Isolation And Expression Analysis Of BHLH Homologues In Cineraria And Establishment Of Its Regeneration System

Senecio cruentus L'Herit is one of beautiful perennial flowers, which is flowering from December to next April, belongs to Compositea Senecio. It is an important holiday flower, with a high value in ornamental and economic. Because of its rich color, cineraria is an ideal material for studying of flower color formation mechanism. bHLH transcription factors are important regulators of higher plants, which play important roles in regulation of structural genes in anthocyanin synthesis pathway. There are major results in this study:(1) In this study a stability regeneration system in cineraria was established. We use seeds of five Senecio cruentus'Jester'cultivars with different colors as explants, MS as basal medium to select the optimum concentration of growth regulators for plant regeneration. The results showed that different explants, types of growth regulators and concentration had obvious effect. All the measured traits for leaves explants were superior than petioles. MS+6-BA2.0 mg/L gave the best result for multiplication of shoots. The optimal multiplication of calli and plant regeneration medium were MS+NAA1.0 mg/L +6-BA1.0 mg/L and MS+6-BA2.0 mg/L+NAA2.0 mg/L, respectively, While for rooting medium,1/2MS added with 0.5mg/L NAA was selected. Under favorable conditions we got healthy flowering plant after transplanting. Phenotype test demonstrated that there is no floral characteristics change in regeneration plants from cineraria seeds with five cultivars.(2) In this study, genetic transformation system was discrssed in cineraria. We use the leaves and shoots of pink cineraria as explants, MS+6-BA 2.0 mg/L+NAA 2.0mg/L as medium, edded different concentrations of kanamucin to select the critical concentration of antibiotics. The results showed that the medium edded with 10 mg/L kanamucin can effectively inhibit the differentiation of leaf discs and root development.(2) Using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR), this study isolated two internal segments cDNA from the second development stage in blue cineraria flowers: bHLH-1 (390bp) and bHLH-2 (227bp), which coding 130 and 75 amino acids respectively.(3) The expression patterns of bHLH-1 and bHLH-2 in the first and second developmental stages and different tissues (including roots, stems, leaves and tube flowers) of cineraria with different flower colors were determined for the first time by RT-PCR. The results indicated that these two bHLH transcription factors were both expressed at similar levels in all tissues, which leading to the conclusion that these two bHLH transcription factor regulatory genes were constitutively expressed in cineraria.