A Study On Hereditary Feature And Identification Of Gynostemma Pentaphyllum (Thunb.) Makino In Guang Xi Province By RAPD Analysis

The RAPD method was applied to analyze the genetic diversity of seven samples of medicinal plant Gynostemma pentaphyllum which were collected from different regions and different sources in Guang Xi province.This method also was used to identify the resources of Gynostemma pentaphylum and its spurious breed Cayratia japonica at level of DNA.The results would offer the technological guarantee and molecular biology basis for Gynostemma pentaphyllum resources collection and analysis,classification research, biodiversity protection,genuine drug identification,and further,it would be of great benefit to breeding of medicinal plant resources and safe clinical medication.The main results of this study were as follows:1.The genomic DNA of fresh samples and dry samples of Gynostemma pentaphyllum and Cayratia japonica were extracted successfully by modified CTAB method.2.The reaction system of RAPD-PCR was optimized:With the DNA of Gynostemma pentaphyllum collected from campus as template,the random primer WGS001 for PCR has been done meanwhile RAPD reaction system has been optimized so that it could be determined for the suitable annealing temperature and concentrations of template,Mg²⁺,primer,Taq DNA polymerase, BSA.In the optimized system of 25μL,MgCl₂ 2 mmol/L,primer 0.4μmol/L, template 60 ng,Taq DNA polymerase 1 U,BSA 2μg/μL.The reaction procedure was 94℃in 3 min,then 40 cycle times with 94℃in 30 s,58℃in 30 s,72℃in 80 s,lastly 72℃in 10 min,and preserved at 4℃.3.Based on the optimized system,ten random primers(20 bp)were used for the PCR amplification of genomic DNA.Two of these primers(WGS001, WGS004)could amplify bands which have good reproducibility and high polymorphism.With this two screened random primers,PCR was done for the above mentioned seven fresh samples of Gynostemma pentaphyllum.The RAPD fingerprints were obtained,thirty of thirty-three amplified sites were polymorphic,accounts for 90.91%.It revealed that Gynostemma pentaphyllum existed abundant genetic diversity.The software SPSS16.0 was used to calculate the Jaccard coefficient of similarity and do cluster analysis.The coefficient of similarity of Gynostemma pentaphyllum was in the range of 0.217-0.688.The dendrogram of RAPD cluster analysis indicated that the smaller the geographic distance between two Gynostemma pentaphyllum samples,the smaller their genetic difference,vice versa.4.The fresh samples and dry samples of Gynostemma pentaphyllum and Cayratia japonica from different regions in Guang Xi province were authenticated by two screened primers.According to the different bands of DNA fingerprints,Gynostemma pentaphyllum and Cayratia japonica could be distinguished obviously.Besides,these two primers could amplify characteristic fragments which were common to all Gynostemma pentaphyllum samples studied but not to Cayratia japonica.One common characteristic DNA fragment was about 550 bp by primer WGS001,other was about 500 bp by primer WGS004.The preliminary view was that these two common characteristic DNA fragments could be taken as basis for distinction molecular marker for Gynostemma pentaphyllum and Cayratia japonica in Guang Xi province.5.These seven characteristic DNA fragments(500 bp)amplified by WGS004 were cloned,sequenced and analyzed for bioinformatics.Through bioinformatics analyze we could know these seven DNA sequences had not been recorded in NCBI GenBank before.They were new sequences.It was the first time to apply the RAPD analysis to study hereditary feature of Gynostemma pentaphyllum(Thunb.)Makino in Guang Xi province.This research's result could supply molecular biology basis for genetic diversity of Gynostemma pentaphyllum.It also was the first time to report that RAPD technology could identify both of fresh samples and dry samples between Gynostemma pentaphyllum and its spurious breed Cayratia japonica.The Chinese traditional medicine identification was so easily to be affected by external factors that leaded to inaccurate conclusion.This RAPD method could overcome difficulties in Chinese traditional medicine identification and provide a new thread for identification of Gynostemma pentaphyllum.