| Archidendron clypearia from leguminous plants of the genus Archidendron clypearia dry leaves, has heat-clearing and detoxifying, cooling blood detumescence, antidiarrheal effect and so on. Archidendron clypearia are very versatile, with high economic value. Archidendron clypearia uses a very wide range, with high economic value, and developed a variety of dosage forms, such as Archidendron clypearia tablet, capsule, etc. Over the years, due to the huge demand for resources, many scholars have Archidendron clypearia conducted a series of research, and on a large scale artificial cultivated in many places. Archidendron clypearia similar to the same plant due to the appearance and microstructure, illegible after processing, therefore lead to base source fuzzy, which affect the quality of the medicinal materials. So it is very important to accurately identify Archidendron clypearia species.ObjectiveThrough to the wild Archidendron clypearia samples4period of candidate sequences (chloroplast genes psbA-trnH, rbcL, rpoCl; nuclear gene ITS2sequence) to filter, after screening sequence joint Genbank database of all known than homologous gene sequences for the multiple sequence and genetic relationship analysis, looking for a specific base site. At the same time, from two aspects of intraspecific and interspecific on DNA sequence analysis method to identify the Archidendron clypearia origin is workable.By high performance liquid chromatography (HPLC) method to compare different origin Archidendron clypearia effective component content differences, and combined with DNA barcode, analysis of gene sequence, chemical composition, content, distribution in the three relations, variety breeding and domestication as the Archidendron clypearia as the basis.Method First, DNA sequencing and alignment:all plant material with sterile water is rinsed clean, dry with silica gel drying after cut up,take0.05g each said, after liquid nitrogen grinding with day pillar of centrifugal type of plant genome DNA extraction total genomic DNA extraction kit. The above samples by PCR amplification using primers. Take the PCR amplification product testing on1%agarose gel electrophoresis, and two-way sequencing sequencing company. Sequence splicing and calibration using Codoncode Aligner3.7.1, multiple sequence alignment using the ClustalW Bioedit7, genetic distance was calculated by the K2P models in MEGA5. Construct phylogenetic trees. Using SPSS software for statistical analysis, optimum4segment sequence is used to identify candidate monkey earrings distribution.Second, HPLC detection:can establish simultaneous determination monkey earrings gallic acid and quercetin chromatographic conditions, the determination of two active ingredients in Archidendron clypearia content, draw Archidendron clypearia with the content of effective component differences among regions.Three, gene sequence results and the effective composition content determination results:correlation analysis by SPSS19genotypes with the software of statistical analysis of the relationship type, chemical, and ecological. Preliminary probe into the three intrinsic relations.ResultsFirst, DNA barcode:four sequences, only psbA-trnH and rbcL found mutation loci, Numbers of28and6respectively, and the Numbers of variants in Hainan Archidendron clypearia. Through the statistical analysis, psbA-trnH Archidendron clypearia on the sequence is more suitable for rapid identification of Hainan.Second, Content determination:by building error state chromatographic condition, and concludes that mobile phase of methanol to0.4%acetonitrilephosphate (30:66:4), flow rate of1.0ml/min, detection wavelength at270nm, column temperature:room temperature, at the same time can effectively and accurately determination of gallic acid and quercetin. Content determination results of Archidendron clypearia in Hainan province as effective component content is highest.Three, according to the psbA-trnH sequence to establish genotype, through clustering analysis to establish chemical type, the statistical software SPSS19analysis, come to Hainan samples in psbA-trnH sequence to establish genetic model and chemical model there is a corresponding relationship, this tip of Hainan province Archidendron clypearia is likely formed its own unique ecological.ConclusionInfluenced by Hainan unique environmental factors, and through long-term accumulation of genetic evolution, in psbA-trnH sequence, is likely to form their own unique genotype or ecotype. This prompt, use psbA-trnH sequence is to quickly identify Archidendron clypearia have certain feasibility of hainan province. Samples at the same time, this study of the effective composition content differences influenced by internal and external causes of2factors, internal cause is a genetic factor, external conditions. These two factors influence each other and function, and genetic evolution after a long time, in order to form a unique species. |
