香蕉枯萎病菌TR4原生质体转化和基因敲除体系构建

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-1driselase,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.