Brevibacillus brevis B011次级代谢物中抗青枯菌活性物质的纯化鉴定及生物合成基因簇分析  

Purification,Identification and Biosynthesis Gene Clusters Analysis of Active Substance Against Ralstonia solanacearum in Secondary Metabolites of Brevibacillus brevis B011

作　　者：周向平 陈武[2] 刘天波 王运生[2] 王凯歌 刘峰 李小慧 袁志辉 ZHOU Xiangping;CHEN Wu;LIU Tianbo;WANG Yunsheng;WANG Kaige;LIU Feng;LI Xiaohui;YUAN Zhihui(Yongzhou Company of Hunan Tobacco Company,Yongzhou,Hunan 425100;Plant Protection College,Hunan Agricultural University,Changsha,Hunan 410128;Hunan Institute of Tobacco Science,Changsha,Hunan 410004;College of Chemistry and Bioengineering,Hunan University of Science and Engineering,Yongzhou,Hunan 425199;Hunan Provincial Engineering Research Center for Ginkgo biloba,Yongzhou,Hunan 425199)

机构地区：[1]湖南省烟草公司永州市公司,湖南永州425100 [2]湖南农业大学植物保护学院,湖南长沙410128 [3]湖南省烟草科学研究所,湖南长沙410004 [4]湖南科技学院化学与生物工程学院,湖南永州425199 [5]湖南省银杏工程技术研究中心,湖南永州425199

出　　处：《核农学报》2023年第5期927-935,共9页Journal of Nuclear Agricultural Sciences

基　　金：湖南省教育厅科学研究项目重点项目(21A0519);湖南省重点研发计划项目(2022NK2050);湖南省烟草公司永州市公司科技项目(YZ2022KJ04)。

摘　　要：青枯病是世界上最为严重的植物细菌性病害之一,生物防治是防控其危害的热点研究领域,而分泌抑菌活性物质被认为是生防菌抑制病原菌的主要作用机制。本研究在前期筛选到对青枯菌有良好拮抗活性的短短芽孢杆菌B011(Brevibacillus brevis B011)基础上,采用色谱法分离纯化到抗青枯菌的主要活性物质,通过串联质谱和核磁波谱分析方法鉴定了其结构,并根据B011菌株全基因组序列,预测了活性化合物生物合成相关基因及其合成途径。结果表明,B.brevis B011次生代谢物中抑制青枯菌的主效活性物质为伊短菌素A(edeine A),次效活性物质为N-乙酰基色胺[N-(2-(1H-indol-3-yl)ethyl)acetamide]。这两种化合物对青枯菌的抑制活性为首次报道。基因簇预测结果表明,B011菌株基因组中存在完整的edeine合成基因簇,共包括17个基因,即edeA~edeQ,且基因簇中的基因结构完整,基因序列高度保守。B011基因组中有多个N-乙酰基色胺的生物合成相关基因,包括17个脱羧酶编码基因和54个乙酰转移酶编码基因,可能分别参与色氨酸脱羧反应和色胺的乙酰基转移反应,但具体由哪些基因参与还需进一步的研究。研究结果可为该生防菌的应用提供理论指导,也可为后续解析其生物合成途径及通过合成生物学方法进行定向改造奠定基础。Bacterial wilt caused by plant pathogenic Ralstonia spp.is one of the most important diseases affecting the production of many important crops worldwide.With the advantage of being environmentally friendly,biological control of bacterial wilt is one of the hot research areas,and the secretion of bacteriostatic active substances is considered to be the main mechanism of biocontrol bacteria to inhibit pathogenic bacteria.In present study,Brevibacillus brevis B011,which had good antagonistic activity against Ralstonia solanacearum,was isolated and identified.Membrane filtration,ion exchange chromatography and high performance liquid chromatography were conducted to separate and purify the main active constituents from the broth of B.brevis B011.The chemical structures of purified active compounds were identified by tandem mass spectrometry and nuclear magnetic spectroscopy.On the basis of the complete genome sequence of strain B011,the putative genes involved in the biosynthesis of active compounds and their synthesis pathways were predicted.The results showed that among the secondary metabolites of B.brevis B011,the active constituents for inhibiting bacterial wilt was edeine A,and Nacetyltryptamine[n-(2-(1H-indol-3-yl)ethyl)acetamide].There was a complete gene cluster for edeine synthesis in the genome of strain B011,including 17 genes,edeA-edeQ.The structure of potential biosynthetic genes in the cluster was complete and the sequences were highly conserved.Decarboxylase coding genes of 17 and 54 acetyltransferase coding genes were predicted to be involved in the tryptophan decarboxylation reaction and tryptophan acetyl transfer reaction respectively in B.brevis B011 genome,but the functional genes involved in the biosynthesis of active compounds was needed to be further studied.The results can provide the theoretical guidance for the application of the strain B.brevis B011 and its active compounds,and also lay a foundation for further analysis of its biosynthetic pathway and directional modification of active com

关 键 词：短短芽孢杆菌 伊短菌素A N-乙酰基色胺 生物合成基因簇 

分 类 号：S432[农业科学—植物病理学] S476[农业科学—农业昆虫与害虫防治]