柞蚕真菌蛋白酶抑制剂基因CP8的克隆与表达分析  

Cloning and expression analysis of a fungal protease inhibitor gene CP8 in Antheraea pernyi(Lepidoptera: Saturniidae)

作　　者：钱岑[1] 王芳[1] 李胜[1] 刘朝良[1] 

机构地区：[1]安徽农业大学生命科学学院,合肥230036

出　　处：《安徽农业大学学报》2015年第5期780-785,共6页Journal of Anhui Agricultural University

基　　金：国家农业产业技术体系(CARS-22-SYZ10);国家自然科学基金(31402018)共同资助

摘　　要：蛋白酶抑制剂在昆虫免疫中发挥着重要的作用。首次克隆获得了一个柞蚕真菌蛋白酶抑制剂基因ApCP8的开放阅读框(ORF)序列。该基因的ORF序列全长318 bp,编码了一个由106个氨基酸组成的蛋白(包括19个氨基酸的信号肽)。预测蛋白的等电点和分子量大小分别为9.05和11.3 kD。序列分析和进化树构建结果表明,ApCP8与已知的几种昆虫相应蛋白具有很高的同源性,且与印度柞蚕的CP8聚在同一进化分支上。实时荧光定量PCR(RT-qPCR)结果表明,ApCP8主要在柞蚕脂肪体中高表达;且ApCP8在被白僵菌诱导的初期高表达,推测其可能参与了柞蚕抗真菌的免疫反应。同时,利用pET-32a(+)载体对ApCP8进行了原核表达,并对重组表达蛋白进行了纯化和抗体制备。本研究将为进一步探索ApCP8的抗真菌功能奠定前期基础。Protease inhibitors play important roles in the insect immunity. Here, we isolated a fungal protease inhibitor gene from Antheraea pernyi（Lepidoptera： Saturniidae） for the first time. The gene was named ApCP8. ApCP8 contains 318 nucleotides with a putative open reading frame（ORF） encoding 106 amino acid residues（with a signal peptide of 19 amino acid residues）. The isoelectric point（p I） and predicted molecular weight of ApCP8 protein were 9.05 and 11.3 k Da, respectively. Amino acid sequence and phylogenetic analysis among A. pernyi and other insects represented that these fungal protease inhibitors had a high homology, and ApCP8 existed in one evolutionary branching with Antheraea mylitta＇s. RT-qPCR results showed that ApCP8 displayed the highest expression level in the fat body. In addition, ApCP8 can be upregulated by Beauveria bassiana in 0.5 h after injection, which indicated that it may be involved in the antifungal immune response of Antheraea pernyi. Meanwhile, the recombinant protein of As-ApCP8 was expressed successfully by pET-32a（＋） vector, and the antiApCP8 antibody against rabbit was prepared. This research will provide a preliminary basis for further antifungal immune function exploration of ApCP8.

关 键 词：柞蚕 CP8基因 克隆 原核表达 RT-QPCR 

分 类 号：S885.1[农业科学—特种经济动物饲养]