Cloning and expression of GDP-Mannose-3' ,5'-epimerase cDNA from Camellia sinensis
[Objective] This study cloned GDP-Mannose-3',5'-epimerase (GME) eDNA 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 eDNA was cloned from C.sinensis and prokaryotic expression was successfully accomplished.
Camellia sinensisGMEreal-time PCRprokaryotic expressionSUMO expression system
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