Cloning and Expression of a Cotton <em>Verticillium</em> Wilt Antibacterial Peptide Gene 25a2

doi:10.11963/cs090502

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Cotton Science ›› 2009, Vol. 21 ›› Issue (5) : 346-350. DOI: 10.11963/cs090502

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Cloning and Expression of a Cotton Verticillium Wilt Antibacterial Peptide Gene 25a2

ZHANG Dong-dong, WANG Shi-ying, GUO Xiao-jun, JIANG Jun-po, LIU Hui-juan, ZHU Bao-cheng^*

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In order to clone the gene which encodes the cotton Verticillium wilt antibacterial peptide A2, 15 amino acid residues of N-terminal of the antibacterial peptide A2 was inputted in NCBI to blast and the result showed a protein contained a fragment which was the same with the 15 amino acid residues of A2. Following the potein sequence, the gene of the protein was found and a pair of primers was designed according to the gene. A DNA fragment about 300 bp was obtained by PCR amplification using the genome of strain Bacillus amyloliquefaciens RS-25 as template. The PCR production was ligated into pET-28a plasmid by two restriction enzyme sites of BamHⅠand Hind Ⅲ. After colony PCR and restriction enzyme-digestion analysis, the PCR production was sequenced and sequencing analysis indicated that the PCR production was 354 bp and coded for a protein of 117 amino acid residues. This gene is named 25a2. The constructed expression vector pET-28a-25a2 was transformed into Eschrichia coli BL21 and colony PCR analysis was used to select the positive clones. The positive single clone was inoculated into LB medium and cultivated overnight with the kanamycin concentration about 100 mg·L^-1. The cultivated bacteria was transfered into fresh medium with 10% inoculum and cultured at 37℃without antibiotics until the OD-600- reached 0.5~0.7. Then IPTG was added into the culture with the final concentration about 1 mmol·L^-1 to induce the expression. Five hours later, the cultured cells were collected and ultrasonic crushed. SDS-PAGE analysis on expression product shows the molecular weight is about 15 kDa and coincides with the theory analysis.

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cotton Verticillium wilt / Bacillus amyloliquefaciens / antibacterial peptide A2

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ZHANG Dong-Dong, WANG Shi-Ying, GUO Xiao-Jun, JIANG Jun-Po, LIU Hui-Juan, ZHU Bao-Cheng. Cloning and Expression of a Cotton Verticillium Wilt Antibacterial Peptide Gene 25a2[J]. Cotton Science, 2009, 21(5): 346-350. https://doi.org/10.11963/cs090502

References

[1] 杜威世, 杜雄明, 马峙英. 棉花黄萎病抗性基因SSR标记研究
[J］. 西北农林科技大学学报:自然科学版, 2004, 32(3): 20-24.           DU Wei-shi, Du Xiong-ming, Ma Zhi-ying. Studies on SSR markers of resistance gene of Verticillium wilt in cotton
[J］. Journal of Northwest Sci-tech University of Agriculture and Forestry:Natural Science Edition, 2004, 32(3): 20-24.
[2］陈旭升, 陈永萱, 黄骏麒. 棉花黄萎病菌致病性生理生化研究进展
[J］. 棉花学报, 2001, 13(3): 183-187.             CHEN Xu-sheng, Chen Yong-xuan, Huang Jun-qi. Advances of studies on physiological and biochemical properties of pathogenesis caused by Verticillium dahliae in Cotton
[J］. Acta Gossypii Sinica, 2001, 13(3): 183-187.
[3］周艳芬, 杜红方, 袁洪水, 等. 棉花黄萎病拮抗蛋白的分离与纯化
[J］. 棉花学报, 2007, 19(2): 98-101.               ZHOU Yan-fen, Du Hong-fang, Yuan Hong-shui, et al. Isolation and purification of antifungal protein from Paenibacillus to Verticillium dahliae
[J］. Cotton Science, 2007, 19(2): 98-101.
[4］贾士荣. 我国棉花基因工程的研究进展
[J］. 中国农业科技导报, 2000, 6(2): 18-22.               JIA Shi-rong. Progress in cotton genetic engineering in China
[J］. Review of China Agricultural Science and Technology, 2000, 6(2): 18-22.
[5］郭荣君, 刘杏忠, 杨怀文, 等. 芽孢杆菌BH-1防治大豆根腐病的效果及机制
[J］. 中国生物防治, 2003, 19(4): 180-184.                   GUO Rong-jun, Liu Xing-zhong, Yang Huai-wen, et al. Mechanism of rhizobacteria BH-1 (Bacillus sp.) to suppress soybean root rot disease caused by Fusarium spp.
[J］. Chinese Journal of Biological Control, 2003, 19(4): 180-184.
[6］王天云, 陈振风, 王福源, 等. 一种促使乳酸芽孢杆菌大量生成芽孢的方法
[J］. 工业微生物, 2001, 31(3): 13-18.               WANG Tian-yun, Chen Zhen-feng, Wang Fu-yuan, et al. A method for facilitate sporulation of Bacillus WTFY1001
[J］. Industrial Microbiology, 2001, 31(3): 13-18.
[7］ GABRIEL E B, Nicolle R, Anette S, et al. Plant-dependent genotypic and phenotypic diversity of antagonistic rhizobacteria isolated from different Verticillium host plants
[J］. Applied and Environmental Microbiology, 2002, 68(7): 3328-3338.
[8］张进霞, 袁洪水, 王士英, 等. 几种氨基酸铜对大丽轮枝菌产生毒素蛋白的影响
[J］. 棉花学报, 2006, 18(2): 79-82.            ZHANG Jin-xia, Yuan Hong-shui, Wang Shi-ying, et al. Effect of cupric complexes of amino acid on secreted toxic protein from Verticillium dahliae
[J］. Cotton Science, 2006, 18(2): 79-82.
[9］张进霞, 袁洪水, 王士英, 等. 几种氨基酸铜对大丽轮枝菌微菌核形成的抑制作用
[J］. 棉花学报, 2006, 18(1): 58-59.            ZHANG Jin-xia, Yuan Hong-shui, Wang Shi-ying, et al. Inhibition of the cupric complexes of amino acid on microsclerotia formation of Verticillium dahliae
[J］. Cotton Science, 2006, 18(1): 58-59.
[10］ LAURA O, Daniel D, Rbert N G M. Variability in ribosomal DNA genic and spacer regions in Verticillium dahliae isolates from different hosts
[J］. Fitopatol Bras, 2004, 29(4): 441-446.
[11］李建武. 生物化学实验原理和方法[M］. 北京: 北京大学出版社, 1994.                   LI Jian-wu. Principle and method of biochemistry experiment[M］. Beijing: Beijing University Press, 1994.
[12］ CHEN Xiao-hua, Koumoutsi A, Scholz R, et al. Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42
[J］. Nat Biotechnol, 2007, 25 (9): 1007-1014.

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2008-11-28	2009-09-15
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