| AP2/ERF transcription factors belong to a huge gene family, which exist in plant kingdom. These proteins are known for its highly conserved AP2 domains composed of 60-70 amino acids. Numerous studies have shown that AP2/ERF transcription factors involved in a variety of biological processes, including the development resistance and secondary metabolism in plant.Salvia miltiorrhiza Bunge, a traditional perennial medicinal plant, is well known for its beneficial effects such as anti-thrombus function, anti-inflammation, anti-tumor, anti-virus, analgesics and so on. It has been widely used in the treatment of cardiovascular and cerebrovascular diseases. Now, many studies have been carried out on genes from S. miltiorrhiza, but little is known about transcription factors, especially for ERFs from S. miltiorrhiza. Based on the analysis of digital expression profiles treated with MeJA or YE, we cloned and characterized SmERF1 (one ERF gene, unigene46389). And then we conducted a basic research of its effects on plant growth and development, stress response and secondary metabolism regulation. The main contents and results of this research are as follows:1. SmERF1 contains a 549 bp ORF, encoding a 182-amino-acid polypeptide. The putative amino acid sequence of SmERF1 had a typical AP2/ERF binding motif, which contained 3 beta turns and 1 alpha helix. Thus, SmERFl may belong to ERF sub-family of AP2 superfamily based on its amino acid sequences. Gene expression pattern analysis showed that SmERFl was significantly induced by MeJA and YE.2. SmERF1 was inserted into pEarleyGate100 with Gateway method for the plant expression vector. Then we generated transgenic positive lines of s. miltiorrhiza and tobacco using Agrobacterium-mediated transformation method. And after detection by qPCR,8 of transgenic s. miltiorrhiza and 4 of tobacco lines with high erxpression levels of SmERFl were obtained. Compared with wild type tobacco, the transgenic tobacco presented many different phenotypes, such as earlier flowering and easy aging of old leaves.3. We had tested the contents of ABA, GA and the expression levels of some key genes involved in ABA, GA biosynthesis in transgenic S. miltiorrhiza and tobacco. Results showed that ABA contents of transgenic S. miltiorrhiza and tobacco are higher than that in the wild type. In contrast, the GA contents are decreased; NtSDR, NtACS and NtACO, which were involved in ABA and ethylene biosynthesis pathways, were expressed highly in transgenic plants; but the expression level of GA20ox which involved in GA3 biosynthesis was decreased. Therefore, we inferred that SmERFl may promote the ABA biosynthesis by regulating the expression of NtSDR. The increase of ABA contents may inhibit the expression of GA20ox and result in the reduction of GA contents. It also may promote the expression of ACS and ACO and cause an increase of ethylene synthesis.4. Thousand kernel weight (TKW) analysis showed that TKW of transgenic tobacco seeds was 0.55-0.63 times heavier than that of wild tobacco. Seed germination experiment showed that the transgenic tobacco seed germinated slower than seed of wild tobacco. According to the results, we speculated that the increase of ABA contents may cause smaller seeds;The slower germination may be affected by seed size or ABA contents.5. With the treatment of salt solution on SmERF1 overexpression lines and the wild lines, the alterations of pathological products and protective enzymes were observed in various lines. The results showed that the growth vigor of SmERF1 overexpression lines was better than that of wild lines after high-salt solution treatment. The contents of proline and SOD, POD activity were higher than wild type. The contents of MDA were decreased compared with wild type. We suggested that SmERF1 can enhance the salt tolerance of S. miltiorrhiza and tobacco through the promotion of ABA biosynthesis.6. The contents of the main active ingredients in transgenic S.miltiorrhiza were detected with HPLC. Results showed that the contents of cryptotanshinone and tanshinone ?A were increased in transgenic line 1,16 compared with wild lines. However, the contents of four main efficacy components we detected was decreased in transgenic line 11. I guessed that SmERF1 may affect the expression of some genes involved in tanshimone biosynthesis by intergrating into S.miltiorrhiza genome.Above all, as a member of ERF transcription factor family, SmERF1 plays an important role in controlling seed size and resisting salt stress, while, SmERF1 may have little contribution to secondary metabolic regulation in salvia miltiorrhiza. |
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