Technical Research Of Regulation Of Tanshinone Synthesis Based On SmMYB1 Transcription Factor

Salvia mitiorrhiza(S.miltiorrhiza),a perennial herb of Salvia Lamiaceae,is used to treat cardiovascular and cerebrovascular diseases.The content of natural tanshinones is low,and the chemical synthesis method is complex and easy to cause environmental pollution.At present,increasing the content of tanshinones by genetic engineering has become a research hotspot.In this study,the SmMYB1 transcription factor of S.miltiorrhiza screened in the early stage of the laboratory was used as the research object.Based on transgenic technology and Agrobacterium rhizogenes-mediated genetic transformation,a hairy root induction culture system with exogenous SmMYB1 gene was successfully constructed.Overexpression SmMYB1 transgenic hairy root were obtained,and their biomass,related gene expression and tanshinone content were detected and analyzed.The effect of SmMYB1 on the biosynthesis and accumulation of tanshinone was preliminarily explored.The main results are as follows :1.The full-length gene coding sequence of SmMYB1 was cloned from S.miltiorrhiza,a total of 1071 bp,encoding 356 amino acids.2.By comparing SmMYB1 with 20 highly conserved MYBs transcription factor sequences in Arabidopsis and other species at the amino acid level,it was found that SmMYB1 was closely related to Pf MYB1-like in Perilla frutescens.3.The expression characteristics of SmMYB1 gene in S.miltiorrhiza were analyzed.The results of tissue expression pattern analysis showed that the expression of SmMYB1 gene in various tissues of S.miltiorrhiza was significantly different,with the highest in leaves,followed by roots,stems and flowers.The results of inducible expression pattern analysis showed that exogenous Me JA and SA treatment significantly promoted the expression of SmMYB1 gene in root tissues of S.miltiorrhiza,reaching a high point at 24 h,which was 1.32 times and 2.86 times of the control.Compared with Me JA induction,SmMYB1 response was more significant after SA induction.Subcellular localization showed that the green fluorescence of SmMYB1 gene appeared in the nucleus.4.A total of 11 SmMYB1 overexpression positive hairy root were obtained by Agrobacterium rhizogenes Ar.Qual-mediated genetic transformation of S.miltiorrhiza leaf disc method,and the transformation efficiency was 36.7 %.After liquid culture,five overexpression lines of SmMYB1-OE3,SmMYB1-OE5,SmMYB1-OE8,SmMYB1-OE16 and SmMYB1-OE20 were obtained.It was found that overexpression of SmMYB1 significantly increased the biomass of hairy roots of S.miltiorrhiza,and the dry weight of SmMYB1-OE16 line was 2.46 times that of the control.5.The expression levels of key enzyme genes in tanshinone biosynthesis pathway were detected by q RT-PCR.The results showed that overexpression of SmMYB1 could significantly up-regulate the expression levels of SmDXS,SmGGPPS,SmKSL1,SmCYP76AK1 and SmCYP71D375.6.The contents of cryptotanshinone and tanshinone IIA in SmMYB1 overexpression hairy roots were detected by UPLC method.The results showed that the contents of cryptotanshinone and tanshinone IIA in SmMYB1 overexpression lines increased significantly.The highest content of cryptotanshinone in SmMYB1-OE16 lines was 0.75 mg/g,and the content of tanshinone IIA in SmMYB1-OE8 and SmMYB1-OE20 lines increased most significantly to 0.59 mg/g and 0.58 mg/g.In summary,this study cloned the SmMYB1 transcription factor in S.miltiorrhiza,and used genetic engineering methods and mature S.miltiorrhiza hairy root culture system to obtain transgenic hairy root materials.It was found that SmMYB1 as an important feedback regulator affected the biosynthesis of tanshinone,promoted the growth of S.miltiorrhiza hairy roots and the accumulation of tanshinone substances in hairy roots.