Analysis For Rapd Polymorphism And18S Rdna Sequences Of Medicinal Plants Uncaria From Guangxi

Medicine Uncaria, includes Uncariarhyncho-phylla(Miq.)Jacks, Uncariamacrophylla Wall.,Uncariahirsuta Havil.Uncariasinensis(Oliv.) Havil., and Uncariasessilifructus Roxb.,is a multi-source Chinese herbal medicine, which belongs to Rubiaceae. The main active ingredient of medicine Uncaria is indole alkaloids.According to Chinese Pharmacopoeia,In addition to the above five species, and more than10types of plant Uncaria never used as medicine. However, development and utization of medicine Uncaria are restricted due to its chaotic genetic background and existing of Adulterants.It is the major aim for the study to analysis the genetic diversity of Medicine Uncaria in different regions of GuangXi by DNA fingerprints with random amplified polymorphic DNA (RAPD) technique and18S rDNA sequences, which is beneficial for molecular breeding and germplasm resources protection. On the other hand, we attempt to adopt DNA markers technique to authenticate of Medicine Uncaria and establish authentication method on molecular level for medicinal plant UncariaThe radom amplified polymorphic DNA (RAPD) analysis was applied to research the polymorphism of three kinds of medicinal plants Uncaria.from Guangxi.The modified extraction kit was used to extract the genomic DNA of several plants, including3regions of Uncaria Macrophylla Wall, Uncaria Sessilifmctus Roxb. and Uncaria Hirsute Havil. Optimized extraction kit can be effective for3Uncaria species genome DNA.With the DNA extracted from several plants as template,18selected random primers with10bp amplified a total of260bands,231bands were polymorphic, polymorphism rate of88.85%, the amplification is effect. NTSYS-pc software were used to compute genetic similarity (GS), then we constructed a dendrogram based on genetic distances by using UPGMA method. The dendogram reflected that5samples clustered into three taxa, and this result was in accordance with their species and geographic origin, but may be different from morphological observed.Based on the analysis of RAPD fingerprints of Medicine Uncaria, we obtained one specific RAPD marker of Uncariamacrophlla Wall.,about length of11OObp, named DG1100. which could be used for authentication of Uncariamacrophyla Wall. The RAPD markers DG1100were cloned, sequenced and analyzed. The bioinformatics analysis revealed that the sequence similarity of DG1100were more than99%and these three DNA sequences were detected for the first time. According to the sequence, we adopted Primer5software to design specific primers and made RAPD markers convert into SCAR markers.In this study,18S rDNA of plant Uncaria(6species,10samples) were be cloned and sequenced. Similarity of the ten sequences was more then96%, internal reference primers could be designed by using conservative sequence. Mac vector software was be used to analysis this10sequences, then executed cluster analysis combined with18S rDNA sequences of other plants Rubiaceae, and established of the phylogenetic tree. The results showed that:in general, different samples in same species could be clustered into one class, which means that different species Uncaria have species-specific sites on18S rDNA sequences.In this study, RAPD technique was be used to obtain the SCAR marker of Uncariamacrophylla Wall., and specific sites of18S rDNA in different species of plant Uncaria would be find out. This study provides some references to develop a new molecular authentication method in different medicinal plants.