Molecular Cloning And Function Analysis Of Genes 4CL And CURS In Curcuma Longa.L

Curcuma longa L.is a perennial herbaceous plant of Zingiberaceae. It is widely distributed in tropical and subtropical regions. The curcumin derived from the dry tuber in Curcuma longa L.is thought to be the main active ingredient. Curcumin has the functions of antioxidation, antitumor, protecting the liver and kidney and important economic value. It has become the focus in the development and utilization of medicinal value.4CL is in the branch point of the benzene propane metabolic pathway of the curumin biosynthetic pathway. It catalyzes the cinnamic acid to generate cinnamoyl-CoA and make the reaction toward the direction of the generation of curcumin; CURS is in the end of the curumin biosynthetic pathway. It catalyzes the ferulic acid-diketone-CoA and ferulic acid-CoA to generate curcumin; In this study, we use homologous cloing and RACE to get the gene sequence of 4CL. We also get the CDS sequence of CURS by homologous cloing. With the fluorescent quantitative PCR(qRT-PCR) technology, we get the expression pattern of 4CL and CURS in different tissues ?stress condition(different concentration of NaCl) and allogenic material(different concentration of SNP) to futher study on the relationship between the two genes and the biosynthesis of curcumin. Finally, we build the plant expression vector of CURS to estabilish the foundation to get the transgenosis plant of CURS. The main research results are as follows:1. We obtained the 4CL gene sequence by homologous cloing and RACE. Results showed that the sequence fragment length is 2109 bp, encoding 574 amino acids.2. Amino acid homology analysis indicated that the 4CL gene of Curcuma longa L.has high homology with Zingiber officinate?Musa acuminata?Morus notabilis?Humulus lupulus?Cannabis sativa and Cinnamomum osmophloeum;According to the analysis of bioinformatics, the 4CL is an unstable, hydrophilic, un-transmembrane protein located in the cytoplasm;Multiple sequence alignment indicated that the amino acid sequence of 4CL shared 8 same actives, 7 same AMP binding sites and 3 same CoA binding sites with other plants.3. Using homology cloing, we get the CDS sequence of CURS. The length of the CDS of CURS is 1173 bp which encodes 390 amino acids.According to the analysis of bioinformatics, the CURS is a stable, hydrophilic, un-transmembrane protein located on the membrane of mitochondria. It's a ?-fold homodimer protein. It has the typical structural domain of Cond_enzymes gene family with three active sites, eleven product binding sites, eight malonyl-Co A binding sites and thirty one chalcone synthase dimer interfaces.4. In this study, we use real-time fluorescent quantitative PCR method to detect the expression pattern of 4CL and CURS in different tissues, different stress and different allogenic material. Results show that the highest gene expression of 4CL is in the root and least in the rhizome, while little difference between the leaf and the flower. Under low concentration of NaCl(25 g·L-1),the highest gene expression of 4CL is found. The lower gene expression of 4CL is found when treated with high concentration of NaCl. The expression of 4CL gradually decreases with the increase of the concentration of SNP; Results show that the highest gene expression of CURS is in the flower and least in the root, while little difference between the leaf and the rhizome. Under the stress of NaCl, the expression of CURS has the highest gene expression when the concentration is 25 g·L-1. Under the effect of SNP, the expression of CURS has the highest gene expression when the concentration is 0.05 mmol·L-1, while the least expression is 1.0 mmol·L-1.5. Add the Nde?and EcoR?in the 5' end of the forward primer and reverse primer respectively. Then we amplificated the CDS sequence of CURS and successfully imported it into the plant vector named PRI-101 AN. Then, we put the plant vector into the agrobacterium tumefaciens named GV3101 to get the plant expression vector. Finally, we using the plant expression vector to infect the leaf discs to get the transgenic tobacco.