利用根癌农杆菌法获得转基因水稻植株及其后代

Obtaining Transgenic Rice Plants and Their Progenies Using Agrobacterium tumefaciens

在100μmol／L乙酰丁香酮（AS）等vir基因诱导分子存在的情况下，用含双元载体pBYT2的根癌农杆菌菌株EHA101同水稻（OrizasativaL．）台北309悬浮培养细胞共培养3天。经过2个月的连续筛选，共从364颗同根癌农杆菌共培养的悬浮培养细胞团中得到17个具有稳定潮霉素抗性和GUS表达的愈伤组织。对从8个转化组织中得到的10株可能的R0代转基因植株及其后代进行外源基因的整合和表达分析，Southern分析表明外源基因已稳定地整合进水稻基因组中并实现了有性遗传传递。杂交结果显示在其中一个转化系的植株中有5个拷贝的T－DNA整合，而其余的转化系则只整合了1个拷贝。转基因水稻细胞及植株中GUS活性的组织化学染色观察和荧光分析表明玉米ubiquitin基因启动子在水稻细胞中能高效启动gus报告基因的表达。ndPAGE－X－Gluc法检测表明转基因水稻细胞中表达的GUS蛋白比Sigma公司的标准GUS蛋白（SigmaCo．G0786）要小，而与来自大肠杆菌HB101（pBI1121）中的GUS蛋白大小相同。结果表明，根癌农杆菌可有效且可靠地介导外源基因转化水稻。

Rice (Oriza sativa L) suspension cells of Taipei 309 were co-cultivated with A.tumefaciens strain EHA101 harbouring binary vector pBYT2 for 3 days in thepresence of vir inducer, 100μmol / L acetosyringone (AS). After 2 months ofcontinuous selection, 17 stable hygromycin-resistant GUS-positive calli were recoveredfrom 364 suspension cell clusters co-cultivated with A. tumefaciens. 10 putativetransgenic R0 plants obtained from 8 transformed calli and their progenies wereanalyzed for the integration and expression of foreign genes. Southem blot analysis ofR0 and R1 generations indicated that foreign genes had been stably integrated in thegenome of transgenic rice and sexaully transmitted. One of the transgenic linesshowed 5 copies of T-DNA integration, while the others had only one copy.Histochemical staining observation and fluorometric assay of GUS activity intransgenic rice cells and plants showed ubiquitin promoter from maize was highlyeffective in driving the expression of gus reporter gene in transgenic rice cells. GUSprotein and its activity were also investigated through ndPAGE-X-Glue staining assay,and it was found that the GUS protein in transgenic rice cells was smaller in sizethan the standard GUS protein (Sigma Co. G0786) but as large as that from E.coliHB101 (pB1121). This study suggested that Agrobacterium-mediated transformation ofplat is an efficient and reliable method to introduce foreign genes into rice.