The Function Study Of Salvia Miltiorrhiza Gene SRP And Arabidopsis Homologous Gene AT2G17350

Salvia miltiorrhiza Bunge is a perennial plant which belongs to Labiatae family and widely cultivated in China.As a traditional herbal medicine,it has been widely used in treatment of clinical cardiovascular disease and various inflammatory.Accompanied by the growing demand of S.miltiorrhiza and gradual reduction of its wild resources,the improvement of its quality has been becoming increasingly concerned.With the development of science and technology,the researchment of molecular biology in S.miltiorrhiza has been carried out gradually,the platform of gene function research has established firstly,which provides a good foundation for secondary metabolites and biomass-related genes in S.miltiorrhiza.In this study,a new highest abundance unigene was screened in the early establishment of EST database in S.miltiorrhiza,the gene expression and characteristics were investigated.Combining with the bioinformatics and mutants of Arabidopsis homolog gene AT2G17350,by using the method of over-expression and RNAi,we obtained a series of transgenic plants of S.miltiorrhiza and A.thaliana,and had studied the difference of phenotypes and the determination total protein.With yeast two-hybrid and BiFC methods,it was clarified the function of the genes for the foundation for further study.The main results were as follows:1.A unigene composed by 376 ESTs for 3.33%were found by screening the EST library of S.miltiorrhiza which sufferred with stress treatments,which named SmSRP(Salvia miltiorrhiza stress-response protein).By sequence alignment,unknown function was found,although it was widespread in plants.SmSRP had two introns while the Arabidopsis homolog AT2G17350 had only one intron.SmSRP encoded 117 amino acids,SmSRP was rich in arginine,glutamic acid and lysine.SmSRP and AT2G17350 can be expressed using prokaryotic expression,which did not have the signal peptide,transmembrane domain and miRNA prediction loci,a-helical was its main secondary structure,accounting for 72.65%.2.By real-time-quantitative PCR,the expression of SmSRP was investigated,and it was found that SmSRP responded to a variety of biological and non-biological stress.Cold and dehydration could stimulate gene expression in a short period,but suppressed in a long period.A long time of darkness and high temperatures treatment could positively regulated the expression of SmSRP,which did not responded to rhythm.In the early period of seed germination,the expression of SmSRP was up-regulated and a high level in cotyledons and leaves.Also,by real-time-quantitative PCR and online database,the determination of Arabidopsis the homologous gene in seed germination and embryo development,AT2G17350 had the highest expression in the cotyledon and was not in response to various stress treatments.3.By DNA Walking,the 5 'flanking sequence of SmSRP was obtained.The sequence of AT2G17350 promoter got from online databases.The cis-elements were found and analyzed,including four endosperm-specific response cis-elements and part of stress and hormonal response cis-elements,only few cis-elements in Arabidopsis.By analysis of deletion promoter,the higher GUS activities of the promoters were obtained as the sequence became longer in transgenic plants.In the analysis of transfor:med Arabidopsis,the flowers had highest expression levels,especially in the receptacle,the pistil styles and stamen flaments pollen and other tissues of leaves and buds.4.By Agrobacterium-mediated transgenic approaches,SmSRP-RNAi plants of S.miltiorrhiza were obtained and transplanting to greenhouses,the aboveground biomass of which was significantly less than the WT,the underground partdifference is not significantly.The contents of total proteins reduced in SmSRP-RNAi plants,as the same phenomenon in Arabidopsis heterozygote,homozygote plants can not be obtained from heterozygote selfed,and the separation ratio of generation(heterozygote:WT)was slightly less than 2:1,No obvious phenotypic differences were found,also in the transgenic over-expression and RNAi Arabidopsis,but the contents of total proteins were increased in SmSRP-overexpression/AT2G17350-overexpression of Arabidopsis,the opposite result was obtained in SmSRP-RNAi of Arabidopsis.5.By yeast two-hybrid,Arabidopsis homologous gene AT2G17350,with AT3G25980(MAD2,spindle assembly checkpoint protein),AT4G02150(a protein transporter and tie proteins IMPA-3),AT4G16143(IMPA-2),AT5G65940(CHY,3-hydroxybutyrate iso-acyl-CoA hydrolase)may have interactions,which was confirmed by BiFC.It were speculated that AT2G17350 disrupted in the embryo development by affecting mitosis and meiosis,or CoA energy of ATP hydrolysis and restore,or the influence of transporter or binding of certain proteins in Arabidopsis,to apply in the growth and development of Arabidopsis.