核盘菌交配型基因mat 1-1敲除载体的构建及转化

J4 ›› 2010, Vol. 48 ›› Issue (1): 140-145.

核盘菌交配型基因mat 1-1敲除载体的构建及转化

安乐¹, 赵文生², 张世宏¹, 刘金亮¹, 彭友良², 潘洪玉¹

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

收稿日期:2009-03-25 出版日期:2010-01-26 发布日期:2010-01-27
通讯作者: 潘洪玉 E-mail:panhongyu@jlu.edu.cn

Construction and Transformation of Knockout Vector for theMating Type mat 1-1 Gene of Sclerotinia sclerotiorum

AN Le¹, ZHAO Wensheng², ZHANG Shihong¹, LIU Jinliang¹, PENG Youliang², PAN Hongyu¹

1. College of Plant Science, Jilin University, Changchun 130062, China|2. Lab of Molecular Plant Pathology of Agricultural Ministry, Chinese Agricultural University, Beijing 100193, China

Received:2009-03-25 Online:2010-01-26 Published:2010-01-27
Contact: PAN Hongyu E-mail:panhongyu@jlu.edu.cn

摘要/Abstract

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mat 1-1进行基因同源重组的敲除对比实验, 获得了缺失mat 1-1基因的转化
菌株. 先利用PCR方法获得mat 1-1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-mat 1-1, 再将ΔPBI-G3CN-mat 1-1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mat11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.

关键词: 核盘菌；mat 1-1基因；敲除载体；遗传转化

Abstract:

We established the gene knockout method using agrobacteriummediated transformation, and carried out contrast experiment of mat 1-1 of Sclerotinia sclerotiorum, and then obtained the transformed strain of mat 1-1 gene that was knocked out. First, the right arm and left arm were cloned by PCR respectively, which were selected from two sides of mat 1-1 gene that gave a great help in the construction of knockout vector, and then constructed the targeting vector ΔPBI-G3CN-mat 1-1, which comprised the two side arms and correctly sequences and resistance gene of fradiomycin.
ΔPBI-G3CN-mat 1-1 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 mat 1-1 gene deleted strain and wild strain in growth speed. But sclerotium and apothecium were not formed in the mat 1-1 gene deleted strain.

Key words: Sclerotinia sclerotiorum, mat 1-1 gene, knockout vector, agrobacteriummediated transformation

中图分类号:

Q939.95

安乐, 赵文生, 张世宏, 刘金亮, 彭友良, 潘洪玉. 核盘菌交配型基因mat 1-1敲除载体的构建及转化[J]. J4, 2010, 48(1): 140-145.

AN Le, DIAO Wen-Sheng, ZHANG Shi-Hong, LIU Jin-Liang, BANG You-Liang, BO Hong-Yu. Construction and Transformation of Knockout Vector for theMating Type mat 1-1 Gene of Sclerotinia sclerotiorum[J]. J4, 2010, 48(1): 140-145.