Molecular Cloning,Function Analysis And Genetic Transformation Of Key Enzyme Genes Of Curcumin Biosynthetic Pathway

Curcuma longa L.is a perennial herbaceous plant of Zingiberaceae. It is widely distributed in tropical and subtropical regions. The curcumin,a fat soluble phenolic pigment extracted from the dry tuber in Curcuma longa L.,is thought to be an natural food additive and the main active ingredient. Curcumins have the functions of anti inflammation, anti-tumor, anti oxidation, protecting kidney and liver. It has become one research hotspot in the world now. Currently, majority of studies are paid attention to the extraction processes and pharmacological activitives of curcumin, but some key enzymes in the biosynthesis pathway of curcumins have not been isolated.C4H is in the branch point of the benzene propane metabolic pathway of the curumin biosynthetic pathway. It catalyzes the trans-cinnamic acid to generate p-coumaric acid and makes the reaction toward the direction of the generation of curcumin. DCS is in the end of the curumin biosynthetic pathway. It catalyzes the ferulic acid diketone-CoA and ferulic acid-CoA so as to further catalyze the formation of curcumins. In this study, homologous cloing and RACE was used for obtaining the gene sequence of C4 H. We also get the CDS sequence of DCS by homologous cloning. With the fluorescent quantitative PCR(qRT-PCR) technology, we get the expression pattern of C4 H and DCS in different tissues, stress conditions(different concentration of NaCl) and allogenic material(different concentrations of SNP). Finally, the obtained the plant expression vector of CURS gene infect tobacco tissue culture seedlings to get the transgenosis plants of CURS by agrobacterium-mediated transformation method. The main research results are as follows:1. Using homology cloing, we get the CDS sequence of DCS. The length of the CDS of DCS is 1173 bp which encodes 390 amino acids. According to the analysis of bioinformatics, the DCS protein is an unstable, hydrophilic, un-transmembrane protein, which is located on the other cell organelles. Besides, it’s a β-fold homodimer protein. It has the typical structural domain of cond_enzymes gene family with three active sites, eleven product binding sites, eight malonyl-CoA binding sites and thirty one chalcone synthase dimer interfaces.2. A pair of degeneracy primer for cloning the conserved sequence of C4 H gene of Curcuma longa is schemed out by using bioinformatics software. Sequencing and NCBI blast analysis prove that C4 H gene is amplified. we use RACE to get the 5’ and 3’ partial gene sequence of C4 H. Results showed that the sequence fragment length is 1842 bp, encoding 446 amino acids. Bioinformatics analysis showed that the deduced turmeric C4 H molecular weight is 51.72 kD, the multipeptide hydrophilic, and presumably protein is unstable, with cytochrome P450 domain. The C4 H gene of Curcuma Longa has very close relationship with Musa acuminate by constructing phylogenetic trees.3. In this study, we use real-time fluorescent quantitative PCR method to detect the expression pattern of C4 H and DCS and C4 H gene in different tissues, different stress and different allogenic material. Results show that DCS gene is expressed in various tissues. The highest gene expression of DCS is in the rhizome and least in the flower. Under low concentration of NaCl(25 g·L-1),the highest gene expression of DCS is found. The lower gene expression of DCS is found when treated with high concentration of NaCl.The highest gene expression of C4 H is in the root, followed by leaves, while little difference between the flower and the rhizome. Under the stress of NaCl, the expression of C4 H has the highest gene expression when the concentration is 75 g·L-1. the lowest gene expression when the concentration is 100 g·L-1. Under the effect of SNP, different concentrations of the SNP are able to improve the the expression of the C4 H gene. The expression of C4 H has the highest gene expression when the concentration is 0.1 mmol·L-1.4. CURS gene was successfully transferred into tobacco tissue culture seedlings by agrobacterium mediated method, and the transgenic tobacco plants were verified by RT-PCR.