香蕉枯萎病菌TR4原生质体转化和基因敲除体系构建

正香蕉枯萎病是一种典型的维管束和土传真菌病害,也是最具毁灭性的植物病害之一~(1])。香蕉一旦感病,很难结果,而且目前尚无有效的防治措施~(1])。因此,研究植物病原菌侵染进程和致病机制,可以拓宽思路以寻求植物病害防治新途径。香蕉枯萎病是由尖孢镰刀菌古巴专化型(Fusarium oxysporum f.sp.cubense,Foc)侵染所致~(1])。对生产影响最大的Foc是侵染包括大蜜哈、

Construction of PEG-mediated genetic transformation and gene knockout system in Fusarium oxysporum f.sp. cubense TR4

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense tropic race 4 (Foc TR4), is a typical vascular and soil-borne disease which has significantly threatened the sustainable development of banana industry. In order to reveal the infection process and pathogenesis of Foc TR4, the young mycelia (66.7 mg·mL^-1) of wild-type strain of Foc TR4 (WT-Foc TR4) cultured for 18-20 h were lysed with enzyme mixture for protoplasting, which consisted of 25 mg·mL^-1 driselase, 0.4 mg·mL^-1 chitinase, 15 mg·mL^-1 lysing enzyme and 1.2 mol·L^-1 potassium chloride. The resulted protoplasts of 2×10⁷cells·mL^-1 were used to test the efficiency of transformation mediated by polyethylene glycol, and up to 9 transformants per μg DNA were obtained. AmCyan, RFP and YFP gene were stably transferred into WT-Foc TR4, separately, using the protoplasts transformation system. The gene FoOCH1 encoding α-1,6-mannosyltransferase in WT-Foc TR4 was knocked out using the split-marker recombination technology. The genetic transformation and gene knockout system in this pathogen laid the foundation for the study of functional genomics and plant-pathogen interactions.