Study On Gene Cloning, Transformation, Expression Of MYB Transcription Factor ODORANT1 From Chimonanthus Praecox L

Wintersweet (Chimonanthus praecox L.) is a traditional fragrant plant endemic to China. Because of its strong fragrance, it is regarded as an ideal raw material for producing essential oil. It’s highly valuable to research its cold resistance and ability to produce rich aroma. RNA of flower buds, full-open flowers and inner and middle tepals of Wintersweet were extracted for transcriptome sequencing using Illumina paired-end sequencing technology. According to the annotation, we have identified two unigenes as the candidate genes of floral-related transcription factor ODORANT1. Full length of cDNA of these two genes were obtained by rapid-amplification of cDNA ends technology (RACE) and they were designated as CpODO1 and CpODO2, respectively. By Agrobacterium-mediated transformation method, CpODO1 and CpODO2 genes were transformed into the model plant Tobacco and Arabidopsis thaliana. The expression pattern of CpODO1 and CpODO2 genes from Wintersweet plants were studied through the Real-time PCR(qPCR) method. The main results are as follows.1. CpODO1 encodes an open reading frame (ORF) of 906nt corresponding to a protein of 302 amino acid; CpODO2 encodes an ORF of 1182nt corresponding to a protein of 394 amino acid. Together with multiple sequence alignment analysis, we compared the database using the BLAST program to determine the homology with the transcription factor ODORANT1 of floral regulation. The physicochemical properties and structural features of the protein encoded by these genes have been analyzed as well and the results indicated that both CpODO1 and CpODO2 might be the unstable hydrophobic protein and located in nucleus.2. CpODO1 and CpODO2 were ligased into expression vector PBI121-CpODO1 and PBI121-CpODO2 respectively for genetic transformation by Agrobacterium-mediated method. Both were cloned into Arabidopsis, while only CpODO1 was cloned into tobacco and 10 PCR positive transgenic tobaccos were obtained.3. Real-time PCR (qPCR) technology was applied to detect CpODO1 and CpODO2 gene expression in different organs and flowering periods of H29 and H64. The highest expression of CpODO1 were observed both in flowers of H29 and H64, while the highest expression of CpODO2 was observed in the flower of H29 and steam of H64.