The Genetic Characteristics Of Danshendao Were Preliminarily Analyzed Based On The “variation Of Origin And Variation” Of Salvia Miltiorrhiza KSL Gene

Salvia miltiorrhiza Bge as a commonly used Chinese herbal medicine,quality has been a hot research.To solve the Dangshen geoherbalism is the core of the quality of the study.But to solve the problem of geoherbalism,it is the key to analyze the genetic mechanism of Danshen.As the main active ingredient of Salvia miltiorrhiza,the generation mechanism of tanshinone has important research meanings.Salvia miltiorrhiza kaunene synthase like as a key enzyme downstream of tanshinone synthesis has a direct impact on the content of active ingredients.The results showed that the SmKSL gene of Salvia miltiorrhiza had obvious characteristics of "habitat variation"(GVT),and the cloning of SmKSL gene from different producing areas was carried out with this key gene as template.The prokaryotic expression method was used to induce protein expression,and then the catalytic reaction was carried out in vitro.The catalytic products were analyzed by GC-MS.The effect of GVT on the biosynthesis of key enzymes was analyzed by comparing the catalytic performance of the enzyme with the peak intensity of the product to explore the genetic characteristics of Rhizoma Dioscorea.Specific research results are as follows:1.Cloning of SmKSL gene and discovering of GVT phenomenonThe molecular weight of the expressed protein is 68 kilodaltons,the SmKSL protein has a certain hydrophilicity,and the structure is more stable.At the same time,it was found that there were 5 mutations in SmKSL,which were located at 246bp,669bp,1047bp,1048bp,1356bp,1491 bp respectively.There were special variation types in Hebei,Inner Mongolia,Shandong and Sichuan.And there were more "non-synonymous mutations" in the mutant loci of "Salvia miltiorrhiza" origin.2.The successful construction of prokaryotic expression systemThe prokaryotic expression system of SmKSL was successfully constructed.The optimal induction temperature was 20?,the induction time was 12h,the concentration of IPTG was 1.0 mM and the A600 was 1.0.After elution with different concentrations of imidazole,the optimal elution concentration was 30mM,Confirmed by PCR amplification and sequencing,proved that the prokaryotic expression system was successfully established.3.Successfully established SmKSL protein expression and enzymatic reaction conditions systemAccording to the reference,the induction temperature was 20?,the induction time was 10 hours,the IPTG concentration was 0.4mM,the OD600 was 0.8,and the SmCPS had a large expression level,according to the SmCPS strain provided by Professor Gao Wei,Capital University of Medical Sciences.4.Successfully tested the function of SmKSL gene and the effect of different genotypes on the catalytic efficiencyThe catalytic reaction of SmKSL was carried out by using GGPP as the reaction substrate,catalyzing the product produced by SmCPS-CPP as the substrate for the next catalytic reaction,and then adding SmKSL enzyme for catalysis.The results show that SmKSL has the function of catalyzing the activity of dibromo-terephthalone diene in cobalt-based pyrophosphate(CPP).Catalytic efficiency shows the highest catalytic performance in Shanxi,followed by Hubei,Shandong,Sichuan,Henan,Inner Mongolia,Hebei.In summary,this study found that SmKSL has a tendency to change the origin of the phenomenon.Its catalytic performance results indirectly show the origin of the catalytic performance of the level,and thus confirm the authentic Danshen.Therefore,this topic can provide a reasonable explanation for the authentic nature of Salvia miltiorrhiza and clarify its genetic mechanism.