论文部分内容阅读
【目的】研究灰葡萄孢菌(Botrytis cinerea)基因组中T-DNA插入位点的整合模式特征。【方法】利用农杆菌(Agrobactirium tumfacience)介导法构建灰葡萄孢菌T-DNA插入突变体库。利用热不对称交错PCR(TAIL-PCR)技术对转化子中T-DNA的旁侧序列进行扩增和克隆,对获得的旁侧序列进行比对分析。【结果】T-DNA插入在灰葡萄孢菌基因组非编码区的占69%,插入在外显子的占30%。T-DNA在插入的过程中发生了碱基缺失、增加等重组现象,其中左边界(left border,LB)整合到基因组碱基缺失较少,有的保持完整,而右边界(right border,RB)及其近邻的T-DNA区域缺失碱基较多。T-DNA的插入位点还发现有额外的序列插入。【结论】对灰葡萄孢菌中插入T-DNA的整合模式的分析为开展该菌的功能基因组学奠定了基础。
【Objective】 The objective of this study was to investigate the integration pattern of T-DNA insertion sites in the genome of Botrytis cinerea. 【Method】 T-DNA insertion mutant library of Botrytis cinerea was constructed by Agrobacterium tumfacience-mediated method. The flanking sequences of T-DNA in the transformants were amplified and cloned by thermal asymmetric interlaced PCR (TAIL-PCR), and the obtained flanking sequences were compared and analyzed. 【Result】 T-DNA insertion accounted for 69% of the non-coding region in the genome of Botrytis cinerea and 30% of the inserted exons. In the process of insertion, T-DNA deletions and increases the recombination phenomenon. The left border (LB) is less integrated into the genome and some remains intact while the right border (RB ) And its neighbor T-DNA region deletion base more. T-DNA insertion sites were also found to have additional sequence insertions. 【Conclusion】 The analysis of integration pattern of T-DNA insertion into Botrytis cinerea lay the foundation for the functional genomics of this strain.