Cloning And Expression Characterization Of The Athocyanin Biosynthesis Key Genes In Eggplant (solanum Melongena L.)

Eggplant (Solanum melongena L.) is an important vegetable in China. In the recent years, there are more and more researches focused on the anthocyanins of eggplants, especially the types and the antioxidant effects. So, the further research focused on the anthocyanin biosynthesis pathway and the reasons of the huge differences of color and anthocyanin content among different eggplant genotypes will support the breeding research and health care development.The research materials were two eggplant genotypes with significant phenotypic differences, Yunnan Long Purple Eggplant (YLP) and Yunnan Round White Eggplant (YRW). After screening 14 anthocyanin related genes in the fruit skins of YLP and YRW at the products mature stage, expression differences were found at the dihydroflavonol 4-reductase (SmDFR) and the anthocyanin 5-O glucosyltransferase (Sm5GT), respectively. Then we cloned the full length of SmDFR cDNA and Sm5GT cDNA, respectively. SmDFR cDNA length was 1503bp, the coding region was from 87 to 1235, the deduced SmDFR protein contained 383 amino acids. Comparing the SmDFR cDNA sequence between YLPSmDFR and YRWSmDFR, two mutant sites were found. The mutation in 632 site was a synonymous mutation, whereas, the mutation in 792 was a missense mutation, changing the amino acid in the 236 site from lysine to glutamate. The deduced amino acid sequence contained the typical NADPH binding motif and the substrate binding motif. At the key site 145, which decided the activity of the enzyme, SmDFR was aspartic acid. In all purple tissues of YLP, the SmDFR expression level was significantly higher than YRWSmDFR. The southern blotting analysis showed the SmDFR was a single copy in both YLP and YRW genomes. Through the prokaryotic expression, we tested the activity of the fusion proteins of YLPSmDFR and YRWSmDFR using the substrates of dihydrokaempferol, dihydroquercetin and dihydromyricetin. The results showed that the activity of SmDFR to dihydromyricetin was higher than dihydrokaempferol and dihydroquercetin, furthermore, the acitivity of YLPSmDFR to dihydromyricetin was higher than YRWSmDFR. These results indicated that the SmDFR may be the key enzyme for the anthocyanin difference. The activity of YRWSmDFR may be weakened by the mutation of amino aicd 236 site.Sm5GT cDNA length was 1789bp, the coding region was from 67 to 1479, the deduced Sm5GT protein contained 470 amino acids. The deduced amino acid sequence contained the typical substrate binding motif. The expression of Sm5GT was found in all the test tissues in YLP and YRW. Only in the purple petals and purple skins at the products mature stage, the expression level of YLPSm5GT was higher than YRWSm5GT. These results indicated that Sm5GT may contain a wide substrates range.