苏云金芽胞杆菌cry2Ab34基因的克隆、表达和杀虫活性分析

doi:10.13560/j.cnki.biotech.bull.1985.2017.04.024

摘要/Abstract

摘要： 旨在对苏云金芽胞杆菌cry2Ab34基因进行分子鉴定和生物活测定,从杀虫活性高的Bt BJH500 菌株中提取基因组 DNA,通过 PCR 法克隆得到cry2Ab34基因（GenBank 登录号为KX357382）。这个基因序列已被国际Bt基因命名委员会正式命名为 cry2Ab34,结果显示,对cry2Ab34基因进行表达,获得大小约70 kD的表达产物,将该基因在大肠杆菌E.coli BL21中表达之后进行生物活性测定,结果表明对鳞翅目害虫小菜蛾（Plutella xyllostella）和棉铃虫（Helicoverpa armigera）都具有一定的杀虫活性,其校正死亡率10 µg/mL为31.25 %,100 µg/mL为62.5 %和校正死亡率10 µg/mL为29.4%,100 µg/mL为52.9 %。

关键词: 苏云金芽胞杆菌, cry2Ab34基因, 杀虫晶体蛋白, 杀虫活性, 小菜蛾, 棉铃虫

Abstract: In order to identify and detect insecticidal activities from Bacillus thuringiensis strain,the genomic DNA was extracted from the strain Bt BJH500 with high insecticidal activity,from which the insecticidal gene cry2Ab34（GenBank accession number：KX357382）was cloned by PCR. The gene was designated as cry2Ab34 by International Gene Nomenclature Committee of Bt. Gene cry2Ab34 was expressed and the weight of expressed product was approximate 70 kD. The gene was expressed in Escherichia coli BL21 for the determination of biological activity,results showed that this gene presented certain insecticidal activity to Plutella xyllostella and Helicoverpa armigera of Lepidoptera pests,the corrected mortality of 10 µg/mL was 31.25%,of 100 µg/mL was 62.5 %,of 10 µg/mL was 29.4 %,and of 100 µg/mL was 52.9 %.

Key words: Bacillus thuringiensis, gene cry2Ab34, insecticidal crystal protein, insecticidal activity, Plutella xyllostella, Helicoverpa armigera

张月李海涛刘荣梅高继国. 苏云金芽胞杆菌cry2Ab34基因的克隆、表达和杀虫活性分析[J]. 生物技术通报, 2017, 33(4): 185-190.

ZHANG Yue, LI Hai-tao, LIU Rong-mei, GAO Ji-guo. Cloning,Expression,and Insecticidal Activities of Gene cry2Ab34 from Bacillus thuringiensis[J]. Biotechnology Bulletin, 2017, 33(4): 185-190.

