| The key factor determining the quality of Salvia miltiorrhiza Bge is the authentic character of Salvia miltiorrhiza,and the genetic mechanism of Salvia miltiorrhiza Bge could slove the prolem of Salvia miltiorrhiza’s quality.This study was on the basis of GVT,which was found in the early reseach.Designing of specific primers,cloning the full-length of SmGGPPS from different regions,analysis and comparing of genuine of variation types,constructing of prokaryotic expression system,analysis the "GVT "of Salvia miltiorrhiza effect on the regulation of secondary metabolism in the proces by GS-MS technology,and then provide some guidance in the analysis of Salvia miltiorrhiza genuineness genetic mechanism.GGPPS has the function of regulating carbon flow,and is the key enzyme for its biosynthesis.It plays a key role in plant growth and development,fruit ripening,quality formation and stress resistance.The reseach was on the basis of the prophase research.Producing SmGGPPS polyclonal antibody,establish plant GGPPS detection system,the results will have a direct effect on plant GGPPS,provides a key method of secondary metabolism in ideas,provide valuable research on secondary metabolism.1.Obtain the full length and analysis of SmGGPPS gene and analysis of "GVT"The full length SmGGPPS is 1095bp,encoding a total of 364 amino acids,its encoding protein molecular weight is about 39 kDa.The isoelectric point is 6.13.The residues in the negative charge carrying are 44 residues,carring a positive charge is 39.Hydrophobic hydrophilic coefficient is 0.046,the protein has a certain degree of hydrophobicity.The stability coefficient is 38.51,indicating that the protein is stable.The fat coefficient is 98.16.The signal peptide may be located in 1-30 sites,the protein is likely located in chloroplast transmembrane region.The analysis results showed that the protein may be located outside of the membrane.The secondary structure showed that the protein structure was alpha helix 45.33%,and the irregular curl,extension chain and beta fold were 30.22%,18.13%and 6.32%in turn.Prosite domain of SmGGPPS protein prediction results show that the protein has a terpene synthase domain family(IPR008949,Isoprenoid synthase domain superfamily),polyprenyl synthetase(IPR000092,Polyprenyl,synthetase)of polyprenyl synthetase(IPR033749,polyprenyl and synthetase conserved sites,conserved and site)are located in the protein 149-165 and 282-194 interval.The results suggested that SmGGPPS protein belonged to the Trans IPPS HT enzyme system under Isoprenoid Biosyn C1 superfamily.Phylogenetic tree showed that Salvia miltiorrrhiza and scabrous larynx had the closest relationship.There are 8 sites of "GVT" in the SmGGPPS functional gene.Henan and Sichuan regions in the 120 bp site and 285 bp site showed "G" type,"GVT",at 126 bp locus showed a clear "A" type "GVT" in Hubei,respectively at 126 bp loci and 189 bp loci,284 bp loci and 894 bp loci showed obvious "G" type "GVT",in the 285 bp loci showed "A" type "GVT",showing "G" type "GVT" on the site,in Shanxi,Sichuan region and Hebei region in 189 bp loci and 284 bp loci showed "C" type "GVT",in 285 bp loci showed "G" type "GVT" in the site,Shanxi,Sichuan areas in turn in 195 bp loci and 285 bp loci showing a marked "G" type"GVT",in addition,the region in 705 bp loci and 894 bp loci showed "T" type "GVT,Inner Mongolia,In Hubei,Shandong,and Henan,the "G" type "GVT" was presented at 894 bp.2.Construction of the prokaryotic expression systemPositive clones were obtained and transformed into DH5α,extraction of plasmid was digested,pet of prokaryotic expression system was connected by T4.Most appropriate induced conditions are,induced by temperature is 28℃,the induction time 8h,IPTG concentration of 0.4 mmol-L-1,OD600 was 0.8.After some verifications,the protein was inclusion body protein and the optimal concentration was 90mM eluted with imidazole.3.Verification of catalytic function Sichuan of SmGGPPS and analysis of catalytic effect in different producing areasThe SmGGPPS gene catalyze isopentenyl pyrophosphate and farnesyl phosphate into geranyl geranyl pyrophosphate.Different areas Salvia miltiorrhiza has different kinds of SmGGPPS genotypes,showing different catalytic effect.In different areas,the strongest catalytic effect areas was Shandong,Hubei,Inner Mongolia,Henan region region Hebei areas are diminished in turn.4.The acquisition of polyclonal antibodyThe specific polyclonal antibody was obtained by immunizing New Zealand rabbits and rats by traditional classical immunization.5.Effectiveness,purification and Western-Blot of polyclonal antibodyNew Zealand male rabbit was draw blood after immuned six weeks and two rats was draw blood after immuned five weeks,evaluating its titer by indirect ELISA,showing that New Zealand rabbit polyclonal antibody titer was ELISA 105,rat polyclonal antibody titer was ELISA level 106.The polyclonal antibody of New Zealand rabbits and rats was purified by the purification of staphylococcal protein A,SDS-PAGE verifying,the protein strips are cleaned and single.lg fresh tissues of Panax notoginseng(Bruk.)F.H.Chen,Arabidopsis thaliana,Salvia miltiorrhiza,Solanum tuberosum L and Nicotiana tabacum L were extracted protein.SDSPAGE verificating strip was cleaning,detecting of the concentration of Panax notoginseng(Bruk.)F.H.Chen is 433.15μg/mL,Arabidopsis thaliana is 392.48μg/mL,Salvia miltiorrhiza is 289.78 μg/mL,Solanum tuberosum L is 291.85 μg/mL and Nicotiana tabacum L is 419.74μg/mL.6.Establishment of plant GGPPS protein detection systemThe ELISA system was established by the square matrix titration test,and the rat antibody was established as the standard curve for the detection of antibodies,R2=0.9907.Arabidopsis thaliana,Nicotiana tabacum L,Panax notoginseng(Bruk.)F.H.Chen,Salvia miltiorrhiza and Solanum tuberosum L samples of total protein were detected GGPPS protein content in different plants according to the GGPPS standard curve detection system.The content of GGPPS protein contained in five different plants was obviously different.The highest content of Arabidopsis thaliana,from high to low were Solanum tuberosum L,Panax notoginseng(Bruk.)F.H.Chen,Salvia miltiorrhiza and Solanum tuberosum L.In total,it is concluded that the presence of "GVT" in SmGGPPS shows the existence of Salvia miltiorrhiza as a catalyst in different areas.This topic provides an effective guidance for the in-depth study of the genetic mechanism of Salvia miltiorrhiza.It is based on the previous experiment,with the help of high purity protein,preparation of specific protein of New Zealand rabbits and rats,purification,titer evaluation,Western-Blot validation,policy titration and plants to verify,obtained the SmGGPPS polyclonal antibody,establishing plant GGPPS protein detection system,the results of the research will have a direct effect towads the GGPPS protein of plants,provides a new way to study towads the functional gene secondary metabolism.This study first discovered the phenomenon of "GVT" of SmGGPPS,and the use of GS-MS technology combined with in vitro analysis to reveal the genetic mechanism of the formation of Salvia miltiorrhiza,which is of great significance for the study of Salvia miltiorrhiza.In addition,this study is the first to prepare high purity,high efficiency and specific SmGGPPS.Clonal antibody was developed and the GGPPS protein detection kit was made successfully.This study will provide new ideas for the further revelation of the genetic mechanism of Salvia miltiorrhiza and the biosynthesis of plant secondary metabolites,which will be of great scientific significance and application value. |
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