龙眼14-3-3基因的克隆与原核表达

 以龙眼(Dimocarpus longan Lour. ) 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1 121 bp, 包括一个783 bp的开放阅读框, 编码261个氨基酸, 序列比较分析显示14-3-3 cDNA具有较高的保守性。半定量RT-PCR分析结果表明, 14-3-3 mRNA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34 kD的可溶性融合蛋白, 经Western blotting验证, 该蛋白为14-3-3蛋白。

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

Full length cDNA of 14-3-3 gene, encoding a plant regulatory factor, was isolated from longan (Dimocarpus longan Lour. ) using homology based cloning and RACE methods. The accession number of the gene in GenBanks is FJ479618 (GI: 218202931). The full length cDNA is of 1 121 bp, with a 783 bp open reading frame encoding 261 amino acids. Homology analysis showed that the longan 14-3-3 cDNA has high consensus regions. Semi-quantitative RT-PCR analysis detected the exp ression of 14-3-3 gene in longan foliarbuds, leaves, mature flowers, and most up2regulated in flower buds. The p rokaryotic exp ression vector was-constructed and was expressed in E.coli; and a recombinant protein with a molecularweight of 34 kD was obtained by SDS-PAGE analysis. Western blotting confirmed the p rotein was 14-3-3 protein.