Cloning And Expression Of Anthocyanin Biosynthesis Related Structure Genes In Vitis Amurensis

Vitis amurensis is an important wild fruit resource. Anthocyanins are the major polyphenol secondary metabolites in V. amurensis, and which are closely related to disease resistance, color, flavor and other indicators. Anthocyanin not only affects the sensory and nutritional value of plant foods, but also has a variety of physiological activity of medicine. In this paper, we constructed V amurensis veraison skin cDNA library, cloned and sequenced the structural genes of V. amurensis anthocyanin biosynthesis. Anthocyanin biosynthesis related gene expression in different V. amurensis tissues, organs and various period of berry were preliminary studied. The major findings are as follows:RNA was isolated from V. amurensis skins and leaves using three methods of extraction. Highly-grade RNA was isolated by using modified CTAB method and suitable to the demands of further molecular biological research. The veraison skins (50% colored) of V. amurensis was used as experimental material, from which the total RNA was extracted. The cDNA was synthesized and the ds cDNA fragment was ligated into the pDNR-LIB vector. The recombinant plasmid was transformed into E.coli DH5a by electroporation. The library qualification evaluation showed:the titer of primary cDNA library was 1.12×106 pfu·mL-1, the titer of amplified library was 2.82×109 pfu·mL-1, the percentage of recombination was about 100%, the fragment size of inserted was 0.5-2 kb. The average insert size was 0.80 kb. The result showed that the cDNA library of V. amurensis veraison skin was constructed successfully with SMARTTM technology.A total of 974 high quality ESTs were obtained from cDNA library of V. amurensis veraison skins. The GenBank accession numbers are GW666520-GW667493. The 974 ESTs were assembled into 710 unigenes. The results of BLAST annotation showed:270 known or putative functional gene,258 unknown gene,182 no hits (maybe new genes). Based on the BlastX annotation and GO analysis,199 ESTs have the cellular component function,313 ESTs were associated with molecular functions, and 341 ESTs with biological process.The full-length cDNA sequences of VAmF3'H, VAmF3'5'H, VAmDFR, VAmLDOX, VAm3GT, VAmOMT, VAmGST4 and VAmOGT genes in V. amurensis were cloned by the technology of RT-PCR and SMARTTM RACE. The GenBank accession number is FJ645766, FJ645767, FJ645768, FJ645769, FJ169463, GU237132, FJ645770, and GU237133. Using bioinformatics analysis, speculated that 8 genes encode 509,508,337,355,456,235,213 and 448 amino acids, with molecular mass of 56.14,56.70,37.50,40.19,50.19,26.35,24.24, and 49.84 kDa. With multiple comparisons to the amino acid sequence of others species related genes, the similarity of each gene and related genes of V. vinifera is more than 90%. On this basis, molecular evolutionary tree of these eight genes and the corresponding genes of other species was constructed, the genetic relationship with related genes of other species was analyzed. Each gene signal peptide prediction results show that the eight genes do not have a signal peptide structure, the various genes were carried out hydrophobicity analysis, preliminary forecast of their secondary structure.Expression of anthocyanin biosynthetic structure genes inⅤ. amurensis young leaves, mature leaves, stems, pulp and skin were preliminary studied. The results of semi-quantitative RT-PCR showed that the expression of VAmF3'H and VAmF3'5'H happened in various period of the skins after flowering and up-regulated expression in veraison. VAmDFR, VAmLDOX and VAmOGT are expressed in each period of skins after flowering, whereas the expression intensity of each period was not significantly different. VAm3GT and VAmGST4 almost have no expression before veraison, and up-regulated expression in veraison period. The expression quantity of VAmOMT is small in two weeks after flowering, then the expression declined and increased at veraison. VAmF3'H, VAmF3'5'H, VAmDFR, VAmLDOX and VAmOGT were expressed in various tissues and organs. However, VAmF3'5'H is expressed in mature leaves at a low level. VAm3GT, VAmOMT and VAmGST4 are expressed in stems, pulp and skins, not in leaves. VAmOMT is expressed in low abundance in the pulp. Northern blot results showed that VAm3GT almost have no expression before veraison, up-regulated expressed at veraison. VAmOGT is expressed at berry maturating.PQE30-Xa-VAm3GT and PQE30-Xa-VAmOGT prokaryotic expression vector was constructed by DNA recombination technology, which was transformed into E.coli 15 expressing 6×His fusion protein. The results showed that the recombination protein expresses at a high level. The molecular mass of two protein purified by Ni-Sepharose FF is 50 kDa and 49.5 kDa. Recombinant protein enzymatic functions and features are being studied.