Cloning And Expression Analysis Of Related Enzyme Genes In Ascorbic Acid Biosynthesis Of Citrus Maxima (Burm.) Merr

Pomelo (Citrus maxima (Burm.) Merr.] belongs to rutaceae,which has a high quality. Ascorbic acid (L-ascorbic acid, AsA),also known as vitamin C,abundant in pomelo. Juice sac granulation seriously affects its edibility and commodity value. Ascorbic acid metabolic pathways was associated with lignin metabolic pathways.lt was reported that ascorbic acid related enzyme genes in AsA biosynthesis of pomelo were cloned. Genes expression levels were analyzed and AsA contents were also determined,using juice sac near column and juice sac away from column of ’Hongroumiyou’.The following contents were the main results: 1.The ORFs of eight ascorbic acid biosynthesis related enzyme genes were cloned from juice sacs of pummelo by RT-PCR.GDP-D-mannose pyrophosphorylase(GMP),the ORF is 1086 bp, which encodes 361 amino acids;GDP-mannose-3’,5’-epimerase(GME),the ORF is 1128 bp,which encodes 375 amino acids;GDP-L-galactose phosphorylase(GGP),The ORF is 1368 bp,which encodes 455 amino acids;L-galactose-1-phosphate phosphatase(GPP),the ORF is 892 bp,which encodes 296 amino acids;L-galactose dehydrogenase,(GalDH),the ORF is 954 bp,which encodes 317 amino acids;L-galactono-1,4-lactone dehydrogenase(GalLDH),the ORF is 1807 bp,which encodes 601 amino acids;Pectinmethylesterase1(PME1),the ORF is 1748 bp,which encodes 582 amino acids;polygalacturonasel (PG1),the ORF is 1337 bp,which encodes 445 amino acids. Analysis indicated that amino acid sequences derived from these genes showed high homology with other species.2. The expresstion levels in different developmental stages and parts of pomelo were analyzed by quantative real-time PCR and AsA contents were determined. Our research indicated that Genes expression and AsA contents of juice sac near column were less than juice sac away from column.Expression levels of CmGMP, CmPME1 and CmGME reduced gradually,after rising first and reached the highest amount in the 170 days.Relative expression levels of CmGPP,CmGalLDH and CmPG1 did not change significantly at the early stage of the fruit development (107 days to 147 days),reduced gradually,after rising first and reached the highest amount in the 191 days.AsA contents reduced gradually,after rising first and reached the highest amount of the 170 days after flowering. AsA content of 170 days was more three times than the 107 days after flowering Independent sample T test of the two parts of the AsA contents, after 117 days,which show significant difference (P<0.05).Differential genes expression of CmGMP、 CmGME、CmGPP、CmGalLDH、CmPME1、CmPG1 were associated with AsA contents,especially genes expression of CmGMP,CmPMEl and CmGME were significantly correlated with AsA contents.L-galactose pathway played an important role in AsA biosynthesis,and D-galacturonic acid pathway may be very important.The two pathways can coexist mutually.