根癌土壤杆菌介导的假禾谷镰刀菌遗传转化体系的优化  

Optimizing of Agrobacterium tumefaciens-Mediated Genetic Transformation Sytem in Fusarium pseudograminearum

作　　者：邢小萍[1] 张盼盼 丁胜利[1] 袁虹霞[1] 陈琳琳[1] 李洪连[1] 

机构地区：[1]河南农业大学植物保护学院,郑州450002

出　　处：《农业生物技术学报》2017年第11期1887-1894,共8页Journal of Agricultural Biotechnology

基　　金：国家公益性行业(农业)科技专项(No.201503112);河南省基础与前沿研究计划项目(No.152300410073)

摘　　要：假禾谷镰刀菌(Fusarium pseudograminearum)可引起小麦(Triticum aestivum)茎基部腐烂,对生产造成严重危害。研究病原菌与小麦的互作机制,培育抗病品种是防治病害有效措施。本研究以根癌土壤杆菌(Agrobacterium tumefaciens)介导转化假禾谷镰刀菌,获得能够稳定表达绿色荧光蛋白(green fluorescent protein,GFP)的假禾谷镰刀菌的转化菌株,以假禾谷镰刀菌WZ-8A的分生孢子和菌丝体为转化受体,利用携带p CAM-GFP-hyg质粒的根癌土壤杆菌介导转化,对转化体系进行了优化。在转化后,将获得的转化子在不含潮霉素B(hygromycin B,hyg)的PDA培养基上连续培养5代,然后随机选取转化菌株,分别进行hyg引物PCR、hyg探针Southern blot分析、荧光显微镜观察、致病力及稳定性测定。结果表明,优化的假禾谷镰刀菌转化体系为:以10~6个/mL分生孢子悬浮液为转化受体,共培养培养基中Ca Cl2浓度2.6×10^(-2)g/L,在28℃,共培养48 h时转化效率较高(平均转化率为42个转化菌株/106个孢子)。对随机选取的转化菌株进行分子生物学和荧光显微验证及致病性测定,证实携带GFP基因的T-DNA质粒以单拷贝的形式插入假禾谷镰刀菌的基因组,转化菌株的致病力未减弱。优化的根癌土壤杆菌介导的假禾谷镰刀菌遗传转化体系,可用于病原菌致病机制和小麦品种抗病机制的研究。Crown rot caused by Fusarium pseudograminearum is one of the main devastating diseases in wheat（Triticum aestivum） production.Studying the interaction mechanism between F.pseudograminearum and wheat varieties is the most economical and effective measures to control of wheat crown rot.The objectives of this study are to optimize the Agrobacterium tumefaciens-mediated transformation（ATMT） technology system of F.pseudograminearum,and obtain the transformant strains of F.pseudograminearum（WZ-8A） successfully transformed with with the green fluorescent protein（GFP）.Firstly,the transformation conditions were optimized,and then the GFP gene was transformed into the conidia of F.pseudograminearum strain WZ-8A using the A.tumefaciens strain carrying plasmid p CAM-GFP-Hyg.After transformation,transformants randomly collected were screened and identified through the analysis of the hygromycin B（hyg） resistance,as well as using the fluorescence microscopy techniques.A pathogenicity detection was subsequently conducted.The results showed that a satisfactory transformation with a efficiency of 42 transformants per 1×106 spores could be achieved in a sytem of 1×106 spores per milliliter of F.pseudograminearum spore suspension which were co-cultured with Agrobacterium cells under the culture in the presence of co-culture medium containing CaCl2 at 2.6×10-2 g/L at 28 ℃ for 2 d.The transformants was stable when grown on hygromycin B-free PDA medium for 5 generations.The transformants were found to be hyg-positive by PCR amplification and Southern blot analysis,and to be GFP-positive through the detection using fluorescence microscopy,compared with the wild-type strain of WZ-8A.The subsequent test showed that the transformants really did not lose the pathogenicity.It thus be concluded that the T-DNA bearing GFP gene is successfully inserted into the genome of F.pseudograminearum with the optimized system mediated by A.tumefaciens.The optimized genetic transformation sysytem mediated by A.tumefaciens in F.ps

关 键 词：假禾谷镰刀菌 绿色荧光蛋白 遗传转化 

分 类 号：S432.23[农业科学—植物病理学]