MG566057）。Gpec Cu/Zn SOD基因的开放阅读框长726 bp,编码241个氨基酸,编码蛋白质序列与脐橙螟（Amyelois transitella）的一种SOD蛋白基因（Gen Bank登录号：XP_013197347.1）编码氨基酸序列的一致性高达83%。采用邻近相接法构建桑螟与其他昆虫的Cu/Zn SOD系统发生树,结果显示Cu/Zn SOD被分为ec Cu/Zn SOD和ic Cu/Zn SOD两类,主要是随物种进化而进化。采用半定量RT-PCR检测不同温度处理桑螟5龄幼虫的Gpec Cu/Zn SOD基因表达情况,结果显示幼虫体内的Gpec Cu/Zn SOD在高温（40℃）和低温（4℃）条件下表达量最高。构建p Cold-Gpec Cu/Zn SOD重组表达质粒,并在大肠埃希菌中实现异源表达。利用镍柱亲和层析法分离纯化重组Gpec Cu/Zn SOD蛋白,采用WST-1法测定重组Gpec Cu/Zn SOD蛋白在40℃时的酶活性最高,并且经60～70℃处理后仍然具有较高的酶活性。采用牛津杯法分析重组Gpec Cu/Zn SOD的抗氧化活性表明：过表达Gpec Cu/Zn SOD的大肠埃希菌抗氧化的能力显著增强,显示Gpec Cu/Zn SOD具有较强的抗氧化功能。研究结果暗示Gpec Cu/Zn SOD在桑螟应对高温胁迫过程中发挥着重要作用。

The mulberry pyralid,Glyphodes pyloalis,is one of the major pests of mulberry. Insect response to various environmental stresses is an important aspect for insect control research. In this study,we found that the expression of extracellular copper zinc superoxide dismutase（ ec Cu/Zn SOD） gene changed significantly when G. pyloalis was exposed to high temperature based on transcriptome data analysis. This gene was cloned,designated as Gpec Cu/Zn SOD（ Gen Bank accession No. MG566057） and characterized to elucidate its function involved in response of G. pyloalis to adverse environmental stress. Sequence analysis indicated that the ORF of Gpec Cu/Zn SOD is 725 bp,encoding 241 amino acids. The putative amino acids share high similarity（83%） with another SOD（ XP_0131-97347. 1） from Amyelois transitella. Phylogenetic tree ofGpec Cu/Zn SOD with other insect Cu/Zn SOD was constructed by using NJ method. The result indicated that Cu/Zn SODs are divided into ec Cu/Zn SOD and ic Cu/Zn SOD,and their evolutions are based on speciation. Expression profile of GpecCu/Zn SOD in 5 th instar larva was obtained by semi-quantitative RT-PCR after treatment at different temperature. The result showed that Gpec Cu/Zn SOD had the maximal expression at high（40 ℃） and low（4 ℃） temperature. Expression vector p Cold-Gpec Cu/Zn SOD was constructed for heterologous expression in E. coli. The expressed recombinant protein was purified by Ni＋affinity chromatography. The purified Gpec Cu/Zn SOD was treated under different temperatures. The highest activity was observed at 40 ℃ and a high activity still remained at 60 ℃ and 70 ℃. Anti-oxidative activity of the recombinant Gpec Cu/Zn SOD was analyzed by Oxford Cup method. Anti-oxidative activity of E. coli with overexpressed Gpec Cu/Zn SOD was significantly enhanced,showing that Gpec Cu/Zn SOD may function as a strong antioxidant. It was suggested that Gpec Cu/Zn SOD plays an important role in response of G. pyloalis to high temperature stress.