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Application of nucleotide sequence of rDNA (recombinant deoxyribonucleic acid) ITS (internal transcribed spacer)-D3 region in establishment of DNA (deoxyribonucleic acid) bar code identification system for medicinal plants

An ITS-D3, nucleotide sequence technology, applied in the field of identifying medicinal plants using nucleotide sequences, can solve the problems of slow sequence evolution rate, lack of conserved regions, large workload, etc., achieving strong species differentiation ability and saving The effect of measuring time and sequence alignment is accurate

Inactive Publication Date: 2011-09-21
GUANGZHOU UNIVERSITY OF CHINESE MEDICINE +1
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Problems solved by technology

However, this standard scheme has the following disadvantages in the identification of plant species: 1. It needs to test the two sites of matK and rbcL, which requires a lot of work and high cost; 2. Although rbcL has universal primers, the sequence evolution rate in species is relatively slow. Slow, while matK has a fast sequence evolution rate, but lacks universal primers. These shortcomings make matK+rbcL only distinguish about 70% of plant species, and the identification ability is not ideal
However, the use of ITS2 alone as a plant DNA barcode has the following disadvantages: 1. There are multiple different copies of the ITS2 sequence in many species, and PCR products need to be cloned and multiple positive clones are picked for sequencing; 2. Due to the lack of conserved regions, ITS2 Sequences are difficult to align accurately across broadly taxonomic species
At present, 26S rDNA has been applied to the study of plant phylogeny (Douglas E.Soltis, Anne E.Senters, MichaelJ.Zanis, Sangtae Kim, James D.Thompson, Pamela S.Soltis, Louis P.Ronse De Craene, Peter K.Endress and James S. Farris. Gunnerales are sister to other core eudicots: implications for the evolution of pentamery. American Journal of Botany, 2003, 90: 461-470.), but it has not been seen that the expansion region present on 26S rDNA is used in plants (including medicinal plants) identification report

Method used

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  • Application of nucleotide sequence of rDNA (recombinant deoxyribonucleic acid) ITS (internal transcribed spacer)-D3 region in establishment of DNA (deoxyribonucleic acid) bar code identification system for medicinal plants
  • Application of nucleotide sequence of rDNA (recombinant deoxyribonucleic acid) ITS (internal transcribed spacer)-D3 region in establishment of DNA (deoxyribonucleic acid) bar code identification system for medicinal plants
  • Application of nucleotide sequence of rDNA (recombinant deoxyribonucleic acid) ITS (internal transcribed spacer)-D3 region in establishment of DNA (deoxyribonucleic acid) bar code identification system for medicinal plants

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0024] The design of the primer of the sequence shown in example 1SEQ ID No.1~4

[0025] The specific design method of the primers of the sequence shown in SEQ ID No.1~4 is as follows:

[0026] Reference Soltis et al. (Douglas E. Soltis, Anne E. Senters, Michael J. Zanis, Sangtae Kim, James D. Thompson, Pamela S. Soltis, Louis P. Ronse De Craene, Peter K. Endress and James S. Farris .Gunnerales are sister to other core eudicots: implications for the evolution of pentamery. American Journal of Botany, 2003, 90: 461-470.), download 206 additional taxa of seed plants from the nucleotide sequence database GenBank and EMBL 18S rDNA and 26S rDNA sequences, and downloaded the 9th chromosome sequence of japonica rice (Oryza sativa Japonica Group) (sequence number AP008225), and selected its rDNA complete sequence (from 2898bp to 8680bp, including 18S-ITS1-5.8S-ITS2-25S ). Multiple alignments were performed on the downloaded sequences using clustal X (version 1.83) (www.clustal.org),...

example 2

[0028] Example 2 establishes a DNA barcode database based on the nucleotide sequence of the ITS-D3 region

[0029] This example takes 50 kinds of medicinal plants as an example to illustrate the establishment of a DNA barcode database based on the nucleotide sequence of the ITS-D3 region. The specific steps are as follows:

