提高水稻插入突变体库利用效率的尝试

T-DNA标签法是一种以农杆菌介导的遗传转化为基础来创造插入突变体库, 从而高通量地分离和克隆植物功能基因的方法。但由于种种原因, 水稻插入突变体库的利用效率较低。为了提高水稻插入突变体库的利用效率, 结合水稻一个双拷贝T-DNA插入突变体的发现和鉴定研究, 通过特异PCR检测、侧翼序列与目标性状的共分离分析, 在1个双插入位点均为杂合的植株的后代株系中分拆了2个插入事件, 分离出目标性状存在遗传分离且只带有1个插入事件的后代株系, 为后续的共分离检测和基因克隆研究打下了重要的基础。由此产生了对插入突变体库中的非串联多拷贝插入标签系进行研究的一些思路和方法, 提出来与同行商榷。

Attempting to enhance the efficiency of T-DNA insertional mutant application in rice

T-DNA tagging method is a high throughput system for identifying and cloning novel genes from T-DNA-inserted mutant population created via genetic transformation by Agrobacterium tumefaciens. However, the efficiency of using T-DNA-inserted mutant population to clone genes in rice was much lower than in Arabidopsis. In this study, a rice tagged line with two copies of T-DNA segments inserted independently to each other was screened out via a series of verification tests, including the co-segregated analysis between the mutated character and the sequence of T-DNA or the genomic sequence flanking inserted T-DNA. From this tagged line, two inserted incidents were separated from the progeny population of a plant heterozygous in two tagged sites, and some plants with the target trait and one of the inserted incidents were obtained, which were important basic materials for the subsequently co-segregated analysis between the mutated character and the sequence of inserted T-DNA, and for cloning the mutant gene in future. Based on this study, we have some thoughts about the gene cloning from the T-DNA tagged lines with more than one inserted sequence independently and put forward to discuss with colleagues.