摘要： 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。
关键词:
火把梨,
RACE,
RT-PCR,
14-3-3
Abstract: 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 .
Key words:
Pyrus pyrifolia Nakai cv. Huobali,
Rapid amplification of cDNA ends,
RT-PCR,
14-3-3
中图分类号:
王光勇, 刘迪秋, 李旻, 饶健, 孙兵召, 丁元明. 火把梨14-3-3基因的克隆与序列分析[J]. 华北农学报, 2012, 27(3): 55-61. doi: 10.3969/j.issn.1000-7091.2012.03.011.
WANG Guang-yong, LIU Di-qiu, LI Min, RAO Jian, SUN Bing-zhao, DING Yuan-ming. Isolation and Expression Analysis of a 14-3-3 Gene from Pyrus pyrifolia Nakai cv. Huobali[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2012, 27(3): 55-61. doi: 10.3969/j.issn.1000-7091.2012.03.011.