Study On Rapid Propagation And Establishment Of Genetic Transformation System Of Salvia Miltiorrhiza

Dan shen(Salvia miltiorrhiza Bunge)has being medicine for a long history.One kind of important secondary metabolites derived from S.miltiorrhiza is tanshinone which belongs to diterpenoids,modern medical research has shown that tanshinones possess high medicinal value.At present,the acquisition of tanshinones mainly depends on the extraction from the root of the cultivated S.miltiorrhiza,which can not be produced on a large scale by means of hairy root culture and microbial production.The regulation of related genes in plants to improve the content of secondary metabolites is a common technical method.In the related genes of tanshinone metabolism synthesis,3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR)is located upstream of the mevalonate pathway(MVA),while SmGGPPS1,SmKSL1,and SmCYP76AH1 are close to the downstream of the tanshinone metabolic pathway.The up-regulation of these synthase genes can increase the yield of tanshinone compounds.Therefore,the present study selected S.miltiorrhiza used in medicine as the material,and the primers were designed according to the known gene sequences in National Center of Biotechnology Information(NCBI)databases.The genes SmGGPPS1,SmKSL1 and SmCYP76AH1 of the tanshinone metabolic pathway were cloned,and the obtained genes were ligated into the pCAMBIA3301 vector carrying the herbicide bialaphosyl resistance gene(Bar),pCAMBIA1301 vector carrying the HMGR gene of the Staphylococcus aureus and the hygromycin resistance gene were subjected to genetic transformation mediated by Agrobacterium tumefaciens GV3101 to obtain a transgenic plant.Through the detection,the positive plants with successful genetic transformation are obtained.It is hoped that these genes can be used to provide a basis for subsequent experiments.Some of the results that have been obtained so far are as follows:(1)Through the collation of the use of hormones in all stages of S.miltiorrhiza tissue culture,the rooting culture medium and the one-step seedling culture medium of were explored with reference to public available information.The rooting medium(MS+0.4 mg/L NAA)was obtained,which could better promote the rooting of S.miltiorrhiza buds and stem segments,the one-step seedling culture medium MS+1.5 mg/L 6-BA+0.5 mg/L NAA+0.1 mg/LIBA.The above,the two medium can provided convenience for the genetic transformation of Salvia miltiorrhiza.(2)The SmGGPPS1,SmKSL1,and SmCYP76AH1 genes were obtained by reverse transcription of S.miltiorrhiza RNA using primers designed according to the gene sequence and the three genes were integrated into the expression vector for genetic transformation.Positive plants with anti-herbicide gene Bar and S.miltiorrhiza genes were obtained by identification,PCR analysis of transgenic plants revealed that three plants transgenic with empty expression vector of pCAMBIA3301-linker2 and three plants transgenic with SmKSL1 gene were obtained,five plant transfected with the SmGGPPS1 gene and three plants transfected with the SmCYP76AH1 gene.There are seven plants with hygromycin resistance and HMGR genes.