基于比较基因组学分析苏云金芽胞杆菌防治蛴螬的相关功能基因

doi:10.16409/j.cnki.2095-039x.2018.05.007

摘要/Abstract

摘要： 为挖掘对地下害虫蛴螬高效的苏云金芽胞杆菌（Bacillus thuringiensis，简称Bt）菌株在杀虫及环境适应方面的关键功能基因，本研究对蛴螬高效的菌株261-1、Fcd114、1126-1、HBF-18和78-2以及对鳞翅目害虫高效菌株WBt-2、G03进行了基因组测序，并与对鳞翅目害虫高效菌株HD1、HD12、HD73和对蛴螬高毒力菌株Bt185等基因组进行了比较分析，重点分析比较了杀虫基因、多糖代谢相关基因、与其他微生物竞争和适应性相关的酰基高丝氨酸内酯水解酶（N-Acylhomoserine lactones hydrolase，aiiA）、突破和克服靶标昆虫免疫系统的免疫抑制因子A（Immune Inhibitor A，InhA）、具有抗病并能增效杀虫的双效菌素（Zwittermicin A，ZwA）等五大类基因和基因簇。研究发现，这些菌株按照其所含有的杀虫基因特异性可分为3个类型：对鳞翅目害虫有效的菌株（类型1）、对鞘翅目害虫有效的菌株（类型2）以及同时含有两种害虫有效基因的菌株（类型3）。对地下害虫蛴螬特异（类型2）的Bt菌株除了在杀虫基因上具有特异性之外，所含有的aiiA、InhA基因也有显著特点。相关研究为进一步对地下害虫蛴螬高效Bt菌株的改良提供了新思路。

关键词: 鞘翅目, 蛴螬, 苏云金芽胞杆菌, 比较基因组学

Abstract: In order to discover the key functional genes of insecticidal activity and environmental adaptation from the high toxicity Bacillus thuringiensis (Bt) strains against underground pests, Bt strains 261-1, Fcd114, 1126-1, HBF-18 and 78-2 with high insecticidal activities against Coleoptera were sequenced, as well as lepidoptera-toxic strains WBt-2 and G03. The published genomes of lepidoptera-toxic HD1, HD12, HD73 and Bt185 strains-toxic to white grubs were included in the comparison. Five kind genes and clusters involved in insecticidal, related to the synthesis and degradation of polysaccharide, microbial competition and adaptation, such as aiiA gene (N-Acylhomoserine lactones hydrolase, aiiA), inhA (Immune inhibitor A, inhA) acting on the immune systems of target insects and Zwittermicin A (ZwA), were analyzed and compared respectively. The results indicated that these strains could be divided into three types based on the specific insecticidal genes, they contained:type 1, lepidoptera-specific; type 2, coleoptera-specific; and type 3, both lepidoptera and coleoptera-toxic. Type 2 strains specific to subterranean pests, had specific toxicity-related genes, which were involved in aiiA, and inhA. The results provided a new vision for further improvement of highly effective Bt strains to control underground pests.

Key words: Coleoptera, white grubs, Bacillus thuringiensis, comparative genomics

中图分类号:

S476.1

黄颖, 王奎, 束长龙, 张杰. 基于比较基因组学分析苏云金芽胞杆菌防治蛴螬的相关功能基因[J]. 中国生物防治学报, 2018, 34(5): 693-700.

HUANG Ying, WANG Kui, SHU Changlong, ZHANG Jie. Analysis of Functional Genes of Bacillus thuringiensis Isolates against White Grubs by Comparative Genomics[J]. journal1, 2018, 34(5): 693-700.

