甜菜夜蛾几丁质脱乙酰酶SeCDA7的表达与结合活性分析

Expression and Binding Activity Analysis of Chitin Deacetylase SeCDA7 from Spodoptera exigua Larvae

几丁质脱乙酰酶(Chitin deacetylase,CDA)是昆虫几丁质代谢酶系中的重要组分,是害虫防治的重要靶标。通过RT-PCR技术克隆得到编码甜菜夜蛾几丁质脱乙酰酶secda7基因(GenBank登录号为MG604929),该基因长1431bp,包含开放阅读框长1134bp,SeCDA7蛋白的预测分子量分别为43.156kD。结构域分析显示,SeCDA7具有一个多聚糖乙酰基转移酶催化区,属于第Ⅴ类CDA蛋白。分别构建了原核和真核重组表达载体,利用大肠杆菌和Bac-to-Bac昆虫杆状病毒表达系统转染Sf9昆虫细胞,成功表达了SeCDA7蛋白,纯化SeCDA7蛋白并分析几丁质结合活性,结果表明SeCDA7蛋白具有几丁质结合活性;荧光定量PCR结果显示secda7基因主要在中肠组织表达。本研究实现了甜菜夜蛾几丁质脱乙酰酶基因secda7的外源表达,并鉴定出SeCDA7蛋白具有几丁质结合活性,为深入探究甜菜夜蛾几丁质脱乙酰酶的生理功能提供了理论依据。

Chitin deacetylase(CDA)is an important component of insect chitin metabolism enzymes,thus recognized as the key target in pest control. RT-PCR method was used to clone a gene secda encoding CDA in Spodoptera exigua midgut. The secda was 1 431 bp in length, containing an ORF 1 134bp(GenBank accession number :MG604929). After the signal peptide removed,the secreted SeCDA7 protein was predicted to have a molecular weight 43.156 kD. Domain analysis indicated that SeCDA7 had a polysaccharide acetyltransferase catalytic region belonging to the first class V CDA protein. Then,the prokaryotic and eukaryotic recombinant expression vectors were constructed,and Sf9 insect cells were infected using the Escherichia coli and Bac-to-Bac insect baculovirus expression system,and SeCDA7 was expressed successfully in the insect cells. The purification results of SeCDA7 protein and analysis of chitin binding activity showed that SeCDA7 protein had chitin binding activity. Fluorescence quantitative PCR results demonstrated that secda7 gene mainly expressed in the midgut. In sum,the heterogeneous expression of the chitin deacetylase gene secda7 in S. exigua is achieved and the SeCDA7 is identified to have chitin binding activity,which provides a theoretical basis for further investigation on the physiological function of the chitin deacetylase of S. exigua.