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

摘要/Abstract

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1737bp,在polyA末端上游含有1个多聚腺苷酸信号序列AATAAA,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(chitinbindingdomain,CBD),它们均由6个保守的半胱氨酸残基组成,与Ho-Peritrophin1、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。

Abstract: Holotrichia oblita is an important turfgrass pest, whose peritrophic 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-Peritrophin1 and Ho-Peritrophin2 found in the PM of H. oblita contained only four cysteine 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-Peritrophin1 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.

中图分类号:

周洪旭,李长友,李国勋. 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[J]. 草业学报, 2010, 19(6): 147-153.

ZHOU Hong-xu, LI Chang-you, LI Guo-xun. Molecular cloning and sequencing of cDNAs of the novel protein Ho-Peritrophin3 from the peritrophic matrix of Holotrichia oblita, a turfgrass pest[J]. Acta Prataculturae Sinica, 2010, 19(6): 147-153.

参考文献

[1] 冯书亮, 任国栋. 苏云金芽孢杆菌HBF-1及其在有害金龟治理中的应用[M]. 北京: 中国农业科技出版社, 2005: 1-2.
[2] 吴鹏飞, 刘兴良, 刘世荣. 米亚罗亚高山草甸冬春两季土壤动物群落特征的比较[J]. 草业学报, 2009, 18(5): 123-129.
[3] 李存焕, 杨龙飞, 农向群, 等. 绿僵菌防治高尔夫草坪蛴螬试验[J]. 草业科学, 2008, 25(11): 125-128.
[4] 谢木发. 草坪地下害虫蛴螬及防治[J]. 广东园林, 1999, 1: 46-47.
[5] 马宗仁, 常向前, 郭辉. 高尔夫球场不同功能区草地优势种蛴螬种群生态位分析[J]. 应用生态学报, 2004, 15(8): 1416-1422.
[6] 罗晨, 郭晓军, 张芝利. 京郊草坪蛴螬的种类和为害特点[J]. 昆虫学报, 2008, 51(1): 108-112.
[7] 李帅, 李淑文, 李长友, 等. 鞘翅目害虫-华北大黑鳃金龟围食膜结构的初步研究[J]. 华北农学报, 2008, 23(4): 55-59.
[8] Tellam R L, Eisemann C. Chitin is only a minor component of the peritrophic matrix from larvae of Lucilia cuprina[J]. Insect Biochemistry and Molecular Biology, 2000, 30: 1189-1201.
[9] Bolognesi R, Ribeiro A F, Terra W R. The peritrophic membrane of Spodoptera frugiperda: Secretion of peritrophins and role in immobilization and recycling digestive enzymes[J]. Archives of Insect Biochemistry and Physiology, 2001, 47(2): 62-75.
[10] Lehane M J. Peritrophic matrix structure and function[J]. Annual Review of Entomology, 1997, 42: 525-550.
[11] Tellam R L, Eisemann C, Casu R, et al. The intrinsic peritrophic matrix protein peritrophin-95 from larvae of Lucilia cuprina is synthesized in the cardia and regurgitated or excreted as a highly immunogenic protein[J]. Insect Biochemistry and Molecular Biology, 1996, 30: 9-17.
[12] Tellam R L, Wijffels G, Willadsen P. Peritrophic matrix proteins[J]. Insect Biochemistry and Molecular Biology, 1999, 29: 87-101.
[13] Wang P, Granados R R. Molecular structure of the peritrophic membrane (PM): Identification of potential PM target sites for insect control[J]. Archives of Insect Biochemistry and Physiology, 2001, 47: 110-118.
[14] Shen Z C, Jacobs-Lorena M. A type I peritrophic matrix protein from the malaria vector Anopheles gambiae binds to chitin[J]. The Journal of Biological Chemistry, 1998, 273(28): 17665-17670.
[15] Sarauer B L, Gillott C, Hegedus D. Characterization of an intestinal mucin from the peritrophic matrix of the diamondback moth, Plutella xylostella[J]. Insect Molecular Biology, 2003, 12(4): 333-343.
[16] Tellam R L, Vuocolo T, Eisemann C. Identification of an immuno-protective mucin-like protein, peritrophin-55, from the peritrophic matrix of Lucilia cuprina larvae[J]. Insect Biochemistry and Molecular Biology, 2003, 33: 239-252.
[17] Wang P, Li G X, Granados R R. Identification of two new peritrophic membrane proteins from larval Trichoplusia ni: Structural characteristics and their functions in the protease rich insect gut[J]. Insect Biochemistry and Molecular Biology, 2004, 34: 215-227.
[18] Guo W, Li G X, Pang Y, et al. A novel chitin binding protein identified from the peritrophic membrane of the cabbage looper, Trichoplusia ni[J]. Insect Biochemistry and Molecular Biology, 2005, 35: 1224-1234.
[19] 周洪旭, 谭秀梅, 李长友, 等. 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[J]. 昆虫学报, 2009, 52(1): 10-16.
[20] 郭慧芳, 方继朝, 韩召军. 昆虫病毒增效剂研究进展[J]. 昆虫学报, 2003, 46(6): 766-772.
[21] 王丽娟, 金治平, 王能飞, 等. 羊草叶片cDNA文库的构建及部分表达序列标签的分析[J]. 草业学报, 2009, 18(1): 65-71.
[22] Shi X Z, Chamankhah M, Visal-Shah S, et al. Modeling the structure of the Type I peritrophic matrix: Characterization of a Mamestra configurata intestinal mucin and a novel peritrophin containing 19 chitin binding domains[J]. Insect Biochemistry and Molecular Biology, 2004, 34: 1101-1115.
[23] Wang P, Granados R R. Molecular cloning and sequencing of a novel invertebrate intestinal mucin cDNA[J]. The Journal of Biological Chemistry, 1997, 272(26): 16663-16669.
[24] Shen Z C, Jacobs-Lorena M. Evolution of chitin-binding proteins in invertebrates[J]. Journal of Molecular Evolution, 1999, 48: 341-347.
[25] Elvin C, Vuocolo T, Pearson R, et al. Characterization of a major peritrophic membrane protein, peritrophin-44, from the larvae of Lucilia cuprina: cDNA and deduced amino acid sequences[J]. Journal of Biological Chemistry, 1996, 271: 8925-8935.
[26] Schorderet S, Pearson R D, Vuocolo T, et al. cDNA and deduced amino acid sequences of a peritrophic membrane glycoprotein, ‘peritrophin- 48’, from the larvae of Lucilia cuprina[J]. Insect Biochemistry and Molecular Biology, 1998, 28: 99-111.
[27] Eisemann C H, Wijffels G, Tellam R L. Secretion of the type 2 peritrophic matrix protein, peritrophin-15, from the cardia[J]. Archives of Insect Biochemistry and Physiology, 2001, 47: 76-85.
[28] Wijffels G, Eisemann C. A novel family of chitin binding proteins from insect type 2 peritrophic matrix[J]. The Journal of Biological Chemistry, 2001, 276(18): 15527-15536.