拟南芥AtMYB61基因的克隆及表达载体构建.docVIP

拟南芥AtMYB61基因的克隆及表达载体构建 王 云， 任永兵， 杨 硕， 韩 宇， 陶 杨， 曹树青 (合肥工业大学生物与食品工程学院，安徽合肥230009) 摘要：文章以拟南芥幼苗提取的总RNA为模板，利用RT-PCR技术扩增获得AtMYB61基因的全长cDNA片段，再克隆到pUCm-T载体上，菌落PCR、酶切鉴定和cDNA测序结果表明成功克隆了拟南芥AtMYB61基因。从AtMYB61一pUCm-T载体上，用BstEII和BglII全双酶切切下目的基因片段，将此基因片段连接到植物表达载体pCAMBIA2301中。菌落PCR和酶切鉴定结果表明成功构建了植物表达载体pCAMBIA2301一AtMYB61。利用电转化法将重组表达载体导人根癌农杆菌中，获得了携带AtMYB61基因的根癌农杆菌株，为转基因改良植物抗逆性和进一步研究A￡MyB61基因的抗逆分子机理奠定了基础。 关键词：拟南芥；AtMYB61基因；表达载体 Cloning of Arabidopsis thaliana AtMYB61 gene and the expression vector construction W ANG Yun， REN Yong-bing， YANG Shuo， HAN Yu， TAO Yang， CAO Shu-qing (School of Biotechnology and Food Engineering，Hefei University of Technology，Hefei 230009，China) Abstract：Tota1 RNA was extracted from Arabidopsis thaliana seedlings and used as the template to amplify the full length of eDNA of AtMYB6 1 gene by RT_PCR technology，and the gene fragment was subsequently cloned into plant pUCm-T vector．The results of bacterial colony PCR，enzyme analysis and eDNA sequencing confirmed that the Arabidopsis thaliana AtM YB6 1 gene was successfully cloned．AtMYB61 gene was cut completely from AtMYB61一pUCm-T vector by BstE 1]and BglⅡ，and the gene fragment was cloned into plant expression vector pCAMBIA2301．The results of bacterial colony PCR and enzyme analysis showed the Successfu1 construction of plant expression vector pCAM— BIA2301一AtM YB61．In addition，the recombinant expression vector was carried into Agrobacterium tumefaciens by electrotransformation，and the strain of Agrobacterium tumefaciens carrying AtM YB6 1 gene was obtained．The study provides a basis for improving the resistance of transgenic plants and further exploring the molecular mechanism of AtM YB6 1 gene． Key words：Arabidopsis thaliana；AtMYB6 1 gene；expression vector 0 引 言 随着一些抗逆基因的鉴定和抗逆机理不断深入研究，将外源抗逆基因导人植物的基因工程技术，在提高植物抗逆性方面具有更广泛的应用前景。但植物的抗逆应答是一系列相关基因连续表 达调控的极其复杂的调控网络。在整个调控网络中，转录因子能调控多个相关基因的表达，所以增强转录因子的调控是提高植物抗逆性更有效的方法Ⅲ。MYB转录因子是拟南芥中最大的基因家族。该基因家族主要在植物的生长发育方面发挥重要作用，如植物次级代谢、信号传导、器官形成等方面[3]。另外，也有一部分参与了逆境胁迫介导的细胞应答[4 ]。大量研究