拟南芥HIS1-3基因克隆及表达载体构建

Cloning of Arabidopsis HIS1-3 gene and construction of its expression vector

文章提取拟南芥植株总RNA,通过RT-PCR扩增出HIS1-3基因的cDNA片段,连接到pEASY-T1Simple载体上,转化到Trans1-T1感受态细胞内,筛选阳性单克隆并抽提质粒。利用限制性内切酶Eco91Ⅰ和NcoⅠ对质粒pEASY-T1-HIS1-3和植物表达载体pCAMBIA2301进行完全双酶切,通过电转化法,将重组表达载体pCAMBIA2301-HIS1-3导入根癌农杆菌C58中以期获得携带HIS1-3基因的根瘤农杆菌株,为研究HIS1-3基因的抗逆分子机理以及遗传改良作物抗逆性奠定了基础。

The total RNA was extracted from Arabidopsis seedlings,the cDNA fragments of HIS1-3 gene were amplified by RT-PCR and lighted with pEASY-T1 Simple vector.The pEASY-T1-HIS1-3 was transformed into Trans1-T1 phage resistant chemically competent cells,the single masccline colony was screened and the plasmid was extracted.Then the restriction enzymes of Eco91 Ⅰ and Nco Ⅰ were used to completely double-digeste the pEASY-T1-HIS1-3 plasmid and the pCAMBIA2301 vector.The recombinant pCAMBIA2301-HIS1-3 vector was carried into the C58 strain of Agrobacterium tumefaciens by electrotransformation.Finally,the strains of Agrobacterium tumefaciens carrying HIS1-3 gene were obtained.The study provides the basis for studying the molecular mechanism of stress-tolerance of HIS1-3 gene and improving the stress-tolerance of crops.