紫花苜蓿MsZIP基因超表达载体的构建及烟草转化

Construction and Transformation of Super-Expressing Plasmid of MsZIP Gene from Medicago sativa L.

根据已经克隆得到的MsZIP基因(GenBank序列号:HQ911778),合成编码区cDNA,构建植物超表达载体PBI-MsZIP。将MsZIP基因插入到含有启动子35S和终止子nos的载体PBI121中,酶切鉴定表明,目的基因已经正确的插入到载体中,超表达载体构建成功。采用CaCl2冻融法将其转入农杆菌中,然后采用农杆菌介导的方法转化烟草(Nicotiana tobacum),共得到12株抗性烟草苗,选取其中长势良好的3株进行分析。结果表明:转基因烟草的PCR,RT-PCR和Southern-blot检测均得到了与目的条带大小一致的片段,说明已经成功获得了能够表达MsZIP基因的转基因烟草;进一步对转基因烟草进行组织化学染色分析,该基因在根、茎、叶中都可以表达。本试验为MsZIP基因功能的研究提供了理论依据和试验材料。

According to the MsZIP gene sequence（GenBank number： HQ911778）,cDNA fragment was cloned and the plant expression vector PBI-MsZIP was constructed.The plasmid was transferred into Agrobacterium LBA4404 by CaCl2 freeze-thaw method then transferred to tobacco by Agrobacterium mediated transformation system.Twelve kanamycin resistant plants were obtained.Three vigorous kanamycin resistant plants were sampled to detect target fragment.PCR and Southern-blot identification proved that the recombinant plasmid had been transferred into tobacco.MsZIP gene was successfully over-expressed in transgenic tobacco.The histochemistry testing of regeneration tobaccos showed MsZIP gene expressing in the root,stem and leaf of tested tobacco.This research provided theoretical basic and experimental material for the function study of MsZIP gene.