对一种多基因组装方法的改进

Improvements on a multi-gene assembly system

改建了基于Cre/LoxP重组的2个供体载体和1个兼容Gateway系统的供体载体:pYLd1GW/pYLd2GW、pYLd1GW35s/pYLd2GW35s和pYLd1GWMPK/pYLd2GWMPK。在改建载体的基础上构建了2个供体载体pYLd1GWMdSPDS1和pYLd2GWCBF1,并将2个基因构建于受体载体pYL1305,得到双价表达载体pYL1305MC。采用农杆菌介导的叶盘法转化烟草,经PCR检测和GUS组织化学染色检测发现,双价基因已成功转入烟草。对6株转基因烟草进行荧光定量PCR检测,发现在不同植株中同一基因表达量差异很大,但2个基因表达量差异趋势一致。

By altering two donor vectors of multi-gene assembly vector system based on Cre/LoxP recombination,the donor vectors compatible Gateway system were generated including pYLd1GW/pYLd2GW,pYLd1GW35s/ pYLd2GW35s,pYLd1GWMPK/pYLd2GWMPK.Two donor vectors pYLd1GWMdSPDS1 and pYLd2GWCBF1 were obtained from the modified Gateway compatible vectors.The two genes were cloned in the acceptor vector pYL1305 named pYL1305MC.The double-gene vector was transformed into tobacco through agrobacterium-mediated leaf disc transformation.The analysis of transgenic tobacco with PCR and GUS staining were shown that the double-gene was integrated into tobacco genome.The results of real-time PCR were indicated that the expression level of two transgenes in six transgenic tobacco plants was varied significantly,however,the varying trend of the two genes in expression level was not much different.