根癌农杆菌介导的水稻高效转化系统的建立

A Highly Efficient Transformation System Mediated by Agrobacterium tumefaciens in Rice(Oryza sativa L.)

比较了影响根癌农杆菌转化水稻的各种因素后，建立了农杆菌介导的水稻高效转基因实验体系。按该体系，水稻品种中花11号预培养4d的幼胚经农杆菌EHA105／pCAMBIA1301感染后，具有GUS基因瞬间表达的幼胚比例在50％以上，最高可达90％；按产生潮霉素抗性愈伤和转基因植株的比例计算，转化率分别达到87．6％和64．6％。转基因植株总DNA的Southern杂交分析表明T－DNA上的外源基因已整合进了水稻基因组，且在大多数转基因植株中表现为单拷贝插入；遗传分析证明T1代的表型分离符合孟德尔法则。此转化系统的建立为高效地将有用的外源DNA导入水稻植株奠定了基础。

After the factors influencing transformation were carefully investigated, a highly efficient Agrobacterium-mediated gene transfer system has been developed for rice. Using this protocol, inunature embrys of rice variety Zhongha No. 11 cultured for 4 days were infected and haformd by A. tumefaciens stain EHA105 harboring Ti Plasmid pCAMBIA1301 The results showed that infected embryos gave a high hequency (50% ～ 90%) of GUS gene expression after co-cultivation. The hansformation efficiency of stable integIation of TDNA reached 87. 6% measured as hypomycin-resistan calli produced and 64. 6% measmed as numberof hansemc plants regenerated, respectively. Southem blotting analsis of the total DNA extrated frOm transgenic plants indicated tha the TDNA had been integrated into the rice genom, and most of bosgenic planis had one coPy of TDNA insert. The results of genetic analsis ford tha the bosgenes were segngaed in a Mendelian fashinonin the T1 generation.The establishment of this bosformtion system in rice Provides an efficient way for transferring usend foreign genes to rice plants