核盘菌交配型基因mat1-1敲除载体的构建及转化

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.

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

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.