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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。
Using Dimocarpus longanLour as test material, the full-length cDNA sequence of regulatory protein 14-3-3 was obtained from flower buds by homologous cloning and RACE. The GenBank accession number was FJ479618 (GI: 218202931). The full-length cDNA was 1121bp in length and contained a 783bp open reading frame encoding 261 amino acids. Sequence analysis showed that the 14-3-3 cDNA was highly conserved. Semi-quantitative RT-PCR analysis showed that 14-3-3 mRNA was expressed in leaf buds, leaves, flower buds and mature flowers, but was the highest in flower buds. The prokaryotic expression system of pET14-3-3 was constructed and the full-length cDNA of 14-3-3 was expressed in E.coli. A soluble fusion protein with a molecular weight of about 34kD was obtained. The protein was 14-3-3 protein.