一个耐镉基因过表达载体构建及转基因植株的筛选鉴定

Construction of a Cadmium Tolerant Gene Overexpression Vector and Screening and Identification of Transgenic Plants

[目的]进一步研究MNB1基因在植物应对镉胁迫响应中的功能,构建拟南芥中MNB1基因过量表达载体,通过筛选鉴定最终获得相应的转基因植株。[方法]以野生型拟南芥c DNA为模板,PCR扩增MNB1基因全长,将该基因连接到过量表达载体上;然后将构建成功的重组载体转化至农杆菌菌株GV3101,浸花法转化野生型植株;最后利用转基因筛选与遗传鉴定获得MNB1转基因阳性植株。[结果]MNB1基因CDS全长1 368 bp,酶切连接后转化大肠杆菌鉴定得到阳性菌落,测序结果经比对完全正确。通过抗性筛选及PCR电泳检测获得阳性转基因植株。[结论]MNB1基因过表达载体构建成功并获得相应转基因植株,为进一步研究该基因调控植物镉胁迫响应中的功能及其分子机制奠定基础。

[Objective]In order to further study the function of MNB1 gene in response to cadmium stress in plants,the over-expression vector of MNB1 gene in Arabidopsis was constructed,and the corresponding transgenic plants were finally obtained through screening and identification.[Method]The full-length MNB1 coding sequence was amplified through PCR from cDNA of Arabidopsis(Col-0)using specific primers and cloned into the overexpression vector.Then the resulting construct was transformed into Agrobacterium strain GV3101 for transformation into the Col-0 plants by the floral dip method.The transgenic lines were isolated through antibiotic screening and genetic identification.[Result]The full-length CDS of MNB1 gene was 1 368 bp,which was identified by enzyme digestion and transformed into E.coli.The positive clones were identified and the sequencing results were completely correct.Positive transgenic plants were obtained by resistance screening and PCR electrophoresis.[Conclusion]The construction of MNB1 gene overexpression vector was successful and the corresponding transgenic plants were obtained,which laid the foundation for further study on the function and molecular mechanism of this gene regulating plant stress response to cadmium.