| Salvia miltiorrhiza Bunge, a well-known medicinal plant, is broadly planted in China. As one of the traditional Chinese medicine, its root (called "danshen" in Chinese) has been widely used in China, Japan and the United States for the treatment of menstrual disorders, cardiovascular diseases, 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 tanshinones, and the other is water-soluble phenolic acids including rosmarinic acid, salvianolic acids, lithospermic acid, and 3, 4-dihydroxyphenyllactic acid (Danshensu). In view of their notable pharmacological activities and the conventional use of herbs by decocting with water, the phenolic acids have attracted more and more research attentions recently. Previous studies on other species have suggested that most of the water-soluble constituents of S. miltiorrhiza are derivated from caffeic acid which is synthesized via phenylpropanoid pathway. In order to elucidate the mechanism of the biosynthesis of the water-soluble compounds of S. miltiorrhiza, phenylalanine ammonia-lyase, which is the first key enzyme of phenylpropanoid pathway, was chosen for gene cloning and function analysis by RNAi. We also preliminary discussed the relationship between the expression of PAL gene and the development and metabolism of S. miltiorrhiza. The main results were as follows:1. The entire sequence named SmPAL1 which was 2758bp long was isolated using DNA walking technology. The Genebank accession number was EF462460. The SmPAL1 gene consisted of two extrons and one intron of 622 bp whose position was the same as those reported in other plants. There was consensus sequence in the intron/extron border. Homology analysis showed SmPAL1 shared more than 80% identities with the PAL sequences reported in other species. Southern blot analysis indicated that PAL was a small multigene family and there were at least two members of PAL gene in S. miltiorrhiza.2. Totally, 1.0 kb 5' flanking region of SmPAL1 gene was obtained by DNA walking. The transcription start site and TATA box were predicted. Besides, analysis revealed several putative cis-elements in this region, such as box1 and box2 which were conserved among promoters of genes for the phenylpropanoid pathway, G box, W box, I box, MYB recognition site and ABA responsive element like sequence. Based on this, the expression pattern of SmPAL1 gene was analyzed by real-time quantitative PCR. The results indicated that SmPAL1 gene expressed in all S. miltiorrhiza organs but most highly in leaf. Besides, the expression level of SmPAL1 gene was induced by various environmental factors such as mechanical wounding, ABA and dehydration. However, dark treatment reduced the transcription level of SmPAL1 gene. The effect of low temperature on the transcription level of SmPAL1 gene was not obviously.3. We constructed the recombinant vector pQE30- SmPAL1 and transferred it into E. coli M15 for protein expression. SDS-PAGE analysis revealed that pQE30- SmPAL1 expressed a recombinant protein whose molecular weight was about 77 kDa, a size in good agreement with that predicted by bioinformatics method. Enzyme activity assay suggested that the recombinant protein had high PAL activity. The optima temperature of the enzyme was 60℃and the optima pH in borate buffer was 8.7.4. A 217 bp fragment, located in the 3'end of the coding region of the SmPAL1 gene was chosen for RNAi construct, which was introduced into S. miltiorrhiza by Agrobacterium tumefaciens-mediated gene transformation, and 25 transgenic lines were obtained by PCR analysis. The transformation efficiency was 69.4%. The expression level of SmPAL1 gene was markedly down-regulated in all transgenic lines, whereas SmPAL2 behaved differently in different transgenic plantlets.5. Compared with the control plants, most of the transgenic lines showed abnormal phenotypes, such as dwarfism and delayed root formation. Besides, the leaves and petioles of transgenic plants appeared perished or etiolated spots, and a few individuals died finally during culturing. Moreover, histochemical analysis of lignin by phloroglucinol showed the reduction of lignin content in transgenic plantlets.6. The content of total phenolics was analyzed with Folin-Ciocalteu reagent. The content of total phenolics was decreased by 30%～80% in transgenic lines. Water extracts of SmPAL1-silenced lines were chosen for HPLC analysis. Compared with the control plants, the level of caffeic acid reduced significantly in all transgenic plantlets. However, the content of salvianolic acid B was slightly higher.
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