Transcriptional Profiles Of WRKY Family Genes And Their Putative Roles In The Biosynthesis Of Secondary Metabolites In Salvia Miltiorrhiza

Salvia miltiorrhiza is an important medicinal plant, as well as a model plant for traditional medicinal plant research. Therefore, the research on regulatory mechanism of secondary metabolites biosynthesis and accumulation in S. miltiorrhiza have far-reaching significance and broad application prospects. WRKY transcription factors(WRKY TFs) are one of the largest TFs families in the regulation of secondary metabolism, development, stress response in plants. However, little was known about the types, functions and regulation of secondary metabolites biosynthesis with SmWRKYs in S. miltiorrhiza. In our research, a systematic analysis about the types, functions, expression, and the association with secondary metabolite accumulation of SmWRKYs in S. miltiorrhiza were analysis by methods of bioinformatics, molecular biology and plant chemistry. The study of these WRKYs not only improved the understanding of SmWRKYs, but also lay the foundation for further exploring the function of SmWRKYs and its regulation mechanism for the synthesis of secondary metabolites. The main results were as follows:1. 69 SmWRKYs were indentified from S. miltiorrhiza transcritome. The range of the length of encoded amino acid, isoelectric points(Pi) and molecular weights(Mw) were 129~706, 4.76~9.9 and 15.2~36.2 k Da, respectively. The core structures of SmWRKYs’ not only include a common WRKYGQK domain, but also have WRKYGEK and WRKYGKK. Based on the number of WRKY core domine and the type of zinc-finger, WRKY was divided into three groups(GI, GII and GIII). GII also can be further divided into five subgroups, namely GIIa, GIIb, GIIc, GIId, GIIe. In salvia miltiorrhiza, there are 13 SmWRKYs belong to GI, 46 SmWRKYs belong to GII and 9 SmWRKYs belong to GIII. To further study the structural features of SmWRKYs, the software MEME 5.0 were used to predicted their conserved amino acid sequences. the results indicated different groups of SmWRKYs have specific conserved sequences--Motif 1 and motif 2, which indicated that this structure was the conserved domains of SmWRKYs. The results indicated that the evolution of SmWRKY were divergent and implied that the family genes may be involved in the regulation of many physiological and biochemical processes.2. After cultivated for 18 days, S.miltiorrhiza hairy roots were treated with MeJA(100μmol·L-1). Elicitation was performed on the 18 th day after treatment. The hairy roots were harvested from the culture medium on 0h, 0.5h, 1h, 2h, 4h, 8h, 12 h, 1d, 2d, 3d, 6d, 9d post-treatment. High performance liquid chromatography(HPLC) was used to monitor the content of active compounds in S.miltiorrhiza. And the expression level of key genes in phenolic acids and tanshinones biosynthesis pathways were detected by qRT-PCR. The heatmap diagram was constructed according to gene expression. The results showed most SmWRKYs respond to MeJA. Expression patterns were divided into three categories: upregulated, downregulated, and either upregulated or downregulated expression at different time points. At the same time, there were four tanshinones compounds(tanshinone I, tanshinone IIA, dihydrotanshinone I and cryptotanshinone) and three phenolic acids(rosmarinic acid, salvianolic acid B and caffeic acid) were tested. Results showed the contents of these compounds were changed in a significant level after induction with MeJA. Except for tanshinone I, the accumulation of chemical compounds increased with time. From the beginning of 6d, the accumulation of the treatment group was significantly higher than control group. The correlation analysis of gene and secondary metabolites was performed by SPSS 19.0. The result specualted that 7 SmWRKYs(SmWRKY36, SmWRKY45 etc.) may be involved in the biosynthesis of phenolic acids. 9 SmWRKYs(SmWRKY3, SmWRKY12 etc.) may be involved in tanshinones biosynthesis and 7 SmWRKYs(SmWRKY1, SmWRKY7 etc.) may be involved in the biosynthesis of these two groups of compounds.3. qRT-PCR was used to determine the expression of secondary metabolism key enzymes genes and SmWRKY in different flowering stages, and then contructed the Heatmap. Results showed that 74 gene expression profiles could divided into three kinds of expression patterns. 7 genes belongs to group I expression pattern, and they were predominantly expressed in stems and leaves. So we speculate they may be involved in photosynthesis. Many genes expression pattern belonging to groups II and the genes expression pattern were diversity, which maybe related to the diversification of the WRKY fuction. SmWRKY47 and SmWRKY60 were belong to group III expression pattern. They didn’t express in some tissues. Meanwhile, we found the expression pattern of WRKY in the same taxa is similar. For example, there are respectively 61.%, 37.5%, 44.5%, 66.6% and 55.5% members of I, IIb, IIc, IIe and III of WRKY were clustered together in gene expression spectrum, which maybe the similarity of WRKY sequences in the same kind. In addition, in the gene expression spectrum, there are respectively 50% and 42.8% key enzyme gene of the pathway of tanshinones and phenolic acids was also similar. This may be caused by the similarity of the function of these genes. 4. We have already obtained full-length SmWRKY65 sequence from S. miltiorrhiza transcriptome datebase. After cloning and sequencing, we found the coding region of SmWRKY65 is 867 bp, encoding 288 amino acids. SmWRKY65 belongs to the IIa subgroup of WRKY family because of a conserved WRKY domain and a C2H2 zinc finger domain in SmWRKY65 protein, which consistent with the expected results. To investigate the further characteristics of SmWRKY65, we cloned its coding region and got the over-expression vector. Then, SmWRKY65 was transferred into Arabidopsis thaliana by floral dip method, and obtained the positive transgene plant, which lay a foundation for further study on the function.