Construction of PEG-mediated Genetic Transformation and Gene Knockout System in Fusarium oxysporum f. sp.cubense Tropic Race 4  

作　　者：Lei ZHANG Yan GUO Yunyue WANG Weihua TANG Sijun ZHENG 

机构地区：[1]State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan,Ministry of Education Key Laboratory of Agriculture Biodiversity for Plant Disease Management,Yunnan Agricultural University,Kunming 650201,China [2]Agricultural Environment and Resources Institute,Yunnan Academy of Agricultural Sciences,Kunming 650205,China [3]Institute of Plant Physiology and Ecology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai 200032,China [4]Bioversity International,Kunming 650205,China

出　　处：《Agricultural Biotechnology》2020年第1期15-17,21,共4页农业生物技术（英文版）

基　　金：Supported by Yunnan Science and Technology Innovation Talent Program(2015HA034);National Natural Science Foundation of China(NSFC31560505)

摘　　要：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) of wild-type strain of Foc TR4(WT-Foc TR4) cultured for 18-20 h were lysed with enzyme mixture for protoplast formation, which consisted of 25 mg/ml driselase, 0.4 mg/ml chitinase, 15 mg/ml lysing enzyme and 1.2 mol/L potassium chloride. The resulted protoplasts of 2×10~7 cells/ml were used to test the efficiency of transformation mediated by polyethylene glycol, and up to 9 transformants per microgram of DNA were obtained. AmCyan, RFP and YFP genes were stably transferred into the WT-Foc TR4, separately, using the protoplast transformation system. The gene FoOCH1 encoding α-1, 6-mannosyltransferase in the WT-Foc TR4 was knocked out using the split-marker recombination technology. The genetic transformation and gene knockout system in this pathogen lays a foundation for the study of functional genomics and plant-pathogen interactions.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) of wild-type strain of Foc TR4(WT-Foc TR4) cultured for 18-20 h were lysed with enzyme mixture for protoplast formation, which consisted of 25 mg/ml driselase, 0.4 mg/ml chitinase, 15 mg/ml lysing enzyme and 1.2 mol/L potassium chloride. The resulted protoplasts of 2×10~7 cells/ml were used to test the efficiency of transformation mediated by polyethylene glycol, and up to 9 transformants per microgram of DNA were obtained. AmCyan, RFP and YFP genes were stably transferred into the WT-Foc TR4, separately, using the protoplast transformation system. The gene FoOCH1 encoding α-1, 6-mannosyltransferase in the WT-Foc TR4 was knocked out using the split-marker recombination technology. The genetic transformation and gene knockout system in this pathogen lays a foundation for the study of functional genomics and plant-pathogen interactions.

关 键 词：Fusarium wilt of banana Fusarium oxysporum f.sp.cubense PROTOPLASTS Transformation Split-marker recombination 

分 类 号：S436.68[农业科学—农业昆虫与害虫防治]