参考文献

[1] Schnepf E, Crickmore N, Van R, et al. Bacillus thuringiensis and its pesticidal crystal proteins[J]. Microbiology and Molecular Biology Reviews, 1998, 62:775-806.
[2] Estruch J, Warren G, Mullins M, et al. Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects[J]. Proceedings of the National Academy of Sciences of the United States of America,1996, 93(11):5389-5394.
[3] Donovan W P, Engleman J T, Donovan J C, et al. Discovery and characterization of Sip1A:a novel secreted protein from Bacillus thuringiensis with activity against coleopteran larvae[J]. Applied Microbiology and Biotechnology, 2006, 72(4):713-719.
[4] Jouzani G S, Valijanian E, Sharafi R. Bacillus thuringiensis:a successful insecticide with new environmental features and tidings[J]. Applied Microbiology and Biotechnology, 2017, 101(7):2691-2711.
[5] 中华人民共和国农业部. 中国农业统计资料. 2013[M]. 北京:中国农业出版社, 2014.
[6] 张美翠, 尹姣, 李克斌, 等. 地下害虫蛴螬的发生与防治研究进展[J]. 中国植保导刊, 2014, 34(10):20-28.
[7] Yu H, Zhang J, Huang D, et al. Characterization of Bacillus thuringiensis strain Bt185 toxic to the Asian cockchafer:Holotrichia parallela[J]. Current Microbiology, 2006, 53(1):13-17.
[8] Shu C, Liu R, Wang R, et al. Improving toxicity of Bacillus thuringiensis strain contains the cry8Ca gene specific to Anomala corpulenta arvae[J]. Current Microbiology, 2007, 55(6):492-496.
[9] Shu C, Hong Y, Wang R, et al. Characterization of two novel cry8G genes from Bacillus thuringiensis strain BT185[J]. Current Microbiology, 2009, 58(4):389-392.
[10] Shu C, Yan G, Wang R, et al. Characterization of a novel cry8 gene specific to Melolonthidae pests:Holotrichia oblita and Holotrichia parallela[J]. Applied Microbiology and Biotechnology, 2009, 84(4):701-707.
[11] 束长龙, 宋福平, 王容燕, 等. 对鞘翅目害虫高效的苏云金芽胞杆菌cry8H基因、蛋白及其应用[P]. 中国发明专利, 2010, ZL200710120020.2.
[12] 闫贵欣, 束长龙, 张杰, 等. 对鞘翅目害虫高效的苏云金芽胞杆菌cry8Ⅰ基因、蛋白及其应用[P]. 中国发明专利, 2010, ZL 200810226214.5.
[13] Zhang Y, Zheng G, Tan J, et al. Cloning and characterization of a novel cry8Ab1 gene from Bacillus thuringiensis strain B-JJX with specific toxicity to scarabaeid (Coleoptera:Scarabaeidae) larvae[J]. Microbiological Research, 2013, 168(8):512-517.
[14] Bi Y, Zhang Y, Shu C, et al. Genomic sequencing identifies novel Bacillus thuringiensis Vip1/Vip2 binary and Cry8 toxins that have high toxicity to Scarabaeoidea larvae[J]. Applied Microbiology Biotechnology, 2015, 99(2):753-760.
[15] 王奎, 束长龙, 李一梅, 等. 苏云金芽胞杆菌-开放的基因组与多种功能[J]. 植物保护, 2017, 44(4):1-8.
[16] Brasseur K, Auger P, Asselin E, et al. Parasporin-2 from a new Bacillus thuringiensis 4R2 strain induces caspases activation and apoptosis in human cancer cells[J]. PLoS ONE, 2015, 10(8):e0135106.
[17] Wei J, Hale K, Carta L, et al. Bacillus thuringiensis crystal proteins that target nematodes[J]. Proceedings of the National Academy of Sciences of the United of America, 2003, 100(5):2760-2765.
[18] Shrestha A, Sultana R, Chae J C, et al. Bacillus thuringiensis C25 which is rich in cell wall degrading enzymes efficiently controls lettuce drop caused by Sclerotinia minor[J]. European Journal of Plant Pathology, 2015, 142(3):577-589.
[19] Akram W, Mahboob A, Javed A A. Bacillus thuringiensis strain 199 can inducesystemic resistance in tomato against Fusarium wilt[J]. European Journal of Microbiology and Immunology, 2013, 3(4):275-280.
