甜瓜CmERFIV-5基因cDNA的克隆·表达分析及载体构建  

Cloning,Expression Analysis and Vector Construction of the CmERFIV-5 Gene cDNA from Melon(Cucumis melo L.)

作　　者：金乌云 邵琪[1] 郝鑫[1] 鲍牧兰 哈斯阿古拉[1] 

机构地区：[1]内蒙古大学生命科学学院内蒙古自治区牧草与特色作物生物技术重点实验室,内蒙古呼和浩特010021

出　　处：《安徽农业科学》2015年第14期30-32,39,共4页Journal of Anhui Agricultural Sciences

基　　金：国家自然科学基金项目(31360486)

摘　　要：[目的]研究甜瓜Cm ERFIV-5基因的克隆、表达分析及超表达和RNAi载体构建。[方法]根据Gen Bank上登录的甜瓜一个乙烯应答因子(ethylene responsive facter,ERF)基因c DNA序列(登录号:MELO3C024315)设计合成特异性引物。应用RT-PCR技术从甜瓜品种河套蜜瓜(Cucumismelo L.cv.Hetao)成熟果实中克隆得到该基因c DNA序列,命名为Cm ERFIV-5,分析了该基因在甜瓜根、茎、叶及果实发育过程中的表达特性,将其构建到过量表达载体p PZP221中,得到重组植物表达载体p PZP221-Cm ERFIV-5。同时,构建了该基因RNAi载体p ART-27-Cm ERFIV-5。[结果]序列分析显示,所克隆的c DNA长度为808bp,编码255个氨基酸。荧光实时定量PCR分析表明,Cm ERFIV-5基因在甜瓜根、茎、叶及不同发育时期的果实中均有表达,在叶片中表达量最高。[结论]构建了Cm ERFIV-5基因的过量表达载体与RNAi载体,为进一步研究该基因的功能奠定了基础。[Objective] The aim was to study cloning,expression analysis and vector construction of the CmERFIV-5 gene cDNA from melon （Cucumis melo L. ） [ Method] A pair of specific primer was designed according to the cDNA nucleotide sequence in GenBank （accession number： MELO3C024315） of an ethylene responsive facter gene cDNA from melon（Cucumis melo L. cv. Hetao）. The cDNA of the gene was cloned by RT-PCR from mature fruit of melon, which the gene was named CmERFIV-5. The gene expression patterns were characterized in Cucumis melo L. melon root,stem,leaf and fruit of different developmental stage . The cDNA of the CmERFIV-5 was cloned into the overexpression vector pPZP221 to construct the recombinant plant expression vector pPZP221-CmERFIV-5. Meanwhile, the RNAi vector pART-27-CmERFIV-5 was constructed. [ Result] Sequence analysis indicated that the cDNA was 808 bp which incodes a polypeptide of 255 amino acids. Quantitative real- time PCR analysis showed that the CmERFIV-5 expressed ubiquitously in roots, stems, leaves and different developmental stages of fruit and the highest transcription was in leaves. [ Conclusion ] Overexpression vector and RNAi vector of CmERFIV-5 gene was constructed, and laid the foun- dations for the further research of the gene function.

关 键 词：甜瓜 乙烯应答因子 乙烯 表达 CmERFIV-5 

分 类 号：S652[农业科学—果树学]