高效大丽轮枝菌(Verticillium dahliae) 基因敲除体系的构建

High efficient gene knockout in Verticillium dahliae

摘要:

摘要:【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

关键词:关键词:大丽轮枝菌，基因敲除，融合PCR，农杆菌介导转化，致死基因，5-氟脱氧尿苷

Abstract:

Abstract:［Objective］We developed an efficient method of gene knockout in Verticillium dahliae，an important soil-borne fungal pathogen that causes cotton vascular wilt diseases． ［Methods］By using fusion PCR，we constructed gene knockout vectors． By using Agrobacterium tumefaciens-mediated transformation and applying a herpes simplex virus thymidine kinase (HSVtk) gene in T-DNA as a conditional lethal gene to counter-select against ectopic transformants，we developed an efficient method to select gene knockout transformants． ［Results］Gene knockout frequency for ADE4 and ChsV was 87% and 44% ，respectively． ［Conclusion］We developed an efficient tool for gene knockout in Verticillium dahliae，which would help clarify the infection mechanism of this fungal pathogen．

Key words:Keywords: Verticillium dahliae，gene knockout，fusion PCR，Agrobacterium tumefaciens-mediated transformation，lethal gene，5-fluoro-2'-deoxyuridine (F2dU)