核盘菌交配型基因mat1-1敲除载体的构建及转化  

Construction and Transformation of Knockout Vector for the Mating Type mat1-1 Gene of Sclerotinia sclerotiorum

作　　者：安乐[1] 赵文生[2] 张世宏[1] 刘金亮[1] 彭友良[2] 潘洪玉[1] 

机构地区：[1]吉林大学植物科学学院,长春130062 [2]中国农业大学农业部分子植物病理实验室,北京100193

出　　处：《吉林大学学报（理学版）》2010年第1期140-145,共6页Journal of Jilin University:Science Edition

基　　金：国家"十一五"科技支撑计划项目基金(批准号:2006BAD08A08);吉林省科技发展计划项目基金(批准号:20060544);吉林大学农学部重大项目启动基金

摘　　要：利用根癌农杆菌介导真菌遗传转化方法对核盘菌的交配型基因mat1-1进行基因同源重组的敲除对比实验,获得了缺失mat1-1基因的转化菌株.先利用PCR方法获得mat1-1基因的左右两侧片段,将测序正确的两个侧翼片段分别重组到农杆菌转化载体PBI-G3C中,并将新霉素抗性标记引入农杆菌转化载体PBI-G3C中,构建成农杆菌介导转化核盘菌的打靶载体ΔPBI-G3CN-mat1-1,再将ΔPBI-G3CN-mat1-1质粒转化至根癌农杆菌EHA105中.利用核盘菌菌丝进行转化,将得到的转化菌株,利用PCR方法进行验证,证实有敲除菌株存在.通过对敲除菌株进行生理表型的测定发现,缺失mat1-1基因的敲除菌株其生长速度与野生核盘菌菌株相比无明显差异,但敲除菌株不能产生菌核与子囊盘.We established the gene knockout method using agrobacterium-mediated transformation,and carried out contrast experiment of mat1-1 of Sclerotinia sclerotiorum,and then obtained the transformed strain of mat1-1 gene that was knocked out.First,the right arm and left arm were cloned by PCR respectively,which were selected from two sides of mat1-1 gene that gave a great help in the construction of knockout vector,and then constructed the targeting vector ΔPBI-G3CN-mat1-1,which comprised the two side arms and correctly sequences and resistance gene of fradiomycin.ΔPBI-G3CN-mat1-1 was transformed into EHA105.Using hypha of Sclerotinia sclerotiorum,we confirmed the positive knockout strain via PCR.By means of detecting the physiological phenotype,no obvious difference was shown between mat1-1 gene deleted strain and wild strain in growth speed.But sclerotium and apothecium were not formed in the mat1-1 gene deleted strain.

关 键 词：核盘菌 mat1-1基因 敲除载体 遗传转化 

分 类 号：Q939.95[生物学—微生物学] S432.4[农业科学—植物病理学]