以beta-tubulin基因为选择标记的淡紫紫孢菌遗传转化  

Efficient Transformation System of Agrobacterium tumefaciens Mediated Transformation of Purpureocillium lilacinum by Using beta-tubulin as Selectable Marker

作　　者：王曦茁[1] 汪来发[1] 曹业凡 胡坚 汪祥 覃艳 王永春 WANG Xi-zhuo;WANG Lai-fa;CAO Ye-fan;HU Jian;WANG Xiang;QIN Yan;WANG Yong-chun(Key Laboratory of Forest Protection of National Forestry and Grassland Administration,Ecology and Nature Conservation Institute,Chinese Academy of Forestry,Beijing 100091,China;Qipo Forest Farm,Nanning 530000,Guangxi,China;Hangzhou Yisengjian Biotechnology Co.,LTD.,Hangzhou 310000,China)

机构地区：[1]中国林业科学研究院森林生态环境与自然保护研究所,国家林业和草原局森林保护学重点实验室,北京100091 [2]广西壮族自治区国有七坡林场,广西南宁530000 [3]杭州益森键生物科技有限公司,浙江杭州310000

出　　处：《林业科学研究》2023年第4期12-19,共8页Forest Research

基　　金：国家林业局科技成果推广计划项目(201609)。

摘　　要：[目的]建立稳定的食线虫真菌淡紫紫孢菌遗传转化体系,并获得插入突变体。[方法]介导的方法,以淡紫紫孢菌20-7的分生孢子为受体,将新构建的携带beta tubulin基因的质粒转化进入淡紫紫孢菌的细胞中,通过优化诱导乙酰丁香酮(AS)的浓度、诱导培养时间、农杆菌终浓度OD660值、共培养AS的浓度、共培养时间和共培养温度等因子,建立高效遗传转化体系,获得致病力不同的突变体。[结果]共培养过程中使用萌发孢子是成功建立淡紫紫孢菌遗传转化体系的必要条件;淡紫紫孢菌萌发的孢子与农杆菌EHA105在25℃共振荡培养48 h时,且在共培养阶段当乙酰丁香酮浓度为200μg·mL^(−1)(pH5.5)时转化效率最高,转化效率为1200~3200个转化子/106分生孢子,阳性抗性转化子比率为96%;转化子PCR表明,T-DNA已整合到淡紫紫孢菌的基因组中;Southern杂交验证表明,83.3%的转化子为T-DNA单拷贝插入;成功建立了可靠的淡紫紫孢菌的遗传转化体系,并从20个转化子中筛选到16个致病力变异的突变体。[结论]本研究成功构建了农杆菌介导的、以beta-tubulin基因为选择标记的淡紫紫孢菌高效遗传转化体系,并获得致病力变异的插入突变体,为淡紫紫孢菌的基因功能、致病机制研究及优良菌株选育奠定了基础。[Purpose]To establish an efficient transformation system of the nematopathogenic fungus Purpureocillium lilacinum and obtain its insertional mutagenesis.[Methods]The benomyl resistance gene beta-tubulin being as the selective marker,Agrobacterium tumefaciens-mediated transformation technique was developed to screen different pathogenicity mutants in P.lilacinum.PCR amplification and Southern hybridization were used to verify the transformation events,and Southern blotting of beta-tubulin gene and cloning of transforming DNA(T-DNA)flanking sequences were used to determine insert number and site of T-DNA in the fungal genome,respectively.[Results]A reliable transformation method was established for P.lilacinum.Specifically,pre-germinating spores of P.lilacinum used at co-cultivated period was a prerequisite.P.lilacinum germinating spores co-cultivated with A.tumefaciens EHA105 at 25℃for 48 h achieved the highest transformation efficiency,which was 1200-3200 transformants per 106 spores,and the ratio of positive resistant transformants was 96%.The transformants were cultivated up to 5 generations on beta-tubulin-containing medium and confirmed by PCR and those genetic traits remained stable.Southern hybridization showed that 83.3%of the transformants were single copy insertions of T-DNA,and 16 mutants with virulence variants were screened from 20 transformants.[Conclusion]This study successfully constructed an efficient genetic transformation system mediated by A.tumefaciens with beta-tubulin gene as a selective marker,and obtained an insertion mutant with pathogenicity variation,which was P.lilacinum.It provides insights into studying gene function,pathogenic mechanism and breeding excellent strains.

关 键 词：淡紫紫孢菌 根癌农杆菌 BETA-TUBULIN 插入突变 致病力 

分 类 号：S718.81[农业科学—林学]