草坪害虫华北大黑鳃金龟幼虫围食膜蛋白Ho-Peritrophin3基因克隆及序列分析  

Molecular cloning and sequencing of cDNAs of the novel protein Ho-Peritrophin3 from the peritrophic matrix of Holotrichia oblita,a turfgrass pest

作　　者：周洪旭[1] 李长友[1] 李国勋[1] 

机构地区：[1]青岛农业大学无脊椎动物细胞培养和细胞工程中心,山东青岛266109

出　　处：《草业学报》2010年第6期147-153,共7页Acta Prataculturae Sinica

基　　金：国家973项目(2009CB118902);山东省自然科学基金项目(Y2007D69);青岛农业大学高层次人才基金资助

摘　　要：华北大黑鳃金龟是一种重要的草坪害虫,其幼虫中肠围食膜是害虫生物防治的潜在靶标。本研究利用棉铃虫围食膜蛋白多克隆抗体,从已构建的华北大黑鳃金龟中肠cDNA表达文库中筛选得到一个编码围食膜蛋白的cDNA克隆Ho-Peritrophin3,其cDNA长度为1 737 bp,在polyA末端上游含有1个多聚腺苷酸信号序列AATA-AA,最长开放阅读框(ORF)编码528个氨基酸,与华北大黑鳃金龟围食膜蛋白Ho-Peritrophin2的相似性最高,为64.9%。结构域分析表明,Ho-Peritrophin3只含有较少的O-糖基化位点和N-糖基化位点,不含有类粘蛋白结构域,具有5个几丁质结合功能域(chitin binding domain,CBD),它们均由6个保守的半胱氨酸残基组成,与Ho-Per-itrophin1、Ho-Peritrophin2羧基端的CBD只含有4个半胱氨酸残基明显不同。Ho-Peritrophin3的胰蛋白酶和胰凝乳蛋白酶的作用位点主要位于5个CBD内部,受到其保护,不会被降解;与Ho-Peritrophin1、Ho-Peritrophin2不同的是在第5个CBD下游区域的107个氨基酸中,有36.3%的氨基酸属于胰蛋白酶和胰凝乳蛋白酶的酶切位点。Holotrichia oblita is an important turfgrass pest, whose peritropbic matrix is a potential target for biocontrol of pest insects. The peritrophic matrix proteins of H. oblita were studied by screening with a PM protein polyclonal antiserum from Helicoverpa armigera. One positive cDNA clone named Ho-Peritrophin3, with a size of 1 737 bp and a polyadenylation signal of AATAAA upstream of the polyA tail, was screened and sequenced from the library. The longest open reading frame of Ho-Peritrophin3 coded for 528 amino acids, which were mostly similar to Ho-Peritrophin2 of H. oblita, with a similarity of 64.9%. Ho-Peritrophin3, with a little O-linked glycosylation sites, contained five chitin binding domains, all of which consisted of six conserved cysteine residues, while Ho-Peritrophinl and Ho-Peritrophin2 found in the PM of H. oblita contained only four eysteine residues at the C-terminal. No mucin-like domain was found in the Ho-Peritrophin3 sequence. The cleavage sites of trypsin and chymotrypsin mainly lay inside of CBDs in the Ho-Peritrophin3, and were protected by the intradomain disulfide bonds, so they can resist enzymes and exert a physiological function in the midgut. Compared with Ho-Peritrophinl and Ho-Peritrophin2, 36. 3% of 107 amino acids downstream of the fifth CBD belonged to the cleavage sites of trypsin and chymotrypsin. Further study is necessary to explain why Ho-Peritrophin3 can exist in the midgut in the presence of abundant proteinase.

关 键 词：草坪害虫 华北大黑鳃金龟 围食膜蛋白 免疫筛选 序列分析 

分 类 号：S433[农业科学—农业昆虫与害虫防治]