[20] Kebria D Y, Khodadadi A, Ganjidoust H, et al. Isolation and characterization of a novel native Bacillus strain capable of degrading diesel fuel[J]. International Journal of Environmental Science and Technology, 2009, 6(3):435-442.
[21] Ohtsubo K, Marth J D. Glycosylation in cellular mechanisms of health and disease[J]. Cell, 2006, 126(5):855-867.
[22] Zhang L, Murphy P J, Kerr A, et al. Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones[J]. Nature, 1993, 362(6419):446-448.
[23] Molina L, Rezzonico F, Défago G, et al. Autoinduction in Erwinia amylovora:Evidence of an acyl-homoserine lactone signal in the fire blight pathogen[J]. Journal of Bacteriology, 2005, 187(9):3206-3213.
[24] Fedhila S, Nel P, Lereclus D. The InhA2 metalloprotease of Bacillus thuringiensis strain 407 is required for pathogenicity in insects infected via the oral route[J]. Journal of Bacteriology, 2002, 184(12):3296-3304.
[25] Dalhammar G. Characterization of inhibitor A, a protease from Bacillus thuringiensis which degrades attacins and cecropins, two classes of antibacterial proteins in insects[J]. European Journal of Biochemistry, 1984, 139(2):247-252.
[26] Silosuh L A, Lethbridge B J, Raffel S J, et al. Biological activities of two fungistatic antibiotics produced by Bacillus cereus UW85[J]. Applied and Environmental Microbiology, 1994, 60(6):2023-2030.
[27] Zhao C, Luo Y, Song C, et al. Identification of three zwittermicin a biosynthesis-related genes from Bacillus thuringiensis subsp. kurstaki strain YBT-1520[J]. Archives of Microbiology, 2007, 187(4):313-319.
[28] 王小奇, 束长龙, 蒋健, 等. 对大黑鳃金龟甲幼虫高效的苏云金芽胞杆菌筛选及cry基因鉴定[J]. 植物保护学报, 2016, 43(3):483-492.
[29] Liu G, Song L, Shu C, et al. Complete genome sequence of Bacillus thuringiensis subsp. kurstaki strain HD73[J]. Genome Announcements, 2013, 1(2):e00080-13.
[30] 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, 28(11):197-201.
[31] Lobo. Basic local alignment search tool (BLAST)[J]. Journal of Molecular Biology, 2012, 215(3):403-410.
[32] Kumar S, Stecher G, Tamura K. MEGA7:Molecular evolutionary genetics analysis version 7.0 for bigger datasets[J]. Molecular Biology and Evolution, 2016, 33(7):1870-1874.
[33] Wiwat C, Thaithanun S, Pantuwatana S, et al. Toxicity of chitinase-producing Bacillus thuringiensis sp. kurstaki HD-1(G) toward Plutella xylostella[J]. Journal of Invertebrate Pathology, 2000, 76(4):270-277.
[34] Rocha L O, Tralamazza S M, Reis G M, et al. Correction:Multi-method approach for characterizing the interaction between Fusarium verticillioides and Bacillus thuringiensis subsp. kurstaki[J]. PLoS ONE, 2015, 10(10):e0141522.
[35] Reddy S T, Kumar N S, Venkateswerlu G. Comparative analysis of intracellular proteases in sporulated Bacillus thuringiensis strains[J]. Biotechnology Letters, 1998, 20(3):279-281.
[36] Johnston P R, Crickmore N. Gut bacteria are not required for the insecticidal activity of Bacillus thuringiensis toward the tobacco hornworm, Manduca sexta[J]. Applied and Environmental Microbiology, 2009, 75(15):5094-5099.
[37] 刘建国, 郝再彬, 宋福平, 等. 几种常见Bt菌株中的Zwittermicin A活性比较[J]. 食品研究与开发, 2014, 35(14):91-94.