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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。
The T-DNA fragment was efficiently inserted into the genomic DNA of Zhonghua 11 by Agrobacterium tumefaciens. Utilizing the scutellum callus of the secondary shoots and immature seeds as the starting material for transformation, the conditions affecting the callus induction rate, regeneration rate and transformation rate were explored and optimized. Nearly 10,000 transformed plants were obtained. PCR analysis and Southern blotting proved that foreign genes were integrated into rice genome. Contemporary transgenic plants are resistant to 0.2% herbicide Basta. More than 200 T0 generation of transgenic lines showed mutant phenotypes such as albino seedling, broad leaf, narrow leaf, yellow leaf, tall stalk, dwarfism and weedy, sterile, albino panicles, ear wrinkles and heading delay, Lower. Bar gene analysis of progeny of three transgenic rice plants showed that the bar gene showed a 3: 1 segregation pattern in progenies. In the experiment, it was found that the separation of the mutant progeny of the transgenic progeny coincided with the isolation of the bar gene. T-DNA sequencing analysis of transgenic rice plants with the mutation indicated that these mutations are likely to be related to T-DNA insertion.