Analysis Of Different Species Of Gynostemma By RAPD And Study On Its Anti-bacteria Effect

Five extraction methods(basic method,high salt precipitation,improved SDS,low pH extraction medium with high salt and improved CTAB) of genomic DNA from Gynostemmna pentaphullum were studied.The DNA samples obtained by the above methods were tested by agarose gel electophoresis and RAPD.The results indicated that all methods could be used for Genomic DNA isolation from Gynostemmna.The yield of DNA extracted with basic method was the best,and then the high salt precipitation,improved SDS,low pH extraction medium with high salt and improved CTAB.The difference in yield,quality,time duration of the DNA samples were compared,and low pH extraction medium with high salt was considered to be fast and reliable technique among the five methods.The optimized RAPD analysis with Gynostemmna pentaphullum as templates in RAPD amplication.we used orthogonal experiment combining with single factor experimer to optimize the RAPD reaction conditions.The results showed that optimal conditions in 25uL PCR system were DNA 50ng,random primes 0.6ummol/L,Mg²⁺ 2.0mmol/L,dNTP 0.25mmol/L,Taq polymerase 1.0U.The random amplified polymorphic DNA was conducted with 17 random primes under optimal conditions in five species and 10 random primes showed consistent banding patterns.Totally 66 DNA fragments were amplified with ten random primes.The high reproducible DNA fragments were 55,and it occupied 82%of 66.It could be used to differentiate all cultivars being test and the UPGMA method may be used to analyze the genetic relationship.The results showed that gnetic distance between the five samples varied from 0.3333 to 0.7143 and five species were divided into 3 groups.The results of RAPD were the same as the serology.Extracting the anti-bacteria components from the Gynostemmna with the different solvent,components ectracted with water have the best anti-bacteria effect.The best condition of anti-bacteria components extraction is as follows:After juice of the Gynostemmna extracted,remains of the Gynostemmna and the water are mixed according to 1:30 proportion.The mixture is kept at 60℃and that extraction is repeted one times, the anti-bacteria ingredient in the Gynostemmna is basically extracted.When pH is 4.6～9.0,the anti-bacteria components has the bacteriostatic effect.The anti-bacteria minimum dosage on different flora of Gynostemmna are as follows:Staphylococcus aureus 7.8mg/mL,Micrococcus tetragenus 7.8mg/mL,Bacillus subtilis 31.25mg/mL, Bacillus thuringiensis 31.25mg/mL.The extract couldnot effectively inhabit the growth of Escherichia coli.