拟南芥CDR6基因过表达载体构建及过量表达植株筛选鉴定  

Construction of Arabidopsis CDR6 Over-expression Carrier and Identification of Stably Transgenic Lines

作　　者：董万春[1] 樊婷婷[1] 阳立波 江海坤[2] 张其安[2] 方凌[2] 倪娇娇[1] 管灵霞 曹树青[1] 

机构地区：[1]合肥工业大学生物与食品工程学院,安徽合肥230009 [2]安徽省农业科学院园艺研究所,安徽合肥230009

出　　处：《安徽农业科学》2015年第13期63-64,104,共3页Journal of Anhui Agricultural Sciences

基　　金：安徽省自然科学基金青年基金项目(1308085QC56);安徽省科技攻关项目(1201a0301009)

摘　　要：[目的]以拟南芥为材料克隆CDR6基因,构建CDR6基因的过量表达载体并筛选鉴定获得过表达植株。[方法]提取拟南芥mRNA,反转录成c DNA,并以此为模板克隆CDR6基因CDS全长,通过限制性内切酶切割、T4DNA连接酶连接,将CDR6基因CDS全长连接到带有35S强启动子的p XB094载体上;然后转化至Trans1-T1感受态细胞中,菌落PCR鉴定阳性单克隆并测序确认。将重组质粒转化至根瘤农杆菌GV3101菌株,通过浸花法侵染拟南芥野生型植株,通过抗性筛选和鉴定获得预期的转基因植株。[结果]菌落PCR鉴定和测序结果表明CDR6基因已成功构建至p XB094载体;通过抗性筛选并鉴定获得了过量表达阳性植株。[结论]筛选和鉴定获得的过量表达植株为研究CDR6基因的分子功能奠定了基础。[ Objective ] To build up the carrier of Arabidopsis CDR6 gene over-expression and isolate corresponding transgenic lines. [ Method] Total RNA was extracted from Arabidopsis seedlings and reverse transcribed as PCR template. CDS fragments of CDR6 gene were amplified through RT- PCR. Using the restriction endonuclease and T4 DNA ligase, CDS fragments were subsequently cloned into pXB094 vector, and then were transformed into Trans-T1 phage resistant competent cells. Bacterial colony PCR and DNA sequencing were performed to confirm that CDS of the Arabidopsis CDR6 gene was successfully cloned. The recombinant plasmids were transformed into Agrobacterium GV3101 cells. Wild type plants were transformed using floral-dip method and screened to obtain the desired transgenic plants. [ Result] Bacterial colony PCR and DNA sequencing were performed and recombinant plasmids were confirmed. Stably transgenic lines of CDR6 over-expression were obtained through an- tibiotic screening, and verified through genetic methods. [ Conclusion] Construction of Arabidopsis CDR6 gene over-expression and screening of transgenic plants laid the foundation for functional analysis of CDR6.

关 键 词：拟南芥 CDR6基因 过量表达载体 转基因植株 

分 类 号：S188[农业科学—农业基础科学]