Cloning And Expression Analysis Of ASA Biosynthsis Related Gene In Camellia Sinensis

L-ascorbic acid(AsA), also named vitamin C, is one of the most important antioxidantsand enzyme cofactors of animals and plants. It plays a major role in regulateing growth anddevelopment. Through regulating the expression of genes related in plant ascorbic acidbiosynthetic pathway, it is probable to raise the AsA contents of plants. Tea is an importanteconomic crop and rich in AsA, but there are almost no reports about the biosynthesis andgene regulation of AsA in tea, and all of the genes related in tea ascorbic acid biosyntheticpathway were not been cloned. So this study cloned the genes related in tea AsA biosynthesisand researched the genes expression by real time PCR and prokaryotic expression.The main results were as follows:1. Cloned five genes related in tea AsA biosynthesis:GDP-D-mannose pyrophosphorylase(GMP), GeneBank accession number is KC477765,the full-length cDNA is1510bp, has an ORF of1086bp encoding a polypeptide of361amino acids. The homology analysis demonstrated that GMP shared high similarities ofnucleotides and deduced amino acids with those in other plants over93%and90%;GDP-Mannose-3’,5’-epimerase(GME), GeneBank accession number is KC477767, thefull-length cDNA is1427bp, has an ORF of1131bp encoding a polypeptide of376aminoacids. The homology analysis demonstrated that GME shared high similarities of nucleotidesand deduced amino acids with those in other plants over90%and80%; GDP-L-galactosephosphowlase(GGP), GeneBank accession number is KC505203, the full-length cDNA is2103bp, has an ORF of1359bp encoding a polypeptide of452amino acids. The homologyanalysis demonstrated that GGP shared high similarities of nucleotides and deduced aminoacids with those in other plants all over80%; L-galactose-1-phosphatase(GPP), GeneBankaccession number is KC505202, the full-length cDNA is1199bp, has an ORF of813bpencoding a polypeptide of270amino acids. The homology analysis demonstrated that GPPshared high similarities of nucleotides and deduced amino acids with those in other plants allover80%; L-galactose dehydrogenase(GalDH), GeneBank accession number is KC477766,he full-length cDNA is1276bp, has an ORF of960bp encoding a polypeptide of319aminoacids. The homology analysis demonstrated that GDH shared high similarities of nucleotidesand deduced amino acids with those in other plants all over80%. 2. The result of Real time PCR showed that the expression level of GMP and GME genedecreased along with the leaf maturity and the highest expression level emerged in bud.GDHhas a slight difference. All of them have obvious differences between different varieties.3. Prokaryotic expression.Constructed prokaryotic expression vector pET-GMP、pET-GME and pET-GDH andexpressed in E.coli BL21(DE3). The pET-GMP produced a fusion protein weighted55kD.The pET-GME produced a fusion protein weighted60kD. The pET-GME produced a fusionprotein weighted50kD. The results are consistent with predicted protein molecular weight.