共转化水稻植株T-DNA共整合结构的分子分析

Molecular Characterization of Co-integrated T-DNA Structures in Cotransformed Rice Plants

为研究共转化水稻的T-DNA共整合结构，本研究利用分别含有HPT、GFP和mCherry基因的T—DNA载体共转化水稻，从共转化植株获得了22个由两种T—DNA共整合形成的串联结构和25个关于相同T—DNA的多拷贝结构。序列分析结果表明，相邻T-DNA通过LRRL、RLLR和LRLR模式相互连接，由RB参与形成的T-DNA连接区均发生RB序列的完全缺失，在LB参与的T-DNA连接区，LB发生不同数目的碱基缺失或其3＇末端的1个C碱基发生C→T颠换，因而使T-DNA连接区表现多种变异类型。此外，伴随着RB或LB边界的完全缺失，其边界内侧序列也缺失1-643bp。在共转化水稻植株的T-DNA连接区不存在拟南芥相关报道所述的填充DNA序列。研究结果为深入解析禾谷类植物T—DNA共整合的分子机理奠定了重要基础。

To investigate the co-integrated T-DNA structures in co-transformed rice plants, we co-transformed T- DNA vectors containing each of the HPT, GFP and mCherry genes into rice. Twenty-two tandem T-DNA struc- tures containing two T-DNA types and twenty-five multi-copy structures with one T-DNA type were identified in co-transformed rice plants. Based on the sequencing results, the co-integrated T-DNAs were linked into LRRL, RLLR and LRLR patterns. Deletion of complete RB sequence was observed in all the T-DNA junctions where RB was involved. In T-DNA junctions where LB was involved, the LB sequence lost various numbers of nucleotides or underwent a C---~T transversion at the 3＇ end. For this reason, the LB-involved T-DNA junctions showed different types of sequence variation. Also, the deletion of whole LB or RB sequence was coincident with the loss of 1-643 nucleotides in LB or RB inward sequence. The T-DNA junctions in co-transformed rice plants did not contain any filler DNA that was previously identified in co-transformed Arabidopsis. These results provided important foundation for elucidation of the molecular mechanism of T-DNA co-integration in monocot plants.