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本研究采用人工合成方法合成抗菌蛋白基因AP1,连接到p ET32a(+)表达载体,导入E.coli BL21菌株进行原核表达,表达产物经HIS柱纯化和肠激酶切割后测定抑菌活性。人工合成了分子量为321 bp的抗菌蛋白基因AP1,获得了p ET32a(+)-AP1-BL21基因工程菌株,该菌株在0.1 mmol/L IPTG,25℃诱导2 h,蛋白表达率为43.2%,HIS柱纯化后获得SDS-PAGE电泳一条带分子量为27 k D的融合蛋白,其含量为124.82 mg/L,收率为92%,肠激酶切割后的蛋白能抑制大豆根腐病菌和水稻恶苗病菌的生长。获得高效表达抗菌蛋白AP1的工程菌株,该菌株表达的抗菌蛋白纯化后具有抑菌活性,在植病生防方面具有应用潜力。
Abstract：In this research,we synthesized the antifungal protein gene AP1 by artificial synthetic method,connected the gene to expression vector p ET32a(+),transformed it into E. coli BL21 strain for prokaryotic expression,and detected the antifungal activity of expression product after HIS column purification and enterokinase digestion.We synthesized an antifungal protein gene AP1 with the molecular weight of 321 bp and obtained a gene engineering strain of p ET32a(+)-AP1-BL21. The expression rate of this strain was 43.2% by 0.1 mmol/L IPTG induced at 25℃ for 2 hours. The recombinant protein exhibited one band on SDS-PAGE gel with the molecular weight of27 k D after HIS column purification. Its content was 124.82 mg/L and the recovery rate was 92%. The recombinant protein after enterokinase digestion could inhibit the growth of Fusarium oxysporum and Fusarium moniliforme.We had obtained a gene engineering strain for the efficient expression of antifungal protein AP1. The expressed antifungal protein AP1 had antifungal activity after purification and had potential in biocontrol of plant disease.
Alfonzo A.,Piccolo S.L.,Conigliaro G.,Ventorino V.,Burruano S.,and Moschetti G.,2012,Antifungal Peptides produced by Bacillus amyloliquefaciens AG1 active against grapevin fungal pathogens,Ann.Microbiol.,62:1593-1599
Benitez L.B.,Velho R.V.,Lisboa M.P.,Medina L.F.,and Bran delli A.,2010,Isolation and characterization of antifunga peptides produced by Bacillus amyloliquefaciens LBM5006J.Microbiol.,48(6):791-797
Chu X.M.,Lin Y.X.,Zhang K.,Yan F.,and Lin H.T.,2014,Re search advances of antifungal proteins produced by biocontrol bacterium,Bacillus amyloliquefaciens,Baozhuang Yu Shipin Jixie(Packaging and Food Machinery),32(6):49-54(出晓铭,林毅雄,张珅,严芬,林河通,2014,生防菌解淀粉芽孢杆菌抗菌蛋白的研究进展,包装与食品机械,32(6):49-54)
Hao J.A.,Cao Z.H.,Zhao F.M.,Gao W.H.,Xu H.J.,Bai Y.L.Zhang X.M.,and Qiao M.Q.,2008,Exploring the antifunga activity of Bacillus amyloliquefaciens NK10.BAhja WT,We ishengwuxue Tongbao(Microbiology),35(6):903-908(郝建安,曹志辉,赵凤梅,高卫华,徐海津,白艳玲,张秀明乔明强,2008,解淀粉芽孢杆菌NK10.BAhja WT抑真菌作用的研究,微生物学通报,35(6):903-908)
Kim P.I.,and Chung K.C.,2004,Production of an antifungal pro tein for control of Colletotrichum lagenarium by Bacillu amyloliquefaciens MET0908,234(1):177-183
