| Dendrobium officinale Kimura et is a traditional Chinese medicinal material of the monocotyledonous plant,the orchid family,the tree orchid family,and the Dendrobium genus.The ingredients contained in different varieties are quite different.In the market,D.officinale,which has a shortage of growth years and lacks in nutrients,and Dendrobium of the same genus that do not have both medicine and food,are sold as D.officinale with complete health care properties,which seriously affects the products quality and medicinal efficacy? Therefore,there is an urgent need for a simple and accurate method to identify D.officinale.This study intends to use molecular biology technology to combine the three methods of DNA barcode,HRM technology and Real time PCR technology to establish a DNA-based D.officinale system identification method,which provides a basis for accurate and rapid identification of D.officinale.The main conclusions of this study are as follows:1.Pass 9 commonly used DNA barcodes(chloroplast coding genes mat K,rbc L,ycf1b;chloroplast non-coding genes psb K-psb I,psb A-trn H,trn S-trn G,trn F-trn L sequence fragments;mitochondrial gene nad1 sequence;nuclear gene ITS2 sequence)Sequence sequencing,calibration,calculation of genetic differentiation distance,and use of Wilcoxon rank sum test and establishment of NJ tree analysis methods to evaluate their ability to distinguish D.officinale and other Dendrobium.Among them,the intraspecies maximum genetic distance between rbc L and trn H-psb A is greater than the minimum interspecies genetic distance,the genetic distances of other barcodes all meet the requirement that the maximum intraspecies genetic distance is less than the minimum interspecies genetic distance.The experiment collected 25 kinds of D.officinale and 10 kinds of Dendrobium plants.The identification rate of ITS2 and trn L-trn F for the Dendrobium materials in this experiment reached 90.1%.This experiment recommends ITS2 and trn F-trn L sequences as candidate barcodes for identification of D.officinale and its related species.2.The HRM primers designed based on the ITS2 sequence were used to analyze the melting curve of 35 Dendrobium samples,and successfully distinguished D.officinale from non-office Dendrobium,and the experiment was reproducible.The Tm values of all D.officinale fluctuate between 87.34～87.98 ℃ and all are single peaks.The peak difference between non-office Dendrobium is large.For example,the average Tm value of D.chrysotoxum is 91.25℃,and the average Tm value of other Dendrobium is 88.65 ℃ and 89.23℃.D.officinale and non-office Dendrobium are well distinguished,and the overall difference is 1.50 units of Tm.The HRM analysis method can quickly and high-throughput preliminary identification of D.officinale.Compared with traditional methods,HRM experiment has the advantages of simple operation,closed operation,no need for sequencing electroplate,and stable results,which can provide new ideas for the rapid identification of D.officinale.3.Based on 9 DNA barcode primer sequences,using PCR technology to amplify the required fragments,bidirectional sequencing,sorting,and comparison,to find SNPs in different varieties of D.officinale,and analyze them,design ARMS-q PCR primers for this specific site SYBR Green real-time PCR technology was used to verify the identification of D.officinale.In this experiment,a SNP site corresponding to 4 DNA barcodes was found,and 4 pairs of primers were successfully designed.When a sample containing SNP sites is detected,The Ct value is significantly different from the sample without Ct value.Subsequently,through high-throughput testing of samples,the 05 and 24 D.officinale were successfully locked.SYBR Green real-time PCR technology has the characteristics of high efficiency,time saving,and stability.It can successfully identify D.officinale accurately and provide a new idea for identifying species with higher relatives and distinguishing the quality of medicinal materials. |
PDF Full Text Request