Cloning And Functioal Analysis Of CpCPC In Chimonanthus Praecox

Transcription factor is a kind of DNA binding protein which specifically interacts with cis-acting elements in the promoter region of eukaryotic gene.The transcription factors MYB gene family is the biggest gene family in the plants.The transcription factors in MYB gene family have a common MYB structural domain. According to the number of the MYB structural domain, MYB gene family is divided into three subfamilies,the single domain-MYB,R2R3-MYB,and R1R2R3-MYB. The previous research shows that the functions of MYB gene family are regulating the morphogenesis of plant cell,regulating the Flavonoids metabolism of plant,and participating in the reaction to the plant hormones.The CPC gene belongs to the single domain-MYB subfamilise.Until now,most research on CPC gene focuses on the defferenition of epidermal cell.Transgenic plants overexpressing CPC had more root hairs and fewer trichomes than normal.And some other researchs show that the CPC plays an important role in regulating flavonoid biosynthesis.One CPC gene was cloned by randomly cloning and sequencing, based on the cDNA library constructed from Chimonanthus Praecox flower. Further the full sequence of CPC was developed by5’RACE,named CpCPC..The full-length cDNA of the CPC gene was acquired through Degenerate PCR and RACE PCR technology,and named CpCPC.And the bioinformatics, eukaryotic expression, and real-time quantitative PCR were used to analyze its potential biological functions. The main results are as follows:1. Molecular characteristics of CpCPCBased on the methods of degenerate PCR and RACE technique, the full-length.cDNA of CPC was cloned from Chimonanthus praecox.The full length of CpCPC cDNA sequence is640bp, with an open reading frame (ORF) of228bp encoding a putative polypeptide of75amino acid residues.It cloned1700bp secquence used DNA template. This sequence contains three intron used sequence analysis, the intron belong to GT-AG type. Sequence alignments shows that the large protein was persenting in the chloroplast, no signal peptide, composed with52.63%random coil and46.05%α-helix, and there are9serine phosphorylation sites,2threonine phosphorylation sites,and1tyrosine phosphorylation sites.2. Transcriptional expression of CpCPCIt is showed that the expression level of CpCPC in tender leaves is higher than that in roots, stamen, and pistil by Real-time fluorescence quantitative PCR.And there is a higher e xpression level in outer perianths and medial perianths.In the developmental stages of flower, the expression level of CpCPC in bloom period and wither periodis are more abundant than that in other periods.3. The overexpression of CpCPC gene in tobaccoThe plant over-expression vector of CpCPC named pCAMBIA2301g-CpCPC was constructed and transformed into tobacco plants using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens GV3101(including pCAMBIA2301g-CpCPC recombinant plasmid).Further, in order to validate the fuction of the CpCPC gene in flavonoids synthesis pathway, we measured the terpenoid content of transgenic tobaccoes and control plant (wildtype tobacco).In subsequent molecular detection by qPCR, the high expression level of CpCPC gene in transgenic tobaccoes proved that the content change of flavonoids substances was negatively related to CpCPC gene.Root hairs of transgenic tobacco overexpressing CpCPC were observed using a stereomicroscope and photographed.We found the ectopic expression of CpCPC also affected root hair distribution in transgenic tobacco.