Cloning, Prokaryotic Expression Of VvmybA2Genes And Its Expression Pattern During Grape Berry Development

Flavanols and anthocyanins are important to grape fruit quality (especially to the fruit color) and wine quality(such as color, flavor, taste, nutritional value, etc.). In these years, the structural gene of flavonoids biosynthesis have been cloned, and its expression characteristerics were studied, but its regulation mechanism was reported scarely. In the present study, the full length of MybA2was cloned from grape berries (Vitis vinifera L.Cabemet Sauvignon) via RT-PCR, and the recombinant protein was obtained by prokaryotic expression, and a polyclonal antibody against grape berry MYBA2was prepared; Meanwhile, using the grape berries (Cabernet Sauvignon) as materials, he expression of the grape fruit during development of the gene MybA2were stueided by Western blot hybridization and fluorescence quantitative techniques, respectively; Finally, the expression of the genes involved in flavanols and anthocyainin biosynthesis (LAR1, LAR2, DFR, ANS, ANR and UFGT) were studied. The main results are as follows:1. A798bp bp MybA2cDNA was obtained from grape berry (Cabernet Sauvignon) as expected. Bio-analysis showed that grape MybA2gene has encodes a protein of265amino acids with a predicted molecular mass of40kDa. Protein sequence analysis show that grape MYBA2no tran membrane region does not contain a signal peptide, MYB A2is a non-secreted protein.2. The recombinant plasmid pET-mybA2was highly expressed with0.5mM isopropyl-β-D-thiogalactoside (IPTG) induction at30℃in Escherichia coli BL21(DE3) pLysS cells.Applied Ni-NTA affinity purification recycling MYBA2fusion protein. The purified fusion protein was used as the antigen to prepare ANS polyclonal antibody, which has high sensitivity and specificity.3. Using the Real-Time PCR and Western-blot technique to detect VvmybA2gene expression in different stages of grape fruit development in the level of transcription and translation. The results showed that:VvmybA2in the early development of fruit to keep low level of transcriptional expression, when the grapes began to turn the color, VvmybA2transcriptional expression increased dramatically and reached the maximum at80d after flowering; the expression of MybA2in the level of translation after veraison was higher than the early preiod, and expression reached the maximum level of transcription at90d after flowering.