基于全基因组测序的贝莱斯芽胞杆菌Ba-0321抑菌机制分析及相关功能验证  

Antifungal Mechanism Analysis and Verification of Bacillus velezensis Ba-0321 Based on Whole Genome Sequencing

作　　者：李小杰[1] 邱睿[1] 刘畅[1] 姚晨虓 白静科 陈玉国[1] 李淑君[1] LI Xiaojie;QIU Rui;LIU Chang;YAO Chenxiao;BAI Jingke;CHEN Yuguo;LI Shujun(Tobacco Research Institute of Henan Academy of Agricultural Sciences/Key Laboratory for Green Preservation&Control of Tobacco Diseases and Pests in Huanghuai Growing Area,Xuchang 461000,China)

机构地区：[1]河南省农业科学院烟草研究所/烟草行业黄淮烟区烟草病虫害绿色防控重点实验室,许昌461000

出　　处：《中国生物防治学报》2023年第4期885-894,共10页Chinese Journal of Biological Control

基　　金：河南省农业科学院科技创新团队专项项目(2022TD26);中国烟草总公司河南省公司科技项目(2020410000270012,2022410000240018)。

摘　　要：贝莱斯芽胞杆菌Bacillus velezensis Ba-0321是从健康烟株根际土壤中分离获得的一株具有较强抑菌活性的根际促生细菌,具有较好的生防潜力和应用前景。本研究采用菌丝生长速率法测定菌株Ba-0321无菌滤液对烟草尖孢镰刀菌Fusariumoxysporum和疫霉菌Phytophthoranicotianae的抑制效果,采用二代illumina与三代Nanopore相结合的测序技术,对菌株Ba-0321进行全基因组测序,并对测序数据进行基因组组装、基因功能注释及次级代谢产物合成基因簇预测等。结果表明,菌株Ba-0321无菌滤液对尖孢镰刀菌和疫霉菌均具有抑制效果,其中含有10%无菌滤液的培养基对两种病原菌菌丝生长的抑制率较高,分别达57.38%和34.30%。全基因组测序结果表明,菌株Ba-0321基因组大小为4099109 bp,共有编码基因3897个,基因组测序数据提交至NCBI获得GenBank登录号为CP101904。在GO、COG和KEGG Pathway数据库中有大量编码酶类、萜类化合物、聚酮化合物代谢途径和参与防御机制相关基因被注释。利用anti-SMASH预测到13个次级代谢产物合成基因簇,编码Surfactin、Fengycin、difficidin、Bacillaene、Bacillibactin、Macrolactin H、Bacilysin等多种抑菌活性物质。通过PCR扩增和序列测定分析,证实了Ba-0321菌株基因组中确实存在抑菌物质合成基因簇及防御机制相关基因。酶活测定结果表明,该菌株具有蛋白酶、几丁质酶和纤维素酶等抗性酶活性。本研究在基因组层面上解析了菌株Ba-0321的抑菌机制,为挖掘抑菌相关基因资源提供了分子基础,对该菌株的后续相关研究及应用具有重要意义。Bacillus velezensis Ba-0321,isolated from the rhizosphere soil of healthy tobacco plants,is a plant growth promoting rhizobacteria with strong antifungal activity.It has a good potential in biocontrol application.In the study,the inhibitory effects of strain Ba-0321 sterile filtrate on Fusarium oxysporum and Phytophthora nicotianae were determined by methods of co-culture.The whole genome of strain Ba-0321 was sequenced using the second-generation Illumina and the third-generation Nanopore platform,then analyzed for genome assembly,gene functional annotation,prediction of secondary metabolite synthetic gene clusters.The results showed that the sterile filtrate of strain Ba-0321 has inhibitory effects on both F.oxysporum and P.nicotianae.The culture medium containing 10%sterile filtrate has a higher inhibitory rate on the mycelial growth of the two pathogens,reaching 57.38%and 34.30%,respectively.The results of whole genome sequencing showed that the genome size of strain Ba-0321 was 4099109 bp,including 3897 coding genes,and the sequencing data are available in the GenBank database(accession No.CP101904).There were a large number of genes encoding enzymes,terpene,polyketone metabolic pathways,and genes involved in defense mechanisms have been annotated in the GO,COG and KEGG databases.Using anti-SMASH software,13 secondary metabolite synthesis gene clusters were predicted encoding antibacterial substances such as Surfactin、Fengycin、difficidin、Bacillaene、Bacillibactin、Macrolactin H、Bacilysin.The existence of biosynthetic gene cluster for antibacterial substances and defence machainsm related genes in Ba-0321 strain genome were verified by PCR amplification and sequencing.The enzyme activity test results showed that the strain has resistant enzyme activities such as protease,chitinase,and cellulase..This study provides a basis for analyzing the antimicrobial mechanism of strain Ba-0321 and mining antifungal related gene resources at the genomic level,which is of a great significance for research and app

关 键 词：贝莱斯芽胞杆菌 全基因组 抑菌机制 功能注释 功能验证 

分 类 号：S476[农业科学—农业昆虫与害虫防治]