Cinnamoyl Coenzyme A Reductase And Caffeic Acid Transferase Reductase Inhibition By Rna Interference On A Single Parameter Of Lignin Deposition And Soluble Secondary Metabolites

Salvia miltiorrhiza Bunge, a well-known medicinal plant, is broadly planted in China. As one of the traditional Chinese medicine, its root has been widely used for the treatment of cardiovascular diseases, menstrual disorders, and the prevention of inflammation. According to the pharmacological investigations, the active constituents of S. miltiorrhiza are divided into two main groups:one is lipid-soluble fractions such as TanshinoelⅠ, TanshinoelⅡA, Cryptotanshinone, Salvia methyl, Dihydro Tan, and the other part is water-soluble phenolic acids including 3,4-dihydroxyphenyllactic acid (Danshensu), lithospermic acid, salvianolic acids and rosmarinic acid. But recent researches have focused on the phenolic acids, due to their notable pharmacological activities and the conventional way of herbs by extracting with water. Previous studies on other species have suggested that Salvia miltiorrhiza water-soluble which were synthesized via tyrosine-derived pathway and phenylpropanoid pathway respectively.In order to elucidate the mechanism of the synthesis of the water-soluble compounds of S. miltiorrhiza, key enzymes involved in phenylpropanoid pathway, caffeic acid O-methyltransferase and cinnamoyl-CoA reductase, were chosen for gene cloning and function analysis by RNAi. We also primarily discussed the relationship between the expression of COMT and CCR genes the Synthesis of lignin and metabolism of S. miltiorrhiza. The main results were as follows:1. The 296 bp and 340 bp fragments, located in the 3'end of the coding region of CCR and COMT genes were chosen for RNAi investigation, which was introduced into S. miltiorrhiza by Agrobacterium tumefaciens-mediated gene transformation, and 18 and 21 transgenic lines were obtained by PCR screening.2. Compared with the controls, histochemical analysis of lignin by phloroglucinol showed the reduction of lignin content in transgenic plantlets.3. Lignin monomer content when using GC-MS analysis CCR-RNAi and COMT-RNAi. The results show that lignin monomer content were reduced in varying degrees in differents transgenic plants.4. Detect the total lignin in transgenic plants, detected the Klason lignin and acid lignin. The results show that:in the CCR-RNAi, Klason lignin was decreased and acid lignin was increased; in the COMT-RNAi, Klason lignin was decreased and most of them acid lignins were increased,small was decreased. The result related to the gene expression. 5. Water extracts of Smpall-silenced lines were chosen for HPLC analysis. The CCR, COMT roots and leaves, compared with control plants, shows difference.1. The 296 bp and 340 bp fragments, located in the 3'end of the coding region of CCR and COMT genes were chosen for RNAi investigation, which was introduced into S. miltiorrhiza by Agrobacterium tumefaciens-mediated gene transformation, and positive transgenic lines were obtained by PCR screening.2. Expression level of lignin was detected by histochemical analysis in transgenic plantlets.3. Lignin monomer content was tested by using GC-MS analysis.4. The total lignin, including the Klason lignin and acid lignin in transgenic plants, were detected UV spectrophotometry and measured the relative content percentage.5. Water extracts of the CCR, COMT roots and leaves were detected with HPLC analysis.In conclusion,18 and 21 transgenic lines were obtained by PCR screening. After RNAi suppression, lignin content decreased faintly. Phenolic acids level increased diverdely in leaves and roots. This study would undoubtedly pave the way for further investigation of the secondary metabolism of phenylpropanoid pathway and improving the water-soluble phenolic acid production.