The Identification Of Salvia Miltiorrhiza GRAS Transcription Factor Family And The Study Of Gene Function Of DELLA Subfamily

In order to adapt to the environment better,plants will accumulate instantaneously a large number of plant hormones under biotic stress and abiotic stress,such as abscisic acid(ABA),salicylic acid(SA),and jasmonic acid(JA),which will result in the regulation of synthesis and accumulation of the secondary metabolites through signal transduction pathways.Studies have shown that methyl jasmonate(MeJA),gibberellin(GA3),and SA can be used as exogenous elicitors to promote the synthesis and accumulation of salvianolic acids,terpenoids and flavonoids in Salvia miltiorrhiza.As one of the specific plant transcription factor,DELLA(GRAS gene family)is a key factors link between the environment and hormones as well as hormones and hormone signaling pathways.It also plays an important role in the hormone regulatory network,while participating in the synthesis of the secondary metabolism of plants.The research on SmDELLA gene function has significance for secondary metabolism mechanism of Salvia miltiorrhiza.Therefore,SmDELLA genes as the research object,were to elaborate their function by combining genetic engineering techniques with the results of bioinformatics analysis,expression pattern analysis.The main results and conclusions of this study as follows:1.A total of 34 GRAS gene family members were identified in S.miltiorrhiza.It was found that all GRAS proteins have the C-terminal GRAS domain.The results of phylogenetic tree clustering together with GRAS members of Arabidopsis thalian showed that the SmGRAS genes were divided into 11 subfamilies.The results of the gene structure and protein motifs of SmGRAS gene family are consistent with the results of phylogenetic analysis.In order to study the function of SmGRAS gene in S.miltiorrhiza,the cis-acting elements of SmGRAS gene promoter sequence was analyzed firstly.It was found that the SmGRAS gene generally have methyl jasmonate(MeJA),gibberellin(GAs)and salicylic acid(SA)response elements;and then analyzed the expression pattern of SmGRAS gene in various tissues and organs and under different treatment,such as the MeJA,GA3 and SA treatment,the results showed that five SmGRAS genes may regulate secondary metabolic pathways of S.miltiorrhiza.2.In this study,the 4 SmDELLA gene was cloned and the green fluorescent protein(GFP)fusion expression vector pEarleyGate103-SmDELLA was constructed by using the Gateway system.Using gene gun transient transfection of onion epidermis method,we found that the SmDELLA protein was mainly located in the nucleus.3.The yeast expression vector pGBKT7-GW(BD)-SmDELLA was constructed by using Gateway technology,and the toxicity and self activation of four SmDELLA proteins were verified,the results showed that SmDELLA protein was non-toxic to yeast strain AH 109,and the study showed that SmDELLA 1 protein had no self activation,while SmDELLA2-3 protein had.Yeast two-hybrid experiments showed that SmDELLA 1 protein can interact with SmJAZs and SmMYC2,which was similar to Arabidopsis thaliana.However,the SmDELLA 1 protein can also interact with the SmMYB3 6 protein,a negative regulator of the secondary metabolic signal pathway of S.miltiorrhiza,which was different with Arabidopsis,the results suggested that SmDELLA 1 was involved in the synthesis of salvianolic acids signal pathway.4.To further study the function of 4 SmDELLA gene in S.miltiorrhiza,we used the Agrobacterium tumefaciens-mediated leaf disc transformation method to transfect the leaves of S.miltiorrhiza.The results of DNA and RNA level detection showed that the positive interference transgenic lines of SmDELLA were as follows:D1-R1,D1-R8,D2-R3,D2-R4,D3-R2,D3-R3,D4-R1,these transgenic lines can be used for subsequent experiments.And the content of total phenolic acids and total flavonoid in the SmDELLA interference transgenic lines was detected,the results showed that the contents of total phenolic acids and total flavonoids in the SmDELLA interference lines were lower than those in the wild type(WT).Therefore,we speculated that the SmDELLA gene may be a positive regulator of the biosynthetic pathway of total phenolic acids and total flavonoids in S.miltiorrhiza.