紫花苜蓿MsZIP基因超表达载体的构建及转基因苜蓿检测  

Construction and transformation of an over-expression plasmid of the MsZIP gene from Medicago sativa

作　　者：李燕[1] 孙彦[2] 杨青川[1] 康俊梅[1] 张铁军[1] 房锋[3] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]中国农业大学动物科技学院,北京100191 [3]中国农业科学院植物保护研究所,北京100193

出　　处：《草业学报》2012年第6期182-189,共8页Acta Prataculturae Sinica

基　　金："十二五"国家科技支撑计划课题(2011BAD17B01-01-3);中央级公益性科研院所专项资金项目(2011cj-14)资助

摘　　要：根据已经克隆得到的MsZIP基因(GenBank序列号:HQ911778),扩增编码区cDNA,构建植物超表达载体PBI-MsZIP。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菌株中,然后采用农杆菌介导的方法,转化紫花苜蓿,共得到11株抗性苗,对其中的4株进行卡那霉素基因PCR检测,均得到了目的条带。同时对这4株抗性苗进行目的基因的RT-PCR检测,均得到了目的条带。说明MsZIP基因已经成功在苜蓿中超表达。为了进一步验证该基因的功能,分别用200mmol/L NaCl和25μmol/LPEG-6000处理转基因苜蓿,3d后进行生理指标的测定。结果表明,MsZIP基因在苜蓿中超表达可以提高苜蓿的耐盐性和耐旱性。Based on the MsZIP gene sequence（GenBank No.HQ911778）,a cDNA fragment was cloned and connected to the PMD18-T vector to construct PMD-MsZIP.The target fragment and linear plasmid were obtained from the cloning vector PMD-MsZIP and from the plant expression vector PBI121 using dual digestion with XbaI and BamHI.The plant over-expression vector PBI-MsZIP was built through directional connections using T4 DNA ligase.The plasmid was transferred to Agrobacterium LBA4404 by the CaCl2 freeze-thaw method and was then transferred into alfalfa by an Agrobacterium-mediated transformation system.Eleven kanamycin-resistant plants were obtained.Four of them were sampled to detect target fragments and PCR identification showed that the recombinant plasmid had been transferred into alfalfa.The MsZIP gene was successfully over-expressed in transgenic Medicago sativa CV.Zhongmu No.1.To test the function of this gene,the transgenic alfalfa was treated with 200 mmol/L NaCl and 25 μmol/L PEG-6000 for three days,and some physiological parameters were measured.The contents of soluble sugar,soluble protein and proline significantly increased,while the MDA content declined.Over-expressed MsZIP gene enhanced the salt and drought tolerance of alfalfa.

关 键 词：紫花苜蓿 MsZIP基因 超表达载体 苜蓿转化 转基因苜蓿检测 

分 类 号：S541.9[农业科学—作物学]