Cloning And Functional Analysis Of Promoter Of GHS Gene And The Related Transcription Factors In Chimonanthus Praecox

Winter Sweet (Chimonanthus praecox L.) is a unique winter flowering plants in China. Although its main color is wax yellow, there are also red varieties. Creating new vireities of different flower color, exploring the cause of color difference and the mechanism of blossom at low temperature are the main concerns of horticulturists. In this study, based on the C. praecox Var. intermedius (H29) transcriptome library, the promoter of chalcone synthase (CHS) gene was cloned by FPNI-PCR, which was designated CpCHSP29. Bioinformatics analysis showed that this sequence contains some typical promoter functional elements. Using the promoter5’end sequence deletion method, we constructed a CpCHSP29deletion expression vector. Then we verified the function of CpCHSP29through instantaneous transformation and Agrobacterium mediated stable transformation, respectively. Based on the CpCHSP29key cis acting element and transcriptome analysis, the complete CDS sequence of the related transcription factors were cloned, named CpMYCl, CpMYC2, CpICE11and CpICE12, respectively. Their structure and function were predicted by bioinformatics techniques. Main results in this study are as follows:1. The815bp fragment upstream of C. praecox Var. intermedius CHS gene was cloned, which named CpCHSP29. It contains some typical cis-elements, such as TATAbox and CAATbox. In addition, it also contains cis acting elements related to stress response:ABRELATERD1, MYCCONSENSUSA, WBOXNT, WRKY710S, and light regulatory elements, such as GATABOX and CACGTGMOTIF, and ABA responsive elements:EBOXBNNAPA, ABRERATCAL. There are also several other cis elements. Bioinformatics analysis showed that the promoter was related with stress, light and abscisic acid induction.3. Using the5’end deletion technique, we construct the expression vector for transient expression. The GUS staining experiments showed that the expression of CpCHSP29can be activated in flower organs. S5expressed barely in the flower organs in Arabidopsis thaliana. The lacking of the key parts of the promoter may result in lackness of the function in this promoter.4. The transgenic CpCHSP29Arabidopsis was gained, which lays the foundation for further functional verification. T3generation plants Gus staining showed that CpCHSP29could be expressed in the whole plant, and is differently regulated at the developmental stages. In stigma and leaves, the expression is strongly induced. In roots, its expression is the strongest at flowering stage, The promoter is regulated by the low temperature and light.5. There are several MYC transcription factors, CpMYC1, CpMYC2, CpICE11and CpICE12, were cloned, which may interact with CpCHSP29cis elements. The full-length of CpMYC1CDS is2372bp, encoding660amino acids; the full-length of CpMYC2CDS is2221bp, encoding627amino acids; The full-length of CpICE11CDS is1868bp, encoding525amino acids; The full-length of CpICE12CDS is1488bp, encoding455amino acids. All of these transcription factors contain typical bHLH domains. Bioinformatics method are used to predicted their properties, structure and function.