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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。
Fusarium pseudograminearum can cause basal decay of wheat (Triticum aestivum), causing serious harm to the production. To study the mechanism of interaction between pathogen and wheat and to cultivate resistant varieties is an effective measure to prevent and control diseases. In this study, Agrobacterium tumefaciens was used to transform Fusarium graminearum to obtain a transformed strain of Fusarium graminearum, which can stably express green fluorescent protein (GFP). Fusarium graminearum The conidia and mycelium of WZ-8A were transformed into the recipient, and the transformation system was optimized by Agrobacterium tumefaciens-mediated transformation with p CAM-GFP-hyg plasmid. After transformation, the obtained transformants were continuously cultured for 5 generations on PDA medium without hygromycin B (hygromycin B), and then the transformed strains were randomly selected and subjected to hyg primer PCR and hyg probe Southern blot analysis , Fluorescence microscopy, pathogenicity and stability determination. The results showed that the optimum transformation system for Fusarium graminearum was as follows: the concentration of CaCl 2 in the culture medium was 2.6 × 10 -2 g / L , The transformation efficiency was higher at 28 ℃ for 48 h (average transformation rate was 42 transformed strains / 106 spores). Molecular biology, fluorescence microscopy and pathogenicity analysis of the randomly selected transformed strains confirmed that the T-DNA plasmid carrying the GFP gene was inserted into the genome of Fusarium graminearum in a single copy to transform the virulence of the strain Not weakened. The optimized Agrobacterium tumefaciens-mediated Fusarium graminearum genetic transformation system can be used for pathogenic mechanism of pathogenic bacteria and wheat disease resistance mechanisms.