茶树GDP-甘露糖-3′,5′-异构酶基因cDNA全长的克隆与表达

Cloning and expression of GDP-Mannose-3′,5′-epimerase cDNA from Camellia sinensis

【目的】对茶树GDP-甘露糖-3′,5′-异构酶(GDP-Mannose-3′,5′-epimerase,GME)基因cDNA全长进行克隆分析及原核表达。【方法】以茶树品种"乌牛早"叶片的cDNA为模板,用RT-PCR和RACE技术克隆GME,并对其进行生物信息学分析。利用pETiteTM N-His SUMO Vector构建GME原核表达载体pET-GME,在大肠杆菌HI-Control BL21(DE3)中进行原核诱导表达。采摘同一茶树嫩茎、芽、叶片以及不同品种茶树叶片样品,提取其RNA,反转录合成cDNA后,通过荧光定量PCR分析GME在不同茶树组织和品种中的表达谱。【结果】克隆获得了长度为1 427bp的GMEcDNA全长序列,其包含长度为1 131bp的开放阅读框,编码376个氨基酸。构建了GME原核表达载体pETGME,其在大肠杆菌HI-Control BL21(DE3)中能诱导表达分子质量约60ku的融合蛋白。荧光定量PCR结果显示,GME基因在芽中的表达量最高,在嫩茎中的表达量最低,在叶片中的表达量随成熟度的增加而逐渐降低;在不同茶树品种之间的表达也存在明显差异。【结论】从茶树叶片中克隆得到了GMEcDNA全长序列,并成功对其进行了原核表达。

【Objective】This study cloned GDP-Mannose-3′,5′-epimerase （GME） cDNA from leaves of Camellia sinensis and analyzed its expression.【Method】GME was isolated from leaves of C.sinensis by RT-PCR and RACE.Then the GME was cloned into pETiteTM N-His SUMO Vector to construct prokaryotic expression vector pET-GME which was expressed in E.coli BL21 （DE3）.Gene expression in different tissues of cultivated species was detected by real time PCR.【Result】GME cDNA sequence with length of 1 427 bp including an ORF of 1 131 bp and a polypeptide of 376 amino acids was obtained.The pET-GME produced a fusion protein with weight of 60 ku in E.coli BL21 （DE3）.Real time PCR showed that the expression level of GME gene decreased gradually as the maturity of leaves.The highest expression level emerged in bud while the lowest expression level emerged in stem.Obvious difference of it exist in different cultivated species.【Conclusion】The complete GME cDNA was cloned from C.sinensis and prokaryotic expression was successfully accomplished.