摘要
[目的]构建拟南芥乙烯受体(ETR1)特异性短发夹环RNA(shRNA)真核表达载体。[方法]从ETR1基因cDNA序列上选取目标序列,设计2对引物,经PCR扩增得到正反向2个DNA片段(S5I和AI),构建含有该正反向互补重复序列DNA片段的中间载体,测序分析正确后再克隆到植物表达载体pCAMBIA1301中的Gus位置并经双酶切证实。[结果]ETR1基因S5I和AI片段被成功克隆进质粒pCAM-BIA1301中,酶切鉴定及测序分析显示,重组质粒shRNA编码序列与设计片段的序列完全一致。[结论]ETR1靶向RNA干扰重组体的成功构建,为进一步获得拟南芥乙烯受体基因的功能缺失突变体奠定良好基础。
[Objective]The eukaryotic expression vector of short hairpin RNA（shRNA） specific for ETR1 receptors was generated.[Methods]Two DNA segments in both sense and anti-sense orientation,namely S5I and AI,specifically corresponding to the ORF（open reading frame） of ETR1 gene by genomic DNA as template were constructed with PCR amplification.The vector that contained the inverted-repeat DNA fragment was constructed through sequencing analysis.Then,the inverted-repeat DNA fragment was cloned into binary vector pCAMBIA1301,in which it replaced -glucuronidase （Gus） gene.The recombinant plasmid was verified by restriction enzyme.[Results]The recombinant was cloned and the sequence of shRNA recombinant plasmid was the same as that of designed fragments.[Conclusion]Successful cloning of the recombinant may start a novel approach to help to search new ethylene response mutant of Arabidopsis thaliana.
出处
《安徽农业科学》
CAS
北大核心
2008年第21期8946-8948,8972,共4页
Journal of Anhui Agricultural Sciences
基金
中国热带农业科学院中央级公益性科研院所基本科研业务费专项资金资助项目
国家自然科学基金资助项目(30060008)
