Cloning And Expression Analysis Of Cinnamate-4-hydroxylase Gene From Caragana Korshinskii Kom

The phenylpropanoid pathway is the specific secondary metabolism pathway in plants, and belongs to one of the three plant’s secondary metabolism pathways. In vascular plants, the phenylpropanoid’s final product is used to synthesize the lignin monomer, and the flavonoid which plays an important role in pigment formation, medicinal ingredients, and the interaction between microbes and plants. Cinnamate4-hydroxylase, which has an important function and catalyzes the second step in phenylpropanoid pathway, to be specific, it converts the trans-cinnamic acid into p-hydroxyl cinnamic acid (p-coumaric acid). In this research, the cinnamate4-hydroxylase encoding gene was cloned for the first time from Caragana Korshinskii Kom and its primary functions were analyzed. The concrete conclusions are as following:1. The cinnamate4-hydroxylase encoding gene was cloned using RT-PCR and RACE technology from C. Korshinskii Kom. The full length cDNA sequence and gDNA sequence were both submitted into GeneBank with the registration number assigned as HQ829860. According to the bioinformatic analysis, the cDNA of the CkC4H gene contains one complete ORF, and encodes505amino acids with the molecular weight at about57850.1D. The structure domain analysis indicates that this protein consists of signal peptide, transmembrane domain, and the typical characters of cellular pigment P450domain. The full length gDNA sequence contains three exons and two introns.2. The expression of the CkC4H gene in different tissues of C. Korshinskii Kom was detected by real time qRT-PCR analysis. The results showed that CkC4H was highly induced in root, while slightly lower in stem, and was much less induced in leaves. After wounding, the transcript of CkC4H accumulated in both leaves and stem, and reached its maximum level around4hr after treatment. The induction level is higher in leaves than in stem.3. The binary vector carrying the CkC4H gene,35S::CkC4H, was constructed and transformed into wild type Arabidopsis Columbia ecotype. Totally8homozygous T3transgenic lines were selected. Expression analysis by qRT-PCR revealed that the transcription level of CkC4H increased differentially in all homozygous lines checked.