参考文献

[1] Ladics GS, Bardina L, Cressman RF, et al. Lack of cross-reactivity between the Bacillus thuringiensis derived protein Cry1F in maize grain and dust mite Der P7 protein with human sera positive for der p7-ige[J]. Regul Toxicol Pharmacol, 2006, 44（2）：136-143.
[2] Ibargutxi MA, Estela A, Ferr J, et al. Use of Bacillus thuringiensis toxins for control of the cotton pest Earias insulana（Boisd. ）（Lepidoptera：Noctuidae）[J]. Applied and Environmental Microbiology, 2006, 72（1）：437-442.
[3] Bravo A, Gómez I, Porta H, et al. Evolution of Bacillus thuringiensis Cry toxins insecticidal activity[J]. Microbial Biotechnology, 2013, 6（1）：17-26.
[4] Bravo A, Likitvivatanavong S, Gill SS, Soberón M. Bacillus thuringiensis：a story of a successful bioinsecticide[J]. Insect Bio-chemistry and Molecular Biology, 2011, 41（7）：423-431.
[5] Schnepf E, Crickmore N, Van RIE J, et al. Bacillus thuringiensis and its pesticidal crystal proteins[J]. Microbiol MolBiol R, 1998, 62（3）：775-806.
[6] Bravo A, GIll SS, Sober NM. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insectcontrol[J]. Toxicon, 2007, 49（4）：423-435.
[7] Cajendra Babu B, Udayasuriyan V, Asia Mariamm M, et al. Comparative toxicity of Cry1Ac and Cry2Aa δ-endotoxins of Bacuillus thuringiensis against Helicoverpa armigera（H. ）[J]. Crop Protection, 2002, 21（9）：817-822.
[8] Schnepf HE, Whiteley HR. Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coil. [J]. Proc Nati Acad Sci, 1981, 78（10）：2893-2897.
[9] Crickmore N, Zelgler DR, Feitelson J, et al. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins[J]. Microbiol Mol Biol Revi, 1998, 62（3）：807-813.
[10] Chen M, Sheltin A, Ye GY. Insect-resistant genetically modified rice in china：From research to commercialization[J]. Annual Review of Entomology, 2001, 56：81-101.
[11] Lecadet MM, Dedonder R. Enzymatic hydrolysis of the crystals of Bacillus thuringiensis by the proteases of Pieris brassicae. I. Preparation and fractionation of the lysates[J]. Journal of Invertebrate Pathology, 1967, 9：310-321.
[12] Johnston KA, Lee MJ, Brough C, et al. Protease activities in the larval midgut of Heliothis virescens：Evidence for trypsin and chymotrypsin-like enzymes[J]. Insect Biochemistry and Molecular Biology, 1995, 25：375-383.
[13] Haider MZ, Ellar DJ. Functional mapping of an entomocidal endotoxin：single amino acid changes produced by site-directed mutagenesis influence toxicity and specificity of the protein[J]. Journal of Molecular Biology, 1989, 208：183-194.
[14] Zhang YJ, Li YH, Zhang Y, et al. Seasonal expression of Cry1Ab and Cry1Ac proteins in transgenic rice lines and their resistance striped rice borer Chilo suppressalis（Walker）[J]. Transgenic Plants & Insects, 2011, 40（5）：1323-1330.
[15] Akhurst RJ, James W, Bird LJ, et al. Resistance to the Cry1Ac dalta-endotoxin of Bacillus thuringiensis in the cotton bollworm, Helicoverpa armigera（Lepidoptera;Noctuidae）[J]. Joumal of Economic Entomology, 2003, 96（4）：1290-1299 .
[16] Park HW, Devera JA, Prins BA, et al. The dual-activity insecticidal protein, Cry2Aa, does not enhance the mosquitocidal activity of Bacillus thuringiensis subsp. Israelensis[J]. Journal of Asia-Pacific Entomology, 2011, 14（4）：429-431.
[17] Winder WR, Whiteley HR, Two highly related crystal proteins of Bacillus thuringiensis serovar kurstaki possess different host range specificities[J]. J Bacteriol, 1989, 171：965-974.
[18] Zheng A, Zhu J, Tan F, et al. Characterisation and expression of a novel haplotype cry2A-type gene from Bacillus thuringiensis strain JF19-2[J]. Ann Microbiol, 2010, 60：129-134.
[19] Wu D, Cao XL, Bai Y, et al. Sequencing of an operon containing a novel d-endotoxin gene from Bacillus thuringiensis[J]. FEMS Microbiol Lett, 1991, 81（1）：31-35.
[20] Karim S, Dean DH. Toxicity and receptor binding properties of Bacillus thuringiensis d-endotoxins to the midgut brush border mem- brane vesicles of the rice leaf folders. Cnaphalocrocis medinalis and Marasmia patnalis[J]. Curr Microbiol, 2000, 41（4）：276-283.
[21] Hire RS, Makde RD, Dongre TK, D’souza SF. Expression, purifica-tion and characterization of the Cry2Aa14 toxin from Bacillus thu-ringiensis subsp[J]. Kenyae Toxicon, 2009, 54（4）, 519-524.
[22] Lenin K, Mariam MA, Udayasuriyan V. Expression of a cry2Aa gene in an acrystalliferous Bacillus thuringiensis strain and toxicity of Cry2Aa against Helicoverpa armigera[J]. World J Microbiol Biotechnol, 2001, 17：273-278.
[23] 王毛, 吴亚飞, 邓拓, 等. 苏云金芽孢杆菌Cry2Ab基因克隆与植物表达载体的构建[J]. 西北林学院学报, 2016, 31（5）：178-181.
[24] 李长友, 张杰, 宋福平, 等. 苏云金芽孢杆菌 B-Pr-88 菌株中cry2Ab4基因的表达和杀虫活性研究[J]. 生物工程学报, 2007, 23（4）：634-638.
[25] 喻子牛. 苏云金芽胞杆菌[M]. 北京：科学出版社, 1990：305-323.
[26] Baroy F, Lecadet MM, Deleluse A. Cloning and sequencing of three new putative toxin genes from Clostridium bifermentans[J]. Gene, 1998, 211（2）：293-299.
[27] 张彦蕊, 束长龙, 宋福平, 等. 一种简单、快速的苏云金芽胞杆菌基因组DNA提取方法[J]. 生物技术通报, 2012（11）：197-201.
[28] 宋福平, 张杰, 黄大昉, 等. 苏云金芽胞杆菌cry基因 PCR-RFLP 鉴定体系的建立[J]. 中国农业科学, 1998, 31（3）：13-18.
[29] 刘东明. 苏云金芽胞杆菌新型cry1基因克隆, 表达及活性分析[D]. 哈尔滨：东北农业大学, 2011.
[30] 王志鑫, 束长龙, 申培立, 等. 含cry8型基因苏云金芽胞杆菌的分离和鉴定. 华北农学报, 2014, 29（6）：149 -154.
[31] 蔡亚君, 袁志明, 胡晓敏, 蔡全信. 苏云金芽胞杆菌几丁质酶基因的克隆及诱导表达[J]. 湖北农业科学, 2011, 50：63-66.
[32] 王继磊, 刘迪秋, 丁元明, 等. Bt转基因抗虫植物研究进展[J]. 生物学杂志, 2010, 27（4）：75-78.
[33] 宋敏, 林祥明, 刘丽军. Cry基因家族的专利分布研究[J]. 生物技术通报, 2010（21）：86-99.
[34] 孟鑫睿, 路杨, 刘艳微, 等. 苏云金芽胞杆菌一新菌株的鉴定及其杀虫活性[J]. 植物保护学报, 2015, 42（3）：410-417.
[35] 邵宗泽, 喻子牛. 苏云金芽胞杆菌杀虫晶体蛋白作用的分子机制研究进展[J]. 生物工程进展, 2001, 21（6）：38-41.
[36] 李海涛, 姚江, 郭巍, 等. 苏云金芽孢杆菌cry2Aa基因的克隆、表达与活性[J]. 农业生物技术学报, 2005, 6：787-791.