Application Study On The Identification Of Dendrobium Officinale With PCR Based On ITS Sequence

Dendrobium officinale is a kind of perennial crawling herbaceous plants. It belongs to orchid. It named by the "iron green" on the surface of the stern. Uncultivated Dendrobium officinale is scarce and expensive because of many restrictive factors. Such as, long growth periods, slow-growing and low survival rate. Some of the kindred plant have simulated Dendrobium officinale sneak into the Chinese herbal drug market.Use a scientific, accurate, fast and effective way to distinguish species is a necessary premise of conducting scientific research and application. It is particularly important in the field of medicinal plant research and application. Especially as in China, with higher utilization rate of the herbal medicine, it’s important to identify each kind of medicinal plants accurately and efficiently, not only relates to every people’s health, but also even affects the life security.This study chooses the Dendrobium officinale as the research object. At first, It clarified the essential differences between the Dendrobium officinale between other Dendrobiums from three aspects:resources distribution of Dendrobium officinale, medical value and clinical researches. At the same time, the thesis expounds the important role of Dendrobium officinale in the application of clinic. Secondly, it points out the deceptive phenomena which exist in the herbal medicine market and the present situation of Dendrobium officinale industry. It proved the urgency of identification of Dendrobium officinale. The study briefly explains the existing identification methods by reading the related literatures. It points out the limitations of plant identification method for Dendrobium officinale. And it also points out the reliability and validity of identification with DNA. Finally, the author chooses the ITS sequences as a DNA molecular marker. Using ITS consensus primer which exists in the ribosomal DNA of plants to do a PCR on the samples of Dendrobium officinale DNA templates which have been defined the provenance. And then recover and sequence the amplified nucleotide sequence. Combined with29different original ITS sequences of Dendrobium officinale which are downloaded from Genbank to do homology comparison analysis, deleted the consensus sequences at the two sides of the sequences. Then take the results of the analysis compare with the ITS sequences of other varieties plants of Dendrobium by bioinformatics soft wares. Make a Phylogenetic tree and estimates of evolutionary divergence between sequences of the51plants of the Dendrobium. Based on the researches, the author tries to find out the specific sequences of Dendrobium officinale in ITS sequences.The main research conclusions are as follows:1. Based on the analysis of ITS sequences of the51species plants of the Dendrobium, the author found that the homology of ITS sequences of the30Dendrobium officinale is extremely high. The evolutionary divergences between sequences are closed. These Dendrobium officinales are classified into a category on the Phylogenetic tree. And it’s obviously different from other varieties.2. The author found four specificity sequences in the nonconservative section of Dendrobium officinales. The length of the four specific sequences is moderate, easy to do the specific primer design.3. Using designed specific primer of Dendrobium officinale to do PCR amplification for two kinds of Dendrobium officinales and one kind of Dendrobium devonianum. The results proved that these specific primers can use the DNA template of Dendrobium officinale to amplified target bars and Dendrobium devonianum failed to amplified the target bars. It’s proved that the primer can be differentiate the Dendrobium officinales and the Dendrobium devonianum.