插入T-DNA片段构建部分水稻突变体株系

The Construction of Rice Mutant Collections by T-DNA Insertional Mutagenesis

通过农杆菌Agrobacterium tumefaciens介导,将T-DNA片段高效插入水稻中花11的基因组DNA中。利用二次枝梗种子和未成熟种子的盾片愈伤组织作为转化起始物,探索并优化了影响愈伤组织诱导率、再生率和转化率的试验条件,得到近10 000株转化植株。经PCR分析和Southern 杂交证明已将外源基因整合到水稻基因组中。当代转基因植株可抗0.2%的除草剂Basta。200余棵T0代转化株系出现白化苗、宽叶、狭叶、黄叶、高秆、矮化并杂草化、不育、白化穗、皱穗和抽穗延迟等突变表型,结实率普遍较低。对3个转基因水稻突变株的子代进行bar基因分析发现,bar基因在子代中呈现3∶1的分离规律。试验中发现了转基因子代的突变征状的分离与bar基因的分离相一致现象。对具有突变征状的转基因水稻植株进行T-DNA 边序分析发现这些突变很可能与T-DNA插入有关。

Insertional mutagenesis with tags to construct a rice mutant pool is an efficient tool in discovery of rice genefunctions. This research established a method by which T-DNA can be inserted with high efficiency into the genome of Oryzasativa L. ssp. japonica cv. Zhonghua11 mediated by Agrobacterium tumefaciens. PCR and Southern blotting were used toindicate that the foreign DNA had been integrated into rice genome DNA. Transgenic rice plants could survive in 0.2% Basta.Approximate 10000 transformed rice plants were obtained up to now and more than 200 of those showed markable mutantcharacters: chlorosis, broad-leaf, narrow-leaf, yellow-leaf, tall-haulm, dwarf-haulm and weediness, sterile, chlorosis-spike, crimple-spike-stalk, heading-delayed etc. Many of the T0 transformed rice plants yielded few seeds. bar gene analysis showed 3∶1segregation in progenies of three transgenic rice plants. We found mutant phenotypes co-segregated with T-DNA tags in transgenicprogenies. The junction fragments of T-DNA and rice genomic DNA from transgenic rice plants were amplified by TAIL-PCRand were analysed in GenBank and the data showed that mutant characters might be involved in T-DNA insertion.