Molecular Cloning And Functional Analysis Of The Two BHLH Transcription Factors About Flower Color From Chimonanthus Praecox L.

Wintersweet(Chimonanthus praecox L.) is a specific plant in china which blossoms in winter. It is popular in China due to its blossom season and special floral color. Because of the different pigments in outer and inner tepals of Chimonanthus praecox var. intemedius, they show yellow and red color, respectively. Therefore, C. praecox is an ideal material to study the expression and regulation of the genes related to flower color. Meanwhile, the research on the molecular mechanism of flower color could lay the foundation for new color variety breeding of the C. praecox.Based on the transcriptome of the H29 and H64 of Chimonanthus praecox, two b HLH transcription factors related to floral color, Cpb HLH1 and Cpb HLH2, were cloned. The expression level of the two transcription factors in two varieties H29 and H64 were identified by using q PCR technology. Through Agrobacterium-mediated transformation method, Cpb HLH1 and Cpb HLH2 genes were transformed into the model plant Nicotiana tabacum and Arabidopsis thaliana to observe phenotypes. The main results are as follows:1. Two b HLH transcription factors Cpb HLH1 and Cpb HLH2 were cloned. By using bioinformatics analysis method,We found that the two transcription factors Cpb HLH1 and Cpb HLH2 were homologs to b HLH transcription factors Vv MYC in Vitis vinifera、Ph AN1 in petunia、TT8、EGL3 in Arabidopsis thaliana which were related to floral color. The protein secondary and tertiary structure were analyzed.2. Real-time PCR(q PCR) technology was applied to detect Cpb HLH1 and Cpb HLH1 gene expression in different organs and flowering periods of H29 and H64. It turned out that the two genes may relate to the formation of flavonols.3. The overexpression vectors of Cpb HLH1 and Cpb HLH2 gene were constructed and transferred into Nicotiana tabacum and Arabidopsis thaliana. At last obtained 39 tansgenic tobacco plants and 20 tansgenic T1 generation arobidopsis plants for phenotypic observation, In tansgenic arobidopsis, the color of the stem turned into purple, then predicted that the Cpb HLH1 and Cpb HLH2 gene regulate the accumulation of the anthocyanin in arobidopsis.