华北大黑鳃金龟两种围食膜蛋白cDNA的分子克隆与序列分析  

Molecular cloning and sequencing of cDNAs of two peritrophic membrane proteins from Holotrichia oblita(Coleoptera:Melolonthidae)

作　　者：周洪旭[1,2] 谭秀梅[2] 李长友[2] 王俊平[2] 孙绪艮[1] 郭巍[3] 李国勋[2] 

机构地区：[1]山东农业大学植物保护学院,山东泰安271018 [2]青岛农业大学植物保护学院,山东青岛266109 [3]河北农业大学,河北省农作物病虫害生物防治工程技术中心,河北保定071000

出　　处：《昆虫学报》2009年第1期10-16,共7页Acta Entomologica Sinica

基　　金：国家重点基础研究发展规划(“973”计划)项目(2009CB118902);山东省自然科学基金项目(Y2007D69)

摘　　要：6本文以华北大黑鳃金龟Holotrichia oblita中肠为材料,依据Stratagene公司文库构建试剂盒方法,构建其中肠cDNA表达文库,该文库滴度为1.9×106pfu/mL,重组率为99.97%。依据现代免疫学原理,利用棉铃虫Helicoverpaarmigera围食膜蛋白多克隆抗体筛选文库,得到两个编码华北大黑鳃金龟围食膜蛋白的cDNA克隆Ho-Peritrophin1与Ho-Peritrophin2,其cDNA长分别为2385bp和1633bp,在polyA末端上游各有3个多聚腺苷酸信号序列AATAAA,最长开放阅读框(ORF)分别编码729个和477个氨基酸,与粉纹夜蛾Trichoplusia niCBP2(chitin bindingprotein2)的相似性最高,分别为21.9%和19.1%。结构域分析表明,Ho-Peritrophin1与Ho-Peritrophin2分别具有9个和6个几丁质结合功能域,只含有较少的O-糖基化位点,不含有类粘蛋白结构域。胰蛋白酶和胰凝乳蛋白酶对两种蛋白的作用位点主要位于几丁质结合功能域(chitin binding domain,CBD)内部,而因受几丁质结合功能域保护,这两种蛋白能够抵抗这些蛋白酶的降解。与正常CBD比较,这两种蛋白C端的CBD只含有4个Cys,只在第1与第3、第4与第5个Cys之间形成两对二硫键,缺少由第2与第6个Cys形成的二硫键。推测其N端还应包括信号肽序列和几丁质结合功能域的未知序列。Peritrophic membrane （PM） proteins of a coleopteran insect Holotrichia oblita were studied by constructing a H. oblita larval midgut cDNA expression library with the cDNA Synthesis Kit of Stratagene Company and screening with a PM protein polyclonal antiserum from Helicoverpa armigera according to the theory of modem immunology. The titer of the primary cDNA expression library was 1.9 ~ 106 pfu/mL and the recombination rate was 99.97%. Two positive cDNA clones named Ho-Peritrophinl and Ho- Peritrophin2, with the sizes of 2 385 and 1 633 bp, were screened and sequenced from the library, respectively. The longest open reading frame of Ho-Peritrophinl and Ho-Peritrophin2 coded for 729 and 477 amino acids respectively, both of which showed the highest similarity to CBP2 of Trichoplusia hi, though their similarities to the latter were only 21.9% and 19.1%, respectively. Ho-Peritrophin! and Ho-Peritrophin2 contained nine and six chitin binding domains （CBDs） respectively with a little O-linked glycosylation sites and no mucin-like domain was found in the two protein sequences. The cleavage sites of trypsin and chymotrypsin are mainly located inside CBDs of the Ho-Peritrophinl and Ho-Peritrophin2, but the two proteins are protected by the intradomain disulfide bonds, so they can resist the enzymes and exert physiological function in the midgut. Compared with the normal CBD, the CBD of carboxylic terminal of the two proteins contains only four cysteines, which form two pairs of disulfide bonds between the 1st and 3rd cysteines and the 4th and 5th cysteines, lacking the one between the 2nd and 6th cysteines. It is inferred that there are one signal peptide and unknown CBDs in the N end of HoPeritrophinl and Ho-Peritrophin2 respectively and these require further study.

关 键 词：华北大黑鳃金龟 中肠 CDNA表达文库 围食膜蛋白 序列分析 

分 类 号：Q966[生物学—昆虫学]