Cloning Of VwANS, VwF3’H And Expression Analysis Of VwANS, VwDFR In Pansy

Pansy is an annual or a perennial flower of the genus Viola in the family Violaceae. Its cultivated hybrid called as garden pansy is an important garden flower, which has a wide range of flower colors, displays large showy face markings, blooms early with blooming period extending to summer. Currently, there are many researches about its structural genes involved in anthocyanin synthesis. Pansy has become an ideal plant for flower blotch research due to its good research background about genetics and anthocyanin composition. However, there were few researches about the principle of pansy flower blotch forming. In this research, yellow pansy with black blotch was used as researching material. It was the first research to clone anthocyanin synthesis structure genes F3’H and ANS. The expression levels of those genes in different time and parts were systematically analyzed by using the technique of quantitative real-time PCR. Meanwhile, transgenic Arabidopsis plants were also used to research the functions of those genes. Construction of DFR and ANS expression vectors, particle bombardment of pansy petals and rose petals were used to study the transient expression. These researches have laid a good foundation for adjusting the pansy flower colors. Major research results are as follows:(1) RT-PCR, homology-based cloning were used to get F3’H and ANS conservative fragment sequences, four full-length cDNA sequences of the ANS and F3’H genes were cloned from the garden pansies for the first time, named respectively as VwF3’H1, VwF3’H2, VwF3’H3, VwF3’H4and VwANS, by designing specific primers and using PCR and RACE technique, Prediction of protein hydrophobic displayed that protein encoded by the gene thereof were hydrophilic, and protein encoded by VwF3’H had transmembrane region, protein encoded by the gene VwANS had no transmembrane region.(2) Quantitative real-time PCR was used to study the expression characteristics of VwF3’H and VwANS genes. The results showed that:among the different tissues of pansies (roots, stems, leaves, flowers), VwF3’Hl, VwF3’H2, VwF3’H3, VwF3’H4and VwANS mainly express in flowers and VwF3’H4only expressed in flowers. During the different developmental stages of pansy petals, expression levels of four VwF3’H genes in non-blotched part in stage Ⅲ, Ⅳ, Ⅴ were obviously higher than in the blotch, with the highest level in stage Ⅳ. Starting from the stage V, gene expression levels of VwF3’H gene declined obviously, indicating that the gene was expressed mainly in the non-blotched part of pansy petals. We speculated that VwF3’H may be the key genes to determine the flower non-boltch forming according to the research result, and VwF3’H4may be specific expression in pansy petals.(3) Transgenic Arabidopsis plants overexpression of VwF3’H1and VwANS genes were obtained, transforming those two genes into Arabidopsis with Agrobacterium-mediated transformation.(4) No color change was observed in flower transformants by transient expression of VwDFR and VwANS genes in pansy and rose petals using particle bombardment.