火把梨14-3-3基因的克隆与序列分析

依据火把梨(Pyrus pyrifolia Nakai)14-3-3蛋白的EST序列设计基因特异引物,采用快速扩增cDNA末端技术(Rapid amplification of cDNA ends,RACE),从云南火把梨中克隆一个新的14-3-3基因(Pp14-3-3)的全长cDNA序列。Pp14-3-3全长cDNA为1 107 bp,具有84 bp 5’非编码区(Untranslated Regions,UTR)、786 bp开放读码框(Openreading frame,ORF),以及237 bp 3’UTR,编码含261个氨基酸的蛋白质。Pp14-3-3与其他物种14-3-3蛋白在中间功能区域高度保守,包含典型的14-3-3结构域。与已知植物14-3-3s家族成员间的氨基酸序列进行聚类分析,将Pp14-3-3聚为非ε大类14-3-3。Pp14-3-3在火把梨接受光照和没有光照的果皮以及幼嫩叶片中大量表达,且表达量稳定。对Pp14-3-3的基因克隆以及表达特性进行了分析,为后期深入研究该新基因的功能奠定了基础。

Isolation and Expression Analysis of a 14-3-3 Gene from Pyrus pyrifolia Nakai cv.Huobali

Based on the EST sequence encoded the 14-3-3,gene-specific primer was designed and used to obtain the full-length cDNA of a novel 14-3-3 gene from Pyrus pyrifolia Nakai cv.Huobali in Yunnan province with the method of rapid amplification of cDNA ends(RACE).This novel gene was named as Pp14-3-3.Pp14-3-3 is 1 107 bp in length with an ORF of 786 bp,a 5′-untranslated region(UTR) of 84 bp,and a 3′-UTR of 237 bp,and the ORF encodes a predicted polypeptide of 261 amino acids.The Pp14-3-3 shares higher homology with the known 14-3-3 proteins,and possesses the basic stucture of 14-3-3 proteins.A phylogenetic analysis of the relationship of the newly identified Pp14-3-3 with some known 14-3-3s from other species grouped the Pp14-3-3 into the class of non-ε 14-3-3s.Pp14-3-3 is abundantly expressed in pericarps of Huobali regardless received sunlight or not,and also expression in the young leaves.Isolation and expression analysis of Pp14-3-3 in this study laid the groundwork for further studying on function of Pp14-3-3.