农杆菌介导的灰葡萄孢T-DNA插入突变体库构建及插入位点分析

Construction of T-DNA insertion mutants of Botrytis cinerea via Agrobacterium tumefaciens mediated transformation and sequence analysis of insertion site

摘要:

摘要：【目的】利用农杆菌(Agrobacterium tumefaciens)介导法对灰葡萄孢（Botrytis cinerea）进行转化，构建T-DNA插入突变体库，为从分子水平上认识灰葡萄孢的致病机制打下基础。【方法】以含有pCAMBIA1390双元载体的农杆菌对灰葡萄孢进行转化，利用潮霉素进行筛选。对抗性稳定的转化子进行生物学和形态学观察，采用离体番茄叶片进行致病性测定。利用TAIL-PCR技术对突变体中T-DNA的旁侧序列进行克隆。【结果】得到了一些突变体，表现为生长速率减缓、产孢能力下降、致病力减弱等。克隆并分析了其中一个突变体中T-DNA插入的位置和旁侧序列。【结论】本实验建立了农杆菌介导的灰葡萄孢转化体系，构建了T-DNA插入的灰葡萄孢突变体库。用TAIL-PCR进行突变体中T-DNA旁侧序列的分析是可行的。

Abstract:

Abstract：【Objective】Construction of Botrytis cinerea T-DNA insertion mutant library through Agrobacterium-mediated transformation and for further understanding of the molecular mechanisms underlying the pathogenicity of Botrytis cinerea.【Methods】Agrobacterium containing the binary vector pCMBIA1390 was used for transformation of Botrytis cinerea. Hygromycin resistant transformants were screened out and subjected to biological and morphological observation. Detached tomato leaves were used for pathogenicity assay. The flanking sequence of T-DNA inserted in mutant genome was cloned and analyzed by using TAIL-PCR.【Results】A variety of mutants were obtained and important phenotypes including reduction in growth rate, loss or reduction in conidiation and loss of pathogenicity were screened out. The T-DNA flanking sequence of one of the mutants was successfully amplified with TAIL-PCR.【Conclusion】Agrobacterium-mediated transformation system of Botrytis cinerea was established and a T-DNA insertion mutants library was constructed. TAIL-PCR was an effective method for isolating the flanking sequence of T-DNA inserted in genome.