Based On The "producing Area Trend Variation" Law Of CMK Gene Of Salvia Miltiorrhiza, The Genetic Characteristics Of The Authenticity Of Salvia Miltiorrhiza Were Analyzed

Recently,the quality of traditional Chinese medicine is a hot topic.Geoherbalism of Danshen(Salvia miltiorrhiza Bge.)plays an important role in the quality of Danshen,but genetic factors of Geoherbalism are the basis of quality of Danshen.This study aimed to clone 4-(cytidine 5’-diphospho)-2-C-methy 1-D-erythritol kinase(CMK)gene for mining different genotypes based on geographical variation tendency(GVT).In this study,CMK gene was considered as an example for further study.The effect of different genotypes on bioactivity of CMK was assessed by invitro expression of CMK and following analysis of LC-MS.This study may provide a new perspective for us to understand genetic mechanism of Geoherbalism of Danshen,which will give a good foundation for solving quality problem of Danshen.Results are as following:1.Clone of SmCMK gene and discovering of GVT phenomenonSmCMK gene,a length of 1191bp,coding 397 amino acids,had the close genetic relataionship of Picrorhiza kurrooa CMK.Its molecular weight(MW)was 43.37 kDa.Isoelectric point(PI)was 6.72.It had 45 nonpolar amino acid residues(Asp+Glu)and 44 polar amino acid residues(Arg+Lys).Its Grand average of hydropathicity is-0.289,and its Instability index is 36.89.It had good hydrophilicity and stable structure.Signal peptide region was predicted from 1 to 53,and was directed to locate in chloroplast.It was also deduced that SmCMK had close relationship with Picrorhiza kurrooa CMK.Meanwhile,we discovered that SmCMK had 6 sites associated with GVT,including sites of 102bp,105bp,115bp,120bp,909bp and 1110bp.Among these,at 102bp,105bp and 115bp,type "G","C’"and "C" geographical variation occurred in Inner Mongolia population.At 909bp and 1110bp,type "T" and "C" geographical variation occurred in Shanxi and Shandong populations.And at 120bp,type "C" geographical variation occurred in Hebei and Shanxi populations.Among the amino acids SmCMK,non-synonymous mutations had been happened in Hebei and Shanxi populations("L"into "F").And non-synonymous mutations had also been happened in Inner Mongolia population and Shandong population("P" and "D").2.Development of prokaryotic expression systemProkaryotic expression systems of SmCMK was successfully developed.Optimal condition of induction was determined as following:tempeture was 15℃,time was 12h、IPTG was 0.25mmol·L-1 and A600 was 0.8.By PCR and sequencing,it was proved that prokaryotic expression systems of SmCMK was successfully developed.By polyacrylamide gel electrophoresi,it was proved that the protein was successfully induced.3.Identification of SmCMK function and the effect of gentypes on catalytic efficiencyWe determined that SmCMK proned to catalyze CDP-ME into CDP-MEP.It was also proven that SmCMK of Sichuan achieved highest catalytic efficiency,followed by Shandong,Shanxi,Hubei,Hebei,Henan and Neimeng.In Contrast,catalytic efficiency of SmCMKs of genuine producing areas was higher,which further confirmed the Geoherbalism of Danshen and the effect of gentypes on catalytic efficiency from the GVT phenomenon.In conclusion,GVT phenomenon was discovered in SmCMK,and catalytic efficiency of SmCMKs also further confirmed the Geoherbalism of Danshen.Therefore,the results will provide a new perspective for us to understand genetic mechanism of Geoherbalism of Danshen.