龙眼14-3-3基因的克隆与原核表达  

Cloning and Prokaryotic Expression of 14-3-3 Gene in Longan

作　　者：游向荣[1] 王平[2] 梁文裕[1] 郑少泉[3] 陈伟[1] 

机构地区：[1]福建农林大学生命科学学院,福州350002 [2]福建农林大学园艺学院,福州350002 [3]福建省农业科学院果树研究所,福州350013

出　　处：《园艺学报》2009年第10期1431-1436,共6页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(30571293);教育部博士点基金项目(200803890009);福建省自然科学基金项目(2007J0045)

摘　　要：以龙眼(Dimocarpus longanLour.)为试材,应用同源克隆和RACE方法从花芽中获得了调控蛋白14-3-3的全长cDNA序列,GenBank登录号为FJ479618(GI:218202931)。该cDNA全长1121bp,包括一个783bp的开放阅读框,编码261个氨基酸,序列比较分析显示14-3-3cDNA具有较高的保守性。半定量RT-PCR分析结果表明,14-3-3mRNA在龙眼叶芽、叶片、花芽和成熟的花中都有表达,但在花芽中表达量最大。构建了pET14-3-3原核表达系统,将14-3-3全长cDNA在大肠杆菌中表达,获得一个分子量约为34kD的可溶性融合蛋白,经Western blotting验证,该蛋白为14-3-3蛋白。Full length cDNA of 14-3-3 gene, encoding a plant regulatory factor, was isolated from longan （Dimocarpus longan Lout. ） using homology-based cloning and RACE methods. The accession number of the gene in GenBanks is FJ479618 （GI： 218202931 ）. The full length eDNA is of I 121 bp, with a 783 bp open reading frame encoding 261 amino acids. Homology analysis showed that the longan 14-3-3 eDNA has high consensus regions. Semi-quantitative RT-PCR analysis detected the expression of 14-3-3 gene in longan foliar buds, leaves, mature flowers, and most up-regulated in flower buds. The prokaryotic expression vector was constructed and was expressed in E. coli; and a recombinant protein with a molecular weight of 34 kD was obtained by SDS-PAGE analysis. Western blotting confirmed the protein was 14-3-3 protein.

关 键 词：龙眼 花芽 14-3-3基因 克隆 原核表达 

分 类 号：S667.2[农业科学—果树学] Q78[农业科学—园艺学]