冷诱导转录因子AtCBF1转化紫花苜蓿的研究

A study on Agrobacterium tumefaciens-mediated transformation of Medicago sativa with the AtCBF1 gene

为研究冷诱导转录因子CBF1(C-repeat-binding-factor)基因对我国优良豆科牧草紫花苜蓿的抗寒性改良作用,本实验以拟南芥基因组DNA为模板,利用PCR方法克隆得到了冷诱导转录因子AtCBF1基因,测序结果表明所克隆的DNA片段长度为642bp,将该序列与GenBank上的CBF1基因序列进行DANMAN序列比较分析,同源性可达99.84%。在此基础上成功构建了含有AtCBF1基因的植物表达载体pBI121-CBF1,并采用根癌农杆菌介导法对紫花苜蓿进行了遗传转化,获得了经卡那霉素筛选的抗性转化植株,进一步经PCR和RT-PCR检测,得到了650bp左右的电泳条带,表明AtCBF1基因已在紫花苜蓿中得到表达。这为选育紫花苜蓿抗寒新品种奠定了良好的基础。

The cold resistance effect of the cold-induced transcription activator CBF1 （C-repeat-binding-factor） gene on Medicago sativa was studied by amplifying an AtCBF1 gene and cloning it by polymerase chain reaction （PCR） from the genomic DNA of Arabidopsis thaliana. The length of the DNA cloned fragment was 642 bp. Compared with the CBF1 sequence in GenBank, the sequence homology rate was 99.84%. On the basis of the CBF1 gene, the plant expression vector pBI121-CBF1 was constructed with the AtCBF1 gene and M. sativa was transformed using Agrobacteriurn tumefaciens. The transformed plants were selected with kanamycin. An electrophoretic band of 650 bp was obtained by PCR and RT--PCR （reverse transcription polymerase chain reaction）. The AtCBF1 gene was expressed in M. sativa. This research provides a good basis for breeding new M. sativa varieties with cold resistance.