Functional Study Of The SmDELLAs Gene Of Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge,a perennial medicinal herb belonging to Salvia family Lamiaceae,is known as a model medicinal plant because of its small genome and efficient transformation system.Roots and rhizomes are medicinal parts of S.miltiorrhiza and the effective ingredients are mainly tanshinone and salvianolic acids.Because of its wide range of pharmacological effects,it can be used to treat coronary heart disease,myocardial infarction,and other cardiovascular and cerebrovascular diseases.In recent years,the research of S.miltiorrhiza mainly focuses on improvinge the percentage of phenolic acids and tanshinone by using genetic engineering technology.DELLA transcription factor involved in the GA3 regulation pathway in plants,and previously study suggested that DELLA genes participate in the biosynthesis of secondary metabolites in various species.Based on the precious research of SmDELLA,his project continue to explore the molecular mechanism of SmDELLA regulating the content of secondary metabolites in S.miltiorrhiza.SmDELLA interference lines have been obtained in the early stage,after that we obtained the SmDELLA over-expression strains of S.miltiorrhiza.The content of secondary metabolites and the expression levels of key enzyme genes in the biosynthetic pathways of secondary metabolites were studied,and its the molecular mechanism of SmDELLA regulating the content of tanshinone and phenolic acid in S.miltiorrhiza were discussed.The main results of this study are as follows:1.We constructed the SmDELLA over-expression vectors and obtained the SmDELLA over-expression transgetic plants by Agrobacterium-mediated leaf transformation.After the level of DNA and RNA verification,four SmDELLA transgenic positive plant were obtained.OE-DEL1-1,OE-DEL1-2,OE-DEL1-5,OE-DEL2-1,OE-DEL2-2,OE-DEL2-3,OE-DEL3-1,OE-DEL3-2,OE-DEL4-1,OE-DEL4-3 strains with the higher expression were selected for the further study the specific functions of SmDELLA in S.miltiorrhiza2 The content of total phenolic acids and flavonoids in SmDELLA were determined.Compared with the control group,the contents of total phenolic acids of OE-DEL1-1,OE-DEL1-5 and OE-DEL2-2 were increased by 2 times,1.75 times and 2.5 times,respectively.The contents of total flavonoids were also significantly increased,OE-DEL1-1 and OE-DEL2-2 were about 2 times and 2.3 times of the control group,respectively.We also determined,the content of salvianolic acid SmDELLA transgenic lines.The results showed that the rosmarinic acid content in OE-DEL1-1,OE-DEL1-2,OE-DEL2-1 and OE-DEL2-3 increased by 2.1 times,2 times,1.7 times and 2 times,respectively.The salvianolic acid B content in OE-DEL1-2,OE-DEL2-2 and OE-DEL4-3 were 1.7 times,2 times and 2 times as compared with the control group.In the SmDELLA interference strains,the content of salvianolic acid B in ir-D2-R8,ir-D2-R12,ir-D3-R4,ir-D4-R4,and ir-D4-R5 were generally 1/2 of the control,while the rosmarinic acid,as the precursor of salvianolic acid B had no significant change.These results indicated that SmDELLA had a positive regulatory effect on the biosynthesis of total phenolic acids,total flavonoids and salvianolic acid B substances.3.We analyzed the expression levels of key enzyme genes in the synthesis pathway in SmDELLA transgenic lines of S.miltiorrhiza qRT-PCR results showed that SmPAL1,SmC4H1,Sm4CL1,SmTAT1 and SmRAS1 in the phenolic acid synthesis pathway were significantly up regulated in the SmDELLA over-expression strain of S.miltiorrhiza,while the opposite results were observed in the interference strain,indicating that the SmDELLA gene regulates the synthesis and accumulation of secondary metabolites by regulating the expression of important enzyme genes in the synthesis pathway.Our results suggested that SmDELLA plays a positive role in regulating the synthesis of salvianolic acid.4.The content of gibberellin in the S.miltiorrhiza and the expression of key enzyme genes in gibberellin synthesis pathway were detected.The gibberellin content in the over-expression strains had no significant change.While in the interference strains,it showed an increasing trend compared with the control group.The detection of the key enzyme gene expression in the gibberellin synthesis pathway showed that the negative regulatory genes SmGA2ox5 and SmGA2ox11 were higher in the overexpression lines than in the control,but generally decreased in the interference lines.SmKO,SmKAO1,and SmKAO2,which play positive regulatory roles,showed a decreasing trend in SmDELLA over-expression strains,and were highly expressed in interference strains.The results indicated that SmDELLA has a negative regulatory effect on gibberellin synthesis.5.The yeast two-hybrid experiment and the two-molecule fluorescence complementation experiment(BiFC)were did to explore the SmDELLA protein-protein interaction.The results proved that four SmDELLA proteins can interact with transcription factors including SmJAZs,SmMYB97,SmMYC2 and other S.miltiorrhiza to regulate secondary metabolism,which showed that SmDELLA proteins can interacts with transcription factors in other regulatory pathways to regulate downstream target genes and affect the accumulation of secondary metabolites in S.miltiorrhiza.6.To further explore the function of DELLA genes in S.miltiorrhiza,this study used the luciferase reporter gene experiment to analyze the effect of SmDELLA1 on the direct activation of key enzyme genes SmPAL1 and SmTAT1 promoters by SmMYB97.We found SmDELLA1 could reduces the level of target gene SmPAL1 activated by SmMYB97 but promoted the activation effect of SmMYB97 on the target gene SmTAT1 the luciferase reporter gene experiment showed that SmDELLA1 regulateds the accumulation of salvianolic acids by regulating the transcription factor SmMYB97,which can directly activate target genes.