Cloning And Expression Analysis Of CpPds From Chimonanthus Praecox Link

Carotenoids is the most abundant terpenes pigment material in nature, widely exists in bacteria, fungi, and plants. So far more than750carotenoids have been found in nature. Carotenoids exercises its functions as an important constituent of the photosynthetic membranes of photosynthetic organisms. They play an important role in photosynthesis. Carotenoids is also the main pigments of many flowers and fruits. Porter and Lincoln have first proposed the carotenoids biosynthetic pathway of higher plants in1950, now almost all of the genes of this biosynthesis process have been isolated and identified. The phytoene dehydrogenase is the major rate-limiting enzyme in the biosynthesis of carotenoids, it can catalytics the colorless phytoene to dehydrogenate, generate lycopene, and then convert to colored carotenoids. The phytoene dehydrogenase is located on the chloroplast thylakoid membrane, plays a special role to protecte on chloroplasts. When the gene of encoding protein is silent, the chloroplast lose the protective effect of this enzyme, leading to the degradation of chlorophyll, chlorophyll-rich leaves and other organizations mottled, faded, yellowing even become white, this phenomenon is called photobleaching. This phenotype is very easy to distinguish, and can be directly observed. So the phytoene dehydrogenase genes are often used as reporter genes in the virus-induced gene silencing system in recent years.In this paper, we cloned a hytoene dehydrogenase gene from Chimonanthus praecox named CpPds (Genbank accession number:KC814175). Bioinformatics analysis, real time quantitative PCR, over-expression in tobacco was used to study the function of CpPds gene. The main results in our rasearch are as follows:1. Cloning and bioinformatics analysis of CpPds geneBased on the methods of degenerate PCR and RACE technique, the full-length cDNA of phytoene dehydrogenase gene named as CpPds was cloned from Chimonanthus praecox. The full-length cDNA of CpPds gene is2242bp contaning a1716bp open reading frame (ORF),encoding a polypeptide of571amino acid. The molecular weight of the encoded protein is64.28kD, predicted theoretical isoelectric point is7.94. BLAST analysis on NCBI showed amino acid sequence of the protein encoding by CpPds gene shares a high similarity with other reported PDS protein and belong to the NAD binding protein. Higher homology of the protein sequence manifested in various species,which can explain phytoene dehydrogenase genes as functional genes which are relatively conserved during evolution. The NJ phylogenetic tree was constructed between the CpPDS protein sequences and other PDS protein sequences, the result showed that CpPDS protein is between dicotyledonous and monocotyledonous plants in evolution, and the genetic distance is near with the two monocots plants PDS protein. Maybe the Chimonanthus praecox PDS protein is more conservative, and is in inferior position in evolution. There are no signai peptide and transmembrane domain in the deduced amino acid sequence, containing38threonine (Thr) phosphorylation sites, which belongs to the relatively stable hydrophilic proteins. Subcellular localization prediction showed that the protein is most likely to play a role in the mitochondrial matrix.2. Transcriptional level analysis of CpPds geneReal-time quantitative PCR analysis showed that:The CpPds gene exhibited different transcription levels in different tissues and had a higher expression in flowers than other tissues. Along with the flowers gradually open to wilt, the gene expression gradually increased. It is suggested that the CpPds gene may associated with flower senescence, in the flower wilting process, regulates the accumulation of carotenoids. The expression level of the external petal, middle petal, inner petal and the stamens are similar and quite higher except the pistil, indicating that CpPds is involved in the development process of flower. Speculate that the gene of CpPds may play an important role in the carotenoid accumulation and coloration of flowers.The plant over-expression vector of CpPds named pCAMBIA2301g-CpPds was consrructed and transformed into tobacco plants using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens GV3101(including pCAMBIA2301g-CpPds recombinant plasmid). Transgenical tobacco plantlets were obtained, in which the transgenic lines were identified by Gus (β-glucuronidase) histochemical staining and PCR analysis. Furtherly, in order to validate the fuction of in lycopene synthesis pathway, we measured the lycopene contents of transgenic tobacco and control plant(wildtype tobacco). In subsequent molecular detection by qPCR, the high expression level of CpPds in transgenic tabacco proved that improved lycopene contene was positively co-related with the expression of CpPds.