柞蚕丝氨酸蛋白酶抑制剂6基因Apserpin-6的克隆及功能分析  

Cloning and Functional Analysis of Serine Protease Inhibitor 6 Encoding Gene (Apserpin-6) from Antheraea pernyi

作　　者：曾军[1] 王国宝[1] 王德意[1] 周敬林[1] 王勇[1] 杨瑞生[1] 秦利[1] 

机构地区：[1]沈阳农业大学生物科学技术学院,沈阳农业大学柞蚕研究所,辽宁省昆虫资源工程技术研究中心,沈阳110866

出　　处：《蚕业科学》2017年第5期764-772,共9页ACTA SERICOLOGICA SINICA

基　　金：现代农业产业技术体系建设专项(No.CARS-18);辽宁省教育厅科学研究项目(No.LSNYB201613)

摘　　要：昆虫的丝氨酸蛋白酶抑制剂(serpin)可以通过抑制丝氨酸蛋白酶的活性调控机体的免疫防御反应。依据柞蚕中肠转录组数据库中一段编码serpin的CDS序列设计正、反向引物,以柞蚕幼虫总c DNA为模板克隆得到一段全长1 239 bp的序列,经序列分析后命名为Apserpin-6(Gen Bank登录号:MF944108)。该基因编码412个氨基酸残基,其中N端第1~17位氨基酸为信号肽序列,在C端第357~391位氨基酸之间有一个反应中心环,第374~375位丝氨酸之间是预测的蛋白质裂解位点。半定量RT-PCR检测Apserpin-6在柞蚕5龄幼虫脂肪体中的表达量最高,在血淋巴和头部组织的表达量次之,在中肠、丝腺和马氏管中的表达量较低。荧光定量PCR检测在受到柞蚕肠球菌(Enterococcus pernyi)、白僵菌(Beauveria bassiana)以及柞蚕核型多角体病毒(Antheraea pernyi nucleopolyhedrovirus)侵染后的柞蚕幼虫血淋巴中,Apserpin-6的表达量均明显上升。利用在原核表达系统表达的重组Apserpin-6蛋白,对柞蚕幼虫血淋巴中的酚氧化酶原(pro PO)和酚氧化酶(PO)活性进行体外抑制试验,结果显示该重组蛋白能够抑制pro PO的活性,但对PO活性没有影响。试验结果提示,Apserpin-6可能作为一种负调控因子参与调控柞蚕血淋巴对于病原菌的免疫防御反应。In insects, serine protease inhibitors regulate the immune defense response of the organism by inhibiting the activity of serine proteases. We here cloned the open reading frame （ 1 239 bp） for a serpin from Antheraea pernyi based on the transcriptome data of midgut in Antheraea pernyi, named as Apserpin-6 （GenBank accession No. MF944108）. The ORF of Apserpin-6 encodes 412 amino acid residues. The predicted signal peptide sequence is located at N-terminal from the first to seventeenth amino acid residues. There is a reaction center loop from the 357th to the 391th amino acid residue at C-terminal. The predicted protein cleavage site is between 374 serine and 375 serine, Apserpin-6 is mainly ex-pressed in fat body detected by semi-quantitative RT- PCR, followed by hemolymph and head, and had the lower expression level in midgut, silk gland and malpighi- an tubule. The results of qRT-PCR showed that the ex- pression level of Apserpin-6 in hemolymph of A. pemyi increased significantly after induced by Enterococcus per- nyi, Beauveria bassiana and Antheraea pernyi nucleo- polyhedrovirus. The recombinant Apserpin-6 protein expr- essed in prokaryotic expression system was used to inhibit the activity of pro-phenoloxidase （proPO） andphenoloxidase （PO） in hemolymph of A. pernyi larvae in vitro. The results showed that Apserpin-6 inhibited proPO activity but did not affect PO activity in A. pernyi hemolymph. Our results suggested that Apserpin-6 acted as a negative regulator to regulate the immune response of A. pernyi hemolymph to pathogens.

关 键 词：柞蚕 丝氨酸蛋白酶抑制剂 基因克隆 组织表达谱 诱导表达 重组蛋白 酚氧化酶活性 

分 类 号：S885.1[农业科学—特种经济动物饲养]