灰葡萄孢菌（Botrytis cinerea）基因组中T-DNA整合模式分析

摘要：【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。

Analysis of T-DNA integration pattern in Botrytis cinerea genome

Abstract: Objective] To analyze the T-DNA integration pattern in the genome of grey mold Botrytis cinerea. Methods] T-DNA (Transfer DNA) inserted mutant library of Botrytis cinerea was created by Agrobactirium tumfacience mediated transformation. By using TAIL-PCR (Thermal asymmetric interlaced polymerase chain reaction), we amplified and cloned the chromosomal regions flanking T-DNA insertions. The obtained T-DNA flanking sequences were subjected to alignment with standard T-DNA border sequence for identification and analysis of integration. Results] Up to 69% T-DNA inserted at noncoding regions and 30% inserted at exons. Recombination including deletion or addition of bases in T-DNA region was observed. The right borders of the T-DNA were frequently truncated, and by contrast the left borders were less prone to degradation and appeared to have been inserted in a relatively integrated manner. Extra sequence additions also occurred in T-DNA integration sites. Conclusion] Analysis of T-DNA integration pattern in B. cinerea genome will stimulate the functional genomics study of this fungus.