论文部分内容阅读
【目的】为了深入研究大丽轮枝菌(Verticillium dahliae)致病基因的功能,构建高效大丽轮枝菌基因敲除体系。【方法】融合PCR构建基因敲除载体;利用农杆菌介导法转化大丽轮枝菌;使用在T-DNA之间加入致死基因的双元载体,使T-DNA随机插入转化子在添加5-氟脱氧尿苷的培养基上不能存活,实现对随机插入转化子的“反向筛选”。【结果】对大丽轮枝菌腺嘌呤合成酶基因和几丁质合成酶基因进行基因敲除验证,基因敲除转化子在总转化子中的比例分别达到87%和44%。【结论】成功构建大丽轮枝菌高效基因敲除体系,为大丽轮枝菌致病基因的功能验证提供了技术平台。
【Objective】 In order to further study the function of virulence genes of Verticillium dahliae and construct a gene knockdown system of Verticillium dahliae, 【Method】 The gene knockout vector was constructed by fusion PCR. Agrobacterium tumefaciens was used to transform Verticillium dahliae. The binary vector with lethal gene was inserted between T-DNA, - fluorodeoxyuridine medium can not survive, to achieve “reverse screening ” for random insertion of transformants. 【Result】 Gene knockdown of adenine synthase gene and chitin synthase gene of Verticillium dahliae was found. The percentage of gene knockout transformants in total transformants reached 87% and 44%, respectively. 【Conclusion】 The successful construction of efficient knock-out system of Verticillium dahliae provides a technological platform for the functional verification of virulence genes of Verticillium dahliae.