菰黑粉菌T-DNA突变体库的构建及分析  

Construction and analysis of the T-DNA insertion mutant library of Ustilago esculenta

作　　者：汤近天 杨芙容 张雅芬 夏文强 崔海峰 叶子弘 TANG Jintian;YANG Furong;ZHANG Yafen;XIA Wenqiang;CUI Haifeng;YE Zihong(Zhejiang Provincial Key Laboratory of Biometrology and Inspection&Quarantine,College of Life Sciences,China Jiliang University,Hangzhou 310018,Zhejiang,China)

机构地区：[1]中国计量大学生命科学学院、浙江省生物计量与检验检疫技术重点实验室,浙江杭州310018

出　　处：《菌物学报》2023年第2期495-506,共12页Mycosystema

基　　金：国家自然科学基金(32100154);浙江省“万人计划”科技创新领军人才(2019R52018);中国计量大学基本科研业务费项目(2021YW64)。

摘　　要：通过建立适用于菰黑粉菌Ustilago esculenta的农杆菌介导遗传转化(Agrobacterium tumefaciens-mediated transformation,ATMT)体系,构建菰黑粉菌T-DNA插入突变体库。针对性地筛选双核菌丝形成缺陷型转化子,并对T-DNA插入位点进行分析,为研究菰黑粉菌二态型转换的分子调控机理打下基础。以构建的菰黑粉菌自融合菌株TSP为出发菌株,以含有遗传霉素(G418)抗性基因(neo)的质粒为载体,通过ATMT构建菰黑粉菌T-DNA突变体库,并对诱导剂乙酰丁香酮(AS)浓度、转化的共培养时间、农杆菌浓度和菰黑粉菌芽孢子浓度等建库影响因素进行单因素条件试验,筛选最优条件;对继代培养的转化子基因组中的遗传霉素抗性基因进行PCR检测,验证转化子遗传稳定性;对突变体库中的转化子双核菌丝生长情况进行观察,测定其双核菌丝形成能力;对上述双核菌丝形成缺陷型转化子进行基因组重测序,分析其T-DNA插入位点。当遗传霉素浓度为75μg/mL时,菰黑粉菌的生长被完全抑制。当AS浓度为100μg/mL、共培养时间为24 h、孢子浓度为1×10^(5)个/mL、农杆菌浓度为OD600=0.3时,转化获得转化子的效率最高,为菰黑粉菌ATMT最优转化体系。在突变体库中随机选取7株转化子在YEPS固体平板上继代培养10代,仍然能够通过PCR的方法在基因组中检测到neo基因片段,说明T-DNA成功插入TSP菌株基因组且稳定遗传。针对部分转化子进行双核菌丝生长能力测定,有5株转化子的菌落边缘没有形成菌丝,而TSP菌株的边缘长出了明显的菌丝,说明这5株转化子双核菌丝形成的能力丧失。对上述双核菌丝形成缺陷型转化子中的其中2个(TSP-1、TSP-23)进行基因组重测序,比对结果显示,TSP-1插入位点位于其交配型基因a位点的(GenBank:MK097140.1)mfa2.1基因的外显子区域,TSP-23插入位点位于两个假定蛋白之间。本研究优化了菰黑粉菌ATMT遗传转化体系,构建了菰黑�Agrobacterium tumefaciens-mediated transformation(ATMT)system was optimized and T-DNA insertion mutant library of Ustilago esculenta was constructed.Mutants defective in fusion and dikaryotic hyphal formation were screened and the T-DNA insertion sites were analyzed for further research on molecular mechanism of dikaryotic hyphal formation of U.esculenta.An artificial modified strain(TSP)with the ability of self-mating and a vector containing resistance gene(neo)of G418 were used for library construction.Optimal conditions of ATMT,including concentration of acetosyringone(AS),co-cultivation time,concentration of A.tumefaciens,and concentration of U.esculenta spores,were screened based on single factor conditional test.Genetic stability of T-DNA in mutant was tested by PCR with the detection of neo target.Mating assay was performed on mutants to test the ability of fusion and dikaryotic hyphal formation.Genome of defective mutants formed in fusion and dikaryotic hyphal formation was sequenced for T-DNA insertion site analysis.When the concentration of G418 was 75μg/mL,the growth of TSP was completely repressed.AS concentration of 100μg/mL,co-cultivation time of 24 h,spore concentration of 1×10^(5)spores/mL,and A.tumefaciens concentration of OD600=0.3,were the optimal ATMT conditions for U.esculenta.Seven mutants were randomly selected and subcultured for ten times on YEPS solid medium.Result of neo target detection of the seven mutants showed T-DNA insertion were stably inherited.Observations showed five mutants had defects in dikaryotic hyphal formation.Whole genome resequencing of two of them(TSP-1 and TSP-23)showed T-DNA inserted in the exon of mfa2.1 a loci(GenBank:MK097140.1)(TSP-1)and intergenic regions of two hypothetical proteins(TSP-23)respectively.In summary,this study optimized the ATMT system and constructed ATMT mutant library of U.esculenta,and dikaryotic hyphal formation defective mutants were screened and the T-DNA insertion sites were analyzed by genome resequencing.Further study on the regulat

关 键 词：菰黑粉菌 T-DNA插入突变体 双核菌丝 二态型转换 

分 类 号：S43[农业科学—农业昆虫与害虫防治]