Cloning And Expression Of Ascorbic Acid-related Enzymes In Camellia Sinesis

L-ascorbic acid(AsA), also known as vitamin C, is one of the most important antioxidants and enzyme cofactors of living body. It is an important antioxidant and redox buffer in plants, playing important roles in metabolism and plant responses to abiotic stresses. As one of the most important economic crops, it contain abundant AsA in tea, however there are few research about synthesize and regulate of AsA in it. This study continue previous research of our group, cloned other two genes related in tea AsA biosynthesis and recycle, the expression of genes were analyze by Real-time PCR and prokaryotic expression. This study also did a preliminary research about the expression of 9 AsA metabolism-related genes under drought and salt as well as the content of AsA after different treatment. The main results were as follows.1. The full-length cDNA of L-galctono-1,4-lactone dehydrogenase(GLDH) and dehydroascorbate reductase(DHAR) were cloned by RT-PCR from the leaves of tea. The full-length of GLDH cDNA is 2052 bp which contain a complete open reading frame(ORF) of 1830 bp, which encodes a polypeptide of 610 amino acids. The accession number of GLDH in GeneBank is KF619448.1. The full-length of DHAR cDNA is 950 bp which contain a complete ORF of 636 bp, which encodes a polypeptide of 212 amino acids. The accession number of DHAR in GeneBank is KF612020.1.2. Constructed prokaryotic expression vector about GDP-L-galactose phosphowlase(GGP), L-galactose-1-phosphatase(GPP), GLDH, DHAR and expressed in HI-Control BL21(DE3) cells. Through the induction of IPTG, those recombinant vectors produce fusion protein about 66 kDa, 45 kDa, 90 kDa, 40 kDa respectively, which consistent with predicted molecular weight of protein.3. Different leaf position show different content of AsA and relative fold expression of genes about AsA-related enzymes in the cultivated species of Longjinchangye. The relative expression of AsA synthesis genes increases along with the leaf maturity to some extent, yet the bud contain the highest relative fold expression of AsA recycle genes, so that the maximum AsA content is in the third leaf under bud while both the bud and the third leaf under bud have the highest content of total AsA. Though the content of AsA and relative fold expression of genes about AsA-related enzymes are various from different species, it is hard to say which gene have the most significant correlation with the content of AsA.4. In the simulating drought stress, the content of AsA and total AsA increasing in 24 hour treatment compared with blank control in the first leaf of the cultivated species of Pingyangtezao by hydroponics experiment, however there were no significant changing regulation in those genes about AsA-related enzymes. Thus through short term experiment could not confirm the regulation mechanism about how AsA-related enzymes genes impact the content of AsA.5. In the high salt stress experiment, the relative fold expression of AsA-related enzymes genes show the tendency of lowered after anterior rised, except for GPP and GDH lowered in the early treatment. Add 4 mmol·L-1 AsA in 500 mmol·L-1 high salt stress solution could futher promote the expression of the genes.