农杆菌介导的香蕉枯萎病菌4号生理小种转化体系的优化  

Optimization of Agrobacterium-mediated Transformation System of Fusarium oxysporum f.sp.cubense race 4

作　　者：高剑[1] 肖苏生[1] 王文华[1] 曾涛[1] 曾会才[1] 

机构地区：[1]中国热带农业科学院热带生物技术研究所,农业部热带作物生物技术重点开放实验室,海口571101

出　　处：《基因组学与应用生物学》2009年第6期1197-1203,共7页Genomics and Applied Biology

基　　金：科技部国际科技合作项目(2007DFA31460);国家自然科学基金(30860160);中央级公益性科研院所中国热带农业科学院基本科研业务费专项资金资助项目(ITBBZX0921);公益性行业(农业)科研专项经费项目任务书(200903049-2)共同资助

摘　　要：香蕉枯萎病是世界范围内香蕉种植区最为严重的病害之一,严重威胁和影响着香蕉产业的发展。本文针对香蕉枯萎病病原菌的4号生理小种,建立了农杆菌介导的转化体系,确定了影响转化效率主要因子的优化体系是:农杆菌在IM培养基诱导前农杆菌OD600为0.15、农杆菌经IM液体培养基诱导的时间为7h、乙酰丁香酮(AS)浓度为150μmol/L、Focr4孢子浓度为1×106个/mL、共培养时间为48h、培养温度为25℃、诱导培养基pH值为5.5。在此条件下,转化效率能达到700～800个转化子/106个香蕉枯萎病菌孢子。PCR验证表明外源的T-DNA已经成功随机地整合到该病原菌基因组中。目前,应用该转化体系已获得2300多个转化子,为后续克隆相关致病基因打下了良好基础。Wilt disease of banana is one of the most devastating diseases in the banana-growing regions around the world,it would seriously threaten and influence on the development of banana industry.In this paper,we successfully established the Agrobacterium-mediated transformation system of Fusarium oxysporum f.sp.cubense(FOC) race4,which was the pathogen of the banana wilt disease,and determined the optimized system of major factors affecting transformation ratio:the Agrobacterium OD600 would be 0.15 before the IM culture,the induction time of Agrobacterium in IM liquid medium would be 7 h,and the concentration of acetosyringone(AS) would be 200 μmol/L,co-cultivation with 1×106 spores per milliliter spore suspension of the Focr4 for 48 h,induction culti vation at pH 5.5 and 25℃,and under this conditions,transformation efficiency might achieve in the range of 700～800 transformants per 106 conidia of FOC race 4 PCR analysis showed that the ectogenous T-DNA had been successfully integrated into the genome of the fungus.A total of 2 300 mutants had been obtained using this transformation system,and may lay a good foundation for subsequent cloning of pathogenicity gene related to banana wilt disease.

关 键 词：香蕉枯萎病 4号生理小种 T-DNA插入 优化体系 

分 类 号：S436.68[农业科学—农业昆虫与害虫防治] Q93[农业科学—植物保护]