贝莱斯芽孢杆菌HN-1突变体文库的构建及Arp基因的鉴定  

Construction of Mutant Library of Bacilus velezensis HN-1 and Identification of Arp Genes

作　　者：王雨 沈钰莹 方渝凯 缪卫国[1] 刘文波[1] 靳鹏飞[1] Wang Yu;Shen Yuying;Fang Yukai;Miao Weiguo;Liu Wenbo;Jin Pengfei(School of Plant Protection,Hainan University,Haikou,570100)

机构地区：[1]海南大学植物保护学院,海口570100

出　　处：《分子植物育种》2023年第8期2626-2634,共9页Molecular Plant Breeding

基　　金：海南省基础与应用基础研究计划(自然科学领域)高层次人才项目(2019RC163);海南省青年科技英才创新计划项目(QCXM201903);国家自然基金地区科学基金项目(31960552);海南大学校内科研启动经费项目(KYQD(ZR)1842);海南省研究生创新科研课题(Hys-2019-96)共同资助。

摘　　要：贝莱斯芽孢杆菌HN-1是一株对香蕉枯萎病4号生理小种尖孢镰刀菌(Fusarium oxysporum f.sp.cubense race 4)具有良好抑制活性的生防芽孢杆菌。本研究旨在构建生防菌HN-1的突变体库,在突变体库的基础上深入研究生防芽孢杆菌的抑菌机理,针对香蕉枯萎病的生物防治夯实理论基础。将携带转座子TnYLB-1的温敏型质粒载体pMarA质粒利用化学法转化进入贝莱斯芽孢杆菌HN-1中,通过50℃高温诱导后构建了由1 800个HN-1突变体组成的贝莱斯芽孢杆菌HN-1突变体文库,质粒消除率达99.7%。通过平板抑菌法筛选得到一株对尖孢镰刀菌Foc4的抑制活性降低的突变体,利用反向PCR技术确定插入破坏的基因与贝莱斯芽孢杆菌模式菌株FZB42 arsenic resistance protein (RBAM-550730)基因同源性为99%,在系统进化树中与贝莱斯芽孢杆菌模式菌株FZB42中的该基因属于同一分支。通过筛选鉴定HN-1突变体文库中抑菌活性丧失突变体发现HN-1中Arp基因与生防芽孢杆菌HN-1抑制尖孢镰刀菌的活性有密切的关系。Bacillus velezensis HN-1 had strong antifungal activity for Fusarium oxysporum.The purpose of this study is to explore the molecular mechanism of Bacillus velezensis antibacterial biocontrol bacteria.Using a tempera ture sensitive plasmid pMarA with a mariner-based transposon TnYLB-1 to transformation of Bacillus velezensis HN-1,TnYLB-1 had inserted into Bacillus velezensis HN-1 gene randomly under the condition of 50℃ high temperature.The insertion mutant library containing 1800 mutants were constructed and HN-1-682 defective lack of bacteriostasis in HN-1 mutant library,pMarA plasmid elimination rate is 99.7%.Using inverse PCR technology to identify the insertion position genes of the bacteriostatic defect mutants.The TnYLB insertion position mutant were identified by comparison with genome sequences of strain HN-1,sequence analysis showed that gene exhibited the highest nucleotide homology(99%)with that of FZB42(RBAM-550730)and they were classified into a same branch in phylogenetic tree.The insertion mutant library containing 1800 mutants were constructed of Bacillus velezensis HN-1,the Arp gene is associated with HN-1 antifungal activity for Fusarium oxysporum.

关 键 词：贝莱斯草芽孢杆菌HN-1 尖孢镰刀菌 pMarA质粒 突变体库 Arp基因 

分 类 号：S476.1[农业科学—农业昆虫与害虫防治]