Cloning And Expression Analysis Of Structural Genes Involved In Anthocyanin Synthesis Of Pansy

Viola×wittrockiana Gams, a kind of important potted plants, belongs to the Family Violaceae. Currently,there are many researches about structural genes involved in anthocyanin synthesis, but it is litte for the principle of the flower blotch forming, which is an important ornamental trait. Pansy is an ideal plant for flower boltch research due to its good research background about genetics and anthocyanin composition. This research try to analyze the principle of flower blotch forming using yellow pansy with black blotch as researching material, according to the anatomical structure and anthocyanin composition of the petals. Then6anthocyanin synthesis structural genes were cloned by homologous cloning method, and their expression levels in different stages of flower development between the blotched area and non-blotched area of petals were investigated through Real-time PCR. The main results as follows.(1)The epidermal cell morphology of the non-blotched of petals was consistent with the blotched area. The upper epidermis cells were conical but the dorsal epidermis were oblateness. The anthocyanin of the blotches was accumulation in the upper epidermal cells, with no anthocyanin in the non-blotches. It showed that the forming of blotches of pansy was due to plentiful anthocyanin in the upper epidermis cells.(2)Analysis of anthocyanin composition of pansy flower by HPLC-MS showed that the non-blotched area of petals does not contain anthocyanin, but two kinds of components were detected in blotched area. Their mass spectrometry signals were m/z=03.0258and m/z=287.0316, the characteristic mass peak of cyanidin and delphinidin. The result indicated that the anthocyanin composition in boltched area of pansy composed of cyanidin and delphinium.(3)The pH differential method was used to determine the anthocyanin content in different development stages of flower petals. Beause blotches of petal have not yet formed in stage I, so it dose not contain anthocyanin. The anthocyanin content reached the peak value in stage V, up to7.37mg/g, then it droped down gradually. The experimental result showed that the flower development in stage Ⅱ-Ⅴ of pansy were the critical periods for anthocyanin accumulating for the forming of blotch.(4) The conserved sequence of six genes of CHS, CHI, F3H, F3’H, DFR and ANS were obtained by RT-PCR.Then RACE was used to obtain the full length gene of CHS (1481bp), CHI(975bp), F3H(1361bp), DFR(1340bp)and gene fragment of F3’H (1105bp), ANS (887bp).(5)The expression of anthocyanin synthesis structure gene like CHS, CHI, F3H, F5’H, DFR and ANS in different flower development stages was analyzed by Real-time PCR. The result showed that the expression levels of the6genes in stage Ⅰ-Ⅴ were higher than other stages, and then droped down rapidly in stage Ⅵ-Ⅶ. The petal dose not coloring in stage Ⅰ, it may due to the low expression levels of CHI and ANS. The expression levels of F3H、F3’H and F3’5’H of blotches were high in stage IV-V that is the critical periods for forming folwer blotch. And the expression levels of DFR and ANS of blotches in stage Ⅳ-Ⅴ were very high, but do not express in non-blotched area. We speculated that DFR and ANS may be the key genes to determine the flower boltch forming according to the research result.