Influence Of BHLH Homologous Gene Expression On Flower Coloration Of Chrysanthemum

Chrysanthemum(Chrysanthemum x morifolium Ramat.) is a traditional Chinese flower. People noticed the rich color variation of chrysanthemum since long time ago, and have bred out all color serials except blue color. Traditional breeding has some limitations, while the regulation of transcription factors as means of molecular breeding for the blue chrysanthemums may open up ideas. There have not been reported about transformation of regulation gene and attaining transgenic lines of color changed in recent studies. Therefore, transform homologous bHLH gene and study its effect on the anthocyanin biosynthesis of chrysanthemum flower is of great significance. In this study, we analysed of expression patterns of homologous bHLH gene in anthocyanin synthesis pathway of chrysanthemum; while induced bHLH homologous gene Lc into chrysanthemum. The difference on gene expression patterns involved in anthocyanin between wild type and transform lines was compared. It is helpful to explain the molecular mechanism of transcription factor in flower coloring of chrysanthemum. This study achieved the following results.(1) Using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) method, isolated one internal bHLH cDNA fragment from chrysanthemum'Riqietaohong'. The sequence comparison showed that the fragment was homology with anthocyanin-related bHLH genes from other species in GenBank. Semi-quantitative RT-PCR results showed that the transcript of this gene was only found in the flower organs in chrysanthemum.(2) The expression patterns of CHS, CHI, F3H, F3'H, DFR, ANS in all six structural genes and MYB, WD40, bHLH regulatory genes in different developmental stages of'Riqietaohong's pink flowers and its white flowers mutant were determined by RT-PCR. The results showed that:compared with the wild type, the transcription signals of key structure genes and regulatory genes were decreased to varying degrees in the mutant inflorescence of white flowers. Expression pattern of bHLH gene in the mutant is not normal, almost undetectable transcripts.(3) Introduced maize Lc into chrysanthemum by Agrobacterium-mediated method. Experiment of 2883 explants, and finally obtained 15 positive lines by PCR amplification, with transformation efficiency of 0.52%. RT-PCR showed that 2 lines were detected the Lc transcript. Compared to the wild type, transgenic lines had deeper color and CIE L*a*b* test results showed that color appears red shift and blue shift. HPLC analysis of ray florets in transgenic pigment content and composition, the results showed a significant increase of anthocyanin content, but no new pigment formation was detected.The main conclusions of this study are as follows:bHLH played an important role as an anthocyanin biosynthesis gene in chrysanthemum flower coloring; Heterologous expression of bHLH gene Lc can increase anthocyanin content in chrysanthemum.