Li B.,Wei X.J.,Li Y.N.,Meng Q.F.,Zhang T.,Liu D.Q.,and Ch G.T.,2013,Prmary analysis of antifungal proteins produced by Bacillus amyloliquefaciens X-278 against Verticillium dahlia,Henan Nongye Kexue(Journal of Henan Agricultur al Sciences),42(7):68-71(黎波,魏学军,李亚宁,孟庆芳张汀,刘大群,赤国彤,2013,解淀粉芽孢杆菌X-278菌株抗菌蛋白初步分析,河南农业科学,42(7):68-71)
Liu T.,Chen Y.P.,Li Q.J.,and Gu Z.F.,2014,Cloning and func tional analyses of Tas A,an antimicrobial protein gene from Bacillus pumilus DX01,Shanghai Jiaotong Daxue Xuebao(Journal of Shanghai Jiaotong University(Agricultural Scie nce)),32(5):48-52(刘通,陈云鹏,李琼洁,顾振芳,2014短小芽胞杆菌抑菌蛋白Tas A基因克隆及功能分析,上海交通大学学报(农业科学版),32(5):48-52)
Lu J.,Xia Q.Y.,Gu W.L.,Sun J.B.,Lu X.H.,Zhang X.,and Wang Y.G.,2013,Isolation,identification of Bacillus amy loliquefaciens LX1 strain against Fusarium oxysporum f.sp cubense and cloning of its antifungal protein gene,Reda Zuowu Xuebao(Chinese Journal of Tropical Crops),34(1)117-124(卢娟,夏启玉,顾文亮,孙建波,卢雪花,张欣,王宇光,2013,拮抗香蕉枯萎病菌的解淀粉芽孢杆菌LX菌株的鉴定及其抗菌蛋白基因的克隆,热带作物学报,3(1):117-124)
Wang Y.G.,Wang J.H.,Quan C.S.,and Fan S.D.,2007,Purifica tion and antifungal characterization of an antifungal sub stance from Bacillus amyloliquifaciens,Zhongguo Shengw Gongcheng Zazhi(China Biotechnology),27(12):41-45(王英国,王军华,权春善,范圣第,2007,解淀粉芽孢杆菌抗菌活性物质的分离纯化及抑菌活性研究,中国生物工程杂志,27(12):41-45)
Wu Y.J.,Lin Y.F.,Lin H.T.,Chen Y.H.,Chen M.Y.,and Jiang P2012,Advances in the researches of biocontrol bacteri Bacillus amyloliquefaciens,Baozhuang Yu Shipin Jixie(Pa ckaging and Food Machinery),30(6):49-52(吴一晶,林艺芬,林河通,陈艺晖,陈梦茵,姜萍,2012,生防菌解淀粉芽孢杆菌研究进展,包装与食品机械,30(6):49-52)
Yao W.L.,Wang Y.S.,Han J.G.,Li L.B.,and Song W.,2004,Pu rification and cloning of an antifungal protein from the ric diseases controlling bacterial strain Paenibacillus polymyx WY110,Yichuan Xuebao(Journal of Genetics and Genom ics),31(9):878-887(姚乌兰,王云山,韩继刚,李潞滨,宋未,2004,水稻生防菌株多粘类芽孢杆菌WY110抗菌蛋白的纯化及其基因克隆,遗传学报,31(9):878-887)
Zhang B.J.,Zhang J.R.,Han J.C.,Liu H.P.,and Wang J.M.2010,Purification and characterization of an antifunga protein from endophytic Bacillus amyloliquefaciens LP-5Zhiwu Baohu Xuebao(Acta Phytophylacica Sinica),37(2)143-147(张宝俊,张家榕,韩巨才,刘慧平,王建明,2010内生解淀粉芽孢杆菌LP-5抗菌蛋白的分离纯化及特性植物保护学报,37(2):143-147)
Zhang S.M.,Wang Y.X.,Meng L.Q.,Cao X.,Zhao X.Y.,Li J.Wang A.X.,and Li J.F.,2013,Study on liquid fermentation conditions of Bacillus amyloliquefaciens TF28,Dongbei No ngye Daxue Xuebao(Journal of Northeast Agricultural Uni versity),44(11):19-24(张淑梅,王玉霞,孟利强,曹旭,赵晓宇,李晶,王傲雪,李景富,2013,内生解淀粉芽孢杆菌TF28液体发酵条件研究,东北农业大学学报,44(11):19-24)
基本信息:
DOI：10.13417/j.gab.035.003083
中图分类号:S476
引用信息:
[1]姜威,李晶,孟利强,等.解淀粉芽孢杆菌TF28抗菌蛋白AP1的表达、纯化与抑菌活性[J].基因组学与应用生物学,2016,35(11):3083-3087.DOI:10.13417/j.gab.035.003083.
基金信息:
黑龙江省科学院学科专项(YXK14ZSM15);黑龙江省科学院学科团队创新能力提升专项(2014sw09)共同资助
2016-11-25
2016-11-25