The Identification Of Gynostemma Pentaphylum And The Differentioation From The Counterfeit Plant, Cayratia Japonica With RAPD Technique

Objetive: To study the variety of Gynostemma pentaphylum and distinguish the counterfeit plant, Cayratia japonica from Gynostemma Pentaphyllum on the Molecular Biology.Methods: The software of Primer Premier 5.0 was used to work out the random primes that the number was 20, and then they were divided into four groups. With the RAPD technique, the improved CTAB method was used to extract the genomic DNA from the medicinal plants that were gathered in distinct places. At the same time, the method of orthogonal design was applied to optimize the reaction system of RAPD. The optimum group of primers was screened and the best reaction system was groped, too.Results: With the improved CTAB method, the extracted genomic DNA from Gynostemma Pentaphyllum, Cayratia japonica, Tobacco and Herba Centellae in distinct places, respectively presented better quality and could be used to progress the amplification of RAPD. The best reaction system of RAPD was groped from the table of orthogonal design. After analyzing the amplification, there were amplified strips to differ Gynostemma Pentaphyllum with the counterfeit plant, Cayratia japonica and other medicinal plants. In the amplified strips of Gynostemma Pentaphyllum from the different places, there were characteristic strips to be amplified, too.Conclusion: The method of extracting the genomic DNA was founded to get rid of the secondary metabolite. The extracted genomic DNA presented better quality and could be used to progress the amplification of RAPD. After optimizing the reaction system of RAPD with the method of orthogonal design, not only we could save material and time so that the experiment was more convenient, but also the optimum reaction system could help us establish the RAPD finger prints of extracted genomic DNA from the medicinal plants. Objetive: To clone the characteristic strips that were progressed with RAPD technique from the genomic DNA that were extracted from the varieties of Gynostemma Pentaphyllum, then align and analyze the feature of sequences. After determining the length of sequence with Vector NTI, we designed 20-mer characteristic primers. At last, construct the finger printing of hereditary feature about the varieties of Gynostemma Pentaphyllum.Methods: The extracted genomic DNA from the seven varieties of Gynostemma Pentaphyllum were progressed by PCR with the optimum reaction system of RAPD again. The progressed characteristic strips were analyzed and then sliced from agarose gel. The objective strips were ligated with the vector pTG19-T, and then the ligated vectors were transformated into Bacterium coli JM109. After purified, the recombinant plasmids were send to the bio-company and the result was aglined and analyzed with Vector NTI. At last, the homologous sequence was inquested with Blast-the software of molecular biology-on GenBank.Results: After aligning and analyzing the sequences with Vector NTI, we could know the extracted genomic DNA from six varieties of Gynostemma Pentaphyllum could be progressed about 826 bp fragment by PCR. And the fragment was close to our calculation (832 bp). But only one Gynostemma Pentaphyllum could be progressed 811 bp fragment and need more study. The homologization of aligning characteristic fragment from the seven varieties of Gynostemma Pentaphyllum was more than 99%.Conclusion: The 20-mer characteristic primers were designed from thecharacteristic sequences of Gynostemma Pentaphyllum. And then the templates that included the seven varieties of Gynostemma Pentaphyllum and other medicinal plants (as Cayratia japonica, Tobacco, Herba Centellae and so on), were progressed by PCR with the 20-mer primers. At last, we could construct the finger printing that could be differed the counterfeit plant, Cayratia japonica and other medicinal plants from Gynostemma Pentaphyllum on molecular biology.