甘蔗镰孢菌插入突变体库的构建、致病变异突变体筛选及突变基因定位  

Construction of Fusarium sacchari Mutant Library,Screening of Pathogenic Mutants,and Identification of Mutated Genes

作　　者：蒙姣荣[1,2] 黄海娟 杨惠贞 曾泉 李珅雨[1] 陈保善 MENG Jiaorong;HUANG Haijuan;YANG Huizhen;ZENG Quan;LI Shenyu;CHEN Baoshan(College of Agriculture,Guangxi University/Guangxi Key Laboratory of Sugarcane Biology,Nanning,Guangxi 530004,China;State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,Nanning,Guangxi 530004,China;College of Life Science and Technology,Guangxi University,Nanning,Guangxi 530004,China)

机构地区：[1]广西大学农学院/广西甘蔗生物学重点实验室,广西南宁530004 [2]亚热带农业生物资源保护与利用国家重点实验室,广西南宁530004 [3]广西大学生命科学与技术学院,广西南宁530004

出　　处：《热带作物学报》2023年第2期233-245,共13页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金项目(No.31960031);广西科技基地和人才专项(桂科AD17129002);广西甘蔗重点实验室自主课题项目(No.2018-266-Z01)。

摘　　要：由镰孢菌复合种引起的甘蔗梢腐病(pokkah boeng disease,PBD)是甘蔗的主要病害之一。在广西,甘蔗镰孢菌(Fusarium sacchari)是PBD的优势病原菌。尽管PBD的研究已有较长的历史,但甘蔗镰孢菌的致病分子机理远未得到阐明。为研究甘蔗镰孢菌的致病分子机理,本研究采用农杆菌介导的遗传转化(Agrobacterium tumefaciens-mediated transformation,ATMT)技术构建甘蔗镰孢菌FF001菌株的T-DNA插入突变体库。采用携带潮霉素B磷酸转移酶基因pTHR1-AH双元载体的农杆菌菌株AGL-1介导转化F.sacchari分生孢子,获得3018个转化子;随机选取12个转化子进行PCR检测和Southern杂交分析,结果显示所有检测突变株均插入潮霉素B磷酸转移酶基因,多数为单拷贝插入。采用离体叶片接种评价转化子的致病力,获得致病力明显减弱21个和致病力增强9个,共30个突变体。通过TAIL-PCR扩增测序并与甘蔗镰孢菌基因组序列比对,确定了其中27个突变株的T-DNA插入位点。观察到多个插入位点伴随有基因组片段缺失,导致1个插入位点可以影响1个以上的功能基因。T-DNA插入位点多为编码区和启动子区,少数为终止子区和间隔区。受影响的基因分别编码α-甘露糖苷酶(alpha-mannosidase)、中性氨基酸渗透酶(neutral amino acid permease)、寡聚高尔基复合体成分4(oligomeric golgi complex component 4)、寡肽转运蛋白(oligopeptide transporter)、酰基辅酶A脱氢酶(acyl-CoA dehydrogenase)、乙酰乙酰-CoA合成酶(acetoacetyl-CoA synthetase)、碳酐酸酶(carbonic anhydrase)、Bud 10蛋白(Bud 10 protein)、类驱动蛋白bimC(kinesin-related protein bimC)、锌指蛋白ASD4(zinc finger protein ASD4)、转录起始因子TFIID(transcription initiation factor TFIID)、角质酶G-box结合蛋白(cutinase G-box binding protein)、吖啶黄素敏感性控制蛋白(acriflavine sensitivity control protein ACR-2)、核类VCP蛋白(nuclear VCP-like protein)、热激蛋白HSP30(heat shock prPokkah boeng disease(PBD)caused by Fusarium complex is one of the major diseases in sugarcane with great impact on the sugarcane industry worldwide.F.sacchari is the prevalent species responsible for PBD in Guangxi.Albeit of a long history of study on the disease,the mechanisms underlying the pathogenicity of the pathogen are still far from clear.To tackle this challenge,we generated an insertional mutant library by transformation of the conidial spores from F.sacchari strain FF001 via Agrobacterium tumefaciens-mediated transformation(ATMT).A total of 3018 hygromycin B-resistant transformants were obtained.Among 12 transformants randomly selected,nine out of 12 trans-formants carried a single copy of T-DNA,as verified by PCR and Southern blot analysis.The mutants were further screened for virulence variation on detached leaves and 30 mutants with obvious changes in virulence were obtained,including 21 mutants with significantly attenuated and 9 mutants with enhanced virulence.The sites of insertion were determined by thermal asymmetric interlaced PCR(TAIL-PCR)and sequence alignment to the genome sequence of F.sacchari.The majority of the insertions were found in coding regions or promotor regions,with a few in terminator or intergenic regions.It was observed that a few insertions were accompanied with genome fragment deletion,resulting in more than one genes being disrupted in a single insertion.Among the 22 mutated genes with annotated functions were those encoding alpha-mannosidase,neutral amino acid permease,oligomeric Golgi complex component 4,oligopeptide transporter 2,acyl-CoA dehydrogenase,acetoacetyl-CoA synthetase,carbonic anhydrase,Bud 10 protein,kine-sin-related protein bimC,zinc finger protein ASD 4,transcription initiation factor TFIID,cutinase G-box binding pro-tein,acriflavine sensitivity control protein ACR-2,nuclear VCP-like protein,heat shock protein 30,thioredoxin,type 2C protein phosphatase(PP2C),exoribonuclease,selenoprotein,DNA polymerase eta,survival factor 1,and aldehyde de-hydrogenase,sev

关 键 词：甘蔗镰孢菌 农杆菌介导 遗传转化 致病力相关基因 侧翼序列 甘蔗梢腐病 

分 类 号：S435.661[农业科学—农业昆虫与害虫防治]