荧光标记基因转化棉花黄萎病菌及标记菌系选育  

Transformation and Selection of Fluorescent Protein Gene Labelled Verticillium dahliae

作　　者：王晓楠[1] 齐宏[1] 张金凤[1] 光杨其 唐灿明[1] 

机构地区：[1]南京农业大学农学院/作物遗传与种质创新国家重点实验室,江苏南京210095

出　　处：《棉花学报》2014年第3期221-227,共7页Cotton Science

基　　金：高等学校博士学科点专项科研基金(20120097110024);国家自然科学基金(31071459)

摘　　要：为了研究黄萎病菌的侵染机制,通过农杆菌介导的转化方法,将绿色荧光蛋白基因sGFP导入落叶型黄萎病菌VD07038,将红色荧光蛋白基因mCherryRFP导入非落叶型黄萎病菌Bp2中,分别获得了具有绿色、红色荧光信号的阳性转化子.经过分子验证和连续继代培养,证明了这些转化子具有遗传稳定的对潮霉素的抗性.通过对转化子的菌落形态、生长速度和致病力进行检测,发现大部分转化子与野生型基本一致,少量转化子发生变异,其中转化子Bp2R-30不能产生微菌核,致病力显著下降.利用荧光显微镜观察了转化子VD07038G-10在感病棉花品种苏棉22幼苗根部的侵染情况.结果表明,在接菌12h后VD07038G-10的孢子可吸附在根表面;接种7～9 d后,菌丝入侵到棉花根部的维管组织.本研究获得的荧光蛋白标记的棉花黄萎病菌VD07038G-10可用于实时观测黄萎病菌侵染棉花根系的过程,并且可以定量鉴定不同棉花品种对该菌系的抗性,为棉花黄萎病菌抗性鉴定提供一种新方法.In order to analyse the infection mechanism of V.dahliae,sGFP encoding green fluorescent protein was transferred into defoliating V.dahliae isolate VD07038 and mCherryRFP encoding red fluorescent protein was transferred into non-defoliating V.dahliae Bp2 via Agrobacterium tumefaciens-mediated transformation.Finally,positive transformants tagged by green fluorescent protein or red fluorescent protein were obtained.The hygromycin B genetic stability of transgenic progenies was tested by series culture and molecular verification.According to colony morphology,growth rate and pathogenicity,we found that traits of most oftransformants were similar to those of wild-type,few transformants tended to form mutants.Tansformants Bp2R-30 did not produce microsclerotia and its pathogenicity decreased significantly.Using the GFP-tagged V.dahliae VD07038G-10 isolate,colonization of the fungus in roots of the susceptible cotton Sumian 22 was investigated.As the results showed that,12hours after inoculation via the root dipping methods,roots surface of Sumian 22 were covered with conidia,7-9 days after inoculation,hyphae from the leading edge of the colony progressed acropetally up the xylem vessels of infected roots.V.dahliae VD07038G-10 tagged by fluorescent protein can be used for observation its infection process in real time on cotton roots and identification of resistance of different cotton varieties to this strain.This study provide a new method for resistance evaluation of V.dahliae.

关 键 词：棉花 大丽轮枝菌 绿色荧光蛋白 红色荧光蛋白 转化 

分 类 号：S435.621[农业科学—农业昆虫与害虫防治]