[0030] 1. Collect the leaves of 50 species of medicinal plants in 19 families from the South China Medicinal Plant Germplasm Bank: Amomum villosum Lour.var.changguo., Alpinia officinarum Hance, Alpinia katsumadai Hayata , Alpinia galanga (Linn) Willd., Hedychium coronarium Koen., Aplinia pumila Hook.f., Crinum asiaticum L.var.sinicum (Roxb.ex Herb.), Hippeastrum vittatum (Ait.) Herb., Asparagus cochinchinensis (Lour.) Merr., Aloe vera L.var.chinesis (Haw.) Berger, Aloe arboresesens.Mill., Asarum sieboldii Miq., Teng San Boussingaultia gracilis Miers.var.baselloides Bail., Cajanus cajan(L.) Millsp., Schizonepeta tenuifolia(Benth.) Briq., Rhododendri mariae...

example 3

[0046] Example 3 Using the nucleotide sequence of the ITS-D3 region to distinguish each medicinal plant in the DNA barcode database and identify the relationship

[0047] In order to verify that the nucleotide sequence of the ITS-D3 region of the present invention has general and accurate beneficial effects as a medicinal plant DNA barcode, the ITS-D3 region nucleus of each medicinal plant in the DNA barcode database established in Example 2 is as follows The number of base differences in the nucleotide sequence is detected and the genetic relationship of each medicinal plant is identified.

[0048] 1. Detection of the number of base differences in paired samples of medicinal plants in the DNA barcode database

[0049] (1) The nucleotide sequences of the ITS-D3 regions of 50 kinds of medicinal plants obtained in Example 2 were multiple-aligned using clustal X (version1.83) (www.clustal.org), and the sequence alignment results were saved as aln file.

[0050] (2) Use MEGA 4 (...

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Abstract

The invention relates to a method for identifying medicinal plants by utilizing nucleotide sequences, in particular to an application of a nucleotide sequence of an rDNA (recombinant deoxyribonucleic acid) ITS (internal transcribed spacer)-D3 region in establishment of a DNA (deoxyribonucleic acid) bar code identification system for medicinal plants. The application comprises the following steps of: firstly, detecting the nucleotide sequences of the ITS-D3 regions of medicinal plants, and establishing a DNA bar code database; then, detecting the nucleotide sequence of the ITS-D3 region of a sample to be identified; constructing a clustering tree by the detected nucleotide sequence and the nucleotide sequences of the ITS-D3 regions in the DNA bar code database; determining the medicinal plant having the closest genetic relationship with the sample to be identified in the DNA bar code database according to the clustering tree, and comparing the difference between the nucleotide sequences of the ITS-D3 regions of the medicinal plant and the sample to be identified; and then, by taking the name of the species of the medicinal plant having the closest genetic relationship with the sample to be identified in the database as a reference and combining information such as morphological characteristics and the like, determining the name of the species of the sample to be identified. In the invention, the DNA bar codes have the characteristics of good primer universality, accurate sequence comparison and strong species distinguishing capability.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to detection and application methods including nucleic acid, in particular to a method for identifying medicinal plants by using nucleotide sequences. Background technique [0002] Medicinal plants are the original plants of Chinese medicinal materials, and they are some plant species with health care and medicinal value. According to statistics, my country has 11,146 kinds of medicinal plants (accounting for 87% of all traditional Chinese medicine resources), belonging to 383 families and 2,309 genera. Rapid and accurate identification of these more than 10,000 medicinal plants is an urgent and arduous task for Chinese medicine workers in my country. The existing identification methods of medicinal plants are mainly based on morphological characteristics. By collecting the morphological characteristics of various organs (such as flowers, fruits, seeds, roots, stems, leaves) or tissues of th...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/10G06F19/24
Inventor 陈蔚文段中岗詹若挺杨锦芬何瑞徐晖
Owner GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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