Cloning And Functional Study Of The MYC2 Gene Of Salvia Miltiorrhiza

MYC2, belonging to bHLH transcription factor family, is a core transcription factor in jasmonate (JA) pathway. It plays an important role in plant defense response, regulation of secondary metabolism, growth and developmental process. In recent years, MYC2 genes have been cloned from many plants, such as Arabidopsis thaliana, Lycopersicon esculentum, Nicotiana tabacum, Catharanthus roseus and the function of MYC2 on secondary metabolites synthesis has been studied. For example, MYC2 can influence the biosynthesis of flavonoids by positively regulating the expression of MYB75, EGL3 in Arabidopsis thaliana; NtMYC2 takes part in the nicotine biosynthesis in Nicotiana tabacum; The content of vinblastine and tabersonine significantly declined in MYC2 interference lines of Catharanthus roseus.As a famous and popular herb, Salvia miltiorrhiza is Labiatae perennial herb and the medicinal parts is dried roots and rhizomes. The active compounds of S. miltiorrhiza are divided into two groups, i.e., lipid-soluble tanshinones and water-soluble phenolic acids, which have the functions of protecting vascular endothelial cells, anti-arrhythmic, anti-atherosclerosis, microcirculation, myocardial protection, increasing coronary blood flow, and protecting liver cells, etc. In recent years, S. miltiorrhiza, known as "mode medicinal plants", becomes an ideal research material for plant secondary metabolic pathways. In this study, MYC2 gene was cloned from S. miltiorrhiza and its regulatory functions in S. miltiorrhiza secondary metabolites were studied, which lays a foundation for further understanding of S. miltiorrhiza secondary molecular mechanism of metabolic regulation.In this study, the main contents and results are listed as follows:1. Based on the S. miltiorrhiza transcriptomes and genome survey sequence, the transcription factor MYC2 gene containing 1809 bp open reading frame (ORF) and its upstream 1500 bp promoter region was cloned and named SmMYC2. The amino acids encoding by the SmMYC2 have a high homology with that of other species. The quantitative real-time PCR analysis showed that SmMYC2 was expressed in roots, stems, leaves and flowers of S. miltiorrhiza, and showed higher expression levels in roots and stems. PlantCARE software analysis showed that the promoter region contained a variety of hormones and stress-related cis-acting elements. In addition, Real-time PCR results showed that the expression of SmMYC2 could be induced by methyl jasmonate (MeJA), light, wounding, gibberellin (GA3) and abscisic acid (ABA), etc.2. SmMYC2 overexpression vector pEarlyGate201-SmMYC2 was successfully constructed by gateway technology. S. miltiorrhiza young leaves have been transformed with the method of Agrobacterium tumefaciens-mediated transformation of leaf discs, and 28 transgenic kanamycin resistant lines of S. miltiorrhiza (OEMs) have been obtained on a selective medium. Based on the DNA and RNA identification of transgenic lines of S. miltiorrhiza,3 positive S. miltiorrhiza transgenic lines (OEM-2, OEM-3, OEM-14) with higher expression levels have been selected to detect the content of total phenol and total flavonoids. The results showed that the content of total phenol and total flavonoids increased significantly in the SmMYC2 overexpression lines, which can reach to 1.7～3.5 times and 1.2～2.4 times of the control lines, respectively.3. The content of phenolic acids (rosmarinic acid, salvianolic acid B) and tanshinones (tanshinone I, Cryptotanshinone) of SmMYC2 overexpression lines was detected by HPLC. The results showed that the content of rosmarinic acid and salvianolic acid B in the roots of OEMs transgenic lines were higher compared with the control lines. The content of rosmarinic acid and salvianolic acid B in OEM-3 was 199.0 and 33.8 times of the control lines respectively, but those in OEM-14 have a small rise and only are 12.5 and 2.4 times of the control lines. While there was no significant difference between the content of tanshinone Ⅰ, cryptotanshinone of OEMs and that of the control lines. Furthermore, the key enzyme genes of phenolic acids, flavonoids and lignin biosynthesis pathway in the OEMs and control lines were analyzed through real-time PCR method. The results showed that expression of phenolic acids biosynthesis genes (TAT, PALI, C4H, etc.) and rosmarinic acid biosynthesis gene (RAS1) increased significantly in the OEMs lines compared with the control lines, while there was no significant difference in the flavonoid pathway genes (CHS, F3’H, FLS, etc.) and lignin pathway genes (CCR, COMT).4. CRISPR Cas9 gene editing technology has been tried to mutate gene in S. miltiorrhiza for the first time and gRNA target primer sequence based on SmMYC2 gene sequence was connected with Cas9/gRNA plant expression vector.25 transgenic resistant lines were obtained through Agrobacterium-mediated transformation of leaf discs and in which 20 transgenic lines were confirmed with exogenous fragment by PCR technology. SmMYC2 genes of the 20 transgenic lines were cloned and sequenced to test the editing efficiency of SmMYC2 gene. Although some SmMYC2 gene mutation has occurred in the transgenic lines, there is no mutation or frameshift mutation near the designed positions. The results indicated that Cas9/gRNA plant expression vector can not be successfully used for gene editing of S. miltiorrhiza.The study lay a foundation for the function research of SmMYC2 gene in S. miltiorrhiza. Meanwhile, it provided some reference for the role of SmMYC2 in regulating the secondary metabolic pathways of S. miltiorrhiza.