有效去除农杆菌和籼稻转化系统优化

Efficient Elimination of A.tumefaciens and Optimization of Agrobacterium-mediated Transformation of Indica Rice

为了提高根农杆菌籼稻转化效率 ,将修饰的豇豆胰蛋白酶抑制剂基因sck和潮霉素磷酸转移酶基因hpt分别置于紧密相连的 2个T -DNA上 ,构建载体 pCDMARUSCK -HPT ,电激法将表达载体转化农杆菌LBA4 40 4获得工程菌。比较了提门叮和国产噻孢霉素对工程菌LBA4 40 4的抑制效果 ,试验得出提门叮在 5 0 0mg/L以内比噻孢霉素能更有效地去除水稻细胞中的农杆菌 ,而且低浓度条件下 (2 5 0mg/L)就能有效地抑制工程菌LBA4 40 4 ,并且有利于水稻转化细胞成功地再生植株。确立了工程菌LBA4 40 4菌液浓度OD值 0 8,浸染时间 5min为明恢 86合适的转化参数。在此基础上 ,采用优化的农杆菌介导法转化杂交籼稻优良恢复系明恢 86 ,获得转基因植株 12 7个克隆 ,转化效率 4 5 %。

In order to improve the transformation efficacy of indica rice mediated by Agrobacterium tumefaciens, the modified cowpea trypsin inhibitor gene (sck) and the hygromycin Phosphotranferase gene (hpt) were separately harbored in two adjacent T-DNA regions, and super vector pCDMARUSCK-HPT was constructed. A. tumefaciens strain LBA4404 integrated the pCDMARUSCK-HPT plasmid was developed by Electroportion. The anti-bacteria capabilities of antibiotics such as Timentin (Tim) and Cefotaxcin (Cef) at various concentrations in controlling LBA4404 strain were tested. Results indicated that Tim was better than Cef at concentration below 500mg/L and still worked effectively at 250mg/L against LBA4404 strain, and the transformed rice calli regenerated more plantlets when using Tim as bacteria inhibitor. The combination of LBA4404 strain concentration at OD 600 0.8 and 5min treatment on Minghui86 (M86) vigorous calli was the suitable parameter in terms of transformation efficiency by the LBA4404 strain in the experiment. 127 independent transgenic lines of M86 at transformation rates 4.5% were obtained exploiting this optimized transformation system.