Gene Cloning And Analysis Of F3’H And Promoter Cloning Of ANS、DFR Of Pansy

Violaxwittrockiana Gams, belonging to the Family Violaceae, is a biennial or perennial herb. It is often cultivated in the open air, and commonly is used in parks,flower beds, the courtyard and the pot. Currently, the international research had been focused on molecular mechanism of anthocyanins, but it is rare for the study on principles of the flower blotch forming, which is an important factor in the visual valuable of plants. To understand the principles of flower blotch forming is of great significance for the expression in the specific parts of plants with artificial pigment control and the creation of new flower types. Pansy is a model plant because of its rich color and solid study base in heredity of flower blotch color. The Pansy was used in this study as experimental materials to measure the overall length of gene F3’H,and constructed the expression vector to lay a good foundation for further plant transformation and expression validation by means of RACE technology. Finally, the principles of pansy flower blotch forming could be seeked through the study on the structural of F3’H gene, then enrich plant anthocyanin gene expression regulation theory. The main results are as follows:1.The methods of extracting total RNA from Pansy petals were optimized and the conclusion showed the improved CTAB was the most efficient method for extracting DNA from Pansy leaves in comparison with CTAB, improved CTAB and SDS method which has highest DNA purity and simples.2.The cDNA which was synthesized with reverse transcription from mRNA and leaf extracted DNA were used as template to clone pansy β-actin gene in order to construct the fragment of599bp and1186bp.The conclusion showed consistency reached95.66%by the homology of amino acid sequence of conservative, This was a further proof that β-actin is highly conserved in higher plants.3. The specific primers were designed according to the conserved sequence of F3’H, mainly using software to splice3’RCAE and5’ RACE gene fragments obtained by the RACR technology,so as to obtain the full-length sequence of F3’ H.Then we took homology comparison through the amino acid nucleic acid and deduced submitted to NCBI. This test validated the sequence belongs to the F3’H gene of Pansy, and the basic characteristics of its protein were analyzed.4. The expression vector in Arabidopsis was successfully constructed through Sma I and Eco RI double enzyme digestion, which insert the F3’H gene into expression vector (pXCS-HAStrep), and then extracted the recombinant plasmid DNA in order to provide the basis of further expression study. 5. Three nested primers were designed using the Tail-PCR technology, according to the conserved sequences of ANS gene and DFR gene of Pansy. Three rounds of PCR reactions were used to explore the cloning method of Pansy flower pigment synthesis key structural genes ANS and DFR upstream promoter. Different size segments of the5’end of the upstream were obtained by agarose gel electrophoresis, in order to do some preparation for the research of further amplification promoter sequences and expression.