Cloning And Functional Initial Characterization Of The Gene Encoding Dihydroflavonol 4-Reductase (DFR) From Lily

Lilium orential’Sorbonne’, one of the world famous cut flower, occupies a large market share in the Chinese cut flower market. If it wants to occupy the highlands of the world flower industry, still needs to constantly innovate to breed of blue and other rare, exotic colors varieties. The formation of flower color is closely related with the kind and content of anthocyanin. Dihydroflavonol 4-reductase gene (DFR) is one of the key genes in the biosynthesis pathway of blue delphinidin. Due to DFR’s specificity of substrate selection, to obtain stable blue lily varieties should first reduction the expression ability of endogenous DFR gene. Therefore, in order to obtain color fade or white flowers of transgenic plants, this study cloned the DFR gene from lily and Analyzed it by bioinformatics methods, then built ihpRNAi plant expression vectors to transformed lily and tobacco, finaly appraised of its function. Several achievements were obtained as follows:1. LoDFRl and LoDFR2 genes were isolated from lily. Two DFR genes were cloned from bud of Lily by RT-PCR and PCR, named LoDFR1 and LoDFR2, both of which contained 1134bp ORF, encoding 377 amino acids, but there are 21 bases difference, and up to 98.2% similarity.2. Using bioinformatics methods, LoDFRl and LoDFR2 genes were analyzed. For the proteins which encoded by LoDFR1 and LoDFR2 genes, their relative molecular weights were forecasted of 42.7kDa and 42.6kDa, the theoretical isoelectric point were 5.87 and 6.25, both the them belonged to the class protein stability. Their N-terminal contained of a 21 amino acid residues highly conserved NADP binding region "VTGASGYVGSWLVMKLLQYGY", and they had a 26 amino acids binding region substrate specificity of "TVNVQEHQMSEYDESSWSDDDFCRRV". Their 134th amino acid was N (ASN) which decided to bind substrate for DHK or DHQ. LoDFRl, LoDFR2 genes had highest homology and closest relationship with the other Oriental Lily LhDFR1, LhDFR2, LsDFR genes, the second is the same branch in Phylogenic tree with TsDFR of Tricyrtis sp. ’Shinonome’ and TfDFR of Tulipa fosteriana.3. Preliminary identification of the function of genes LoDFRl and LoDFR2. First,Using the forward and reverse RNAi fragments (350bp) which were cloned separately from the full-length cDNA of LoDFRl and LoDFR2 genes to construct recombinant pMD 18-Tintron as an intermediate vector, successfully constructed the ihpRNAi expression vectors that contained the RNAi fragments; then, the best callus induction material of Lily was determined to be petiolules and Scales, Kan optimum concentration of resistance to root-resistant callus screening and screening was selected as 100 mg/L. Agrobacterium-mediated transformation was used to transform lily and tobacco. The conversion rate was 7.1% and 15.6%. GUS activity assay showed that pCA-LoDFRl was highly expression in tobacco anthers, petals, style, fruits and seeds expression; Finally, the observation that transgenic lily plants different periods of growth phenotype with no significant difference between the control plants, and changes in gene transfer occurred in the early transformation of tobacco plants leaf morphology, slender and showed green, flowering was delayed for 2-3 days significantly dilute colors, pink corolla into the eaves pale pink. RNAi interference can be determined fragment was integrated into the tobacco genome, the performance of the interference color effects, and there was a certain DFR gene regulation of the tobacco leaf and flower development.