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[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 research aimed to study the cloning, expression analysis and overexpression of Cm ERFIV-5 gene and construction of RNAi vector. [Method] The specific primers were designed and synthesized based on the c DNA sequence of ethylene responsive facter (ERF) gene (accession number MELO3C024315) registered on Gen Bank. The cDNA sequence of this gene was cloned from the mature fruit of melon variety Cucumismelo L.cv.Hetao by RT-PCR and named as Cm ERFIV-5. The gene was analyzed in roots, stems, leaves and fruits of melon During the development, the recombinant plasmid pPZP221-Cm ERFIV-5 was constructed and constructed into overexpression vector pPZP221. At the same time, the gene RNAi vector p ART-27-Cm ERFIV-5 was constructed. [Result] Sequence analysis showed that the cloned cDNA was 808 bp in length and encoded 255 amino acids. Real-time quantitative PCR analysis showed that the Cm ERFIV-5 gene was expressed in the roots, stems, leaves and fruits at different developmental stages, and the expression level was the highest in leaves. [Conclusion] The overexpression vector and RNAi vector of Cm ERFIV-5 gene were constructed, which laid the foundation for further study on the function of this gene.