Method for identifying three original species of rhei radix et rhizoma medicinal material and application

A technology of yellow medicinal materials and medicinal materials, which is applied in the field of identification of source varieties of Chinese medicinal materials, and can solve problems such as cumbersome operations and difficult origin species

Pending Publication Date: 2022-01-07
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Physical and chemical identification is based on the main components of Chinese herbal medicines, but the operation is cumbersome
Compared with traditional identification methods, DNA molecules can provide rich information for species identification. DNA barcodes such as ITS2, psbA-trnH and matK can effectively identify genuine rhubarb from North China rhubarb Rh.franzenbachiiMunt., Hetao rhubarb Rh.hotaoense C.Y.Cheng et Kao and other counterfeit products, but it is difficult to accurately identify the original species

Method used

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  • Method for identifying three original species of rhei radix et rhizoma medicinal material and application
  • Method for identifying three original species of rhei radix et rhizoma medicinal material and application
  • Method for identifying three original species of rhei radix et rhizoma medicinal material and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Screening of three primitive species-specific DNA barcodes of rhubarb medicinal materials

[0022] Taking the annotated chloroplast genome of Rhubarb palmatum as a reference, the chloroplast genomes of Rheum tangutica Rh. figure 1 ). The variation of the intergenic region of the chloroplast genome sequence of the three species is greater than that of the gene region, such as rps16-trnQ, trnT-psbD, psaA-ycf3, trnT-trnL, psbE-petL, ndhF-rpl32, trnN-ycf1, ccsA-ndhD, etc., these high The variable regions may be potential molecular markers for species identification of Rheum tangutica, Rheum officinalis and Rhubarb palmatum.

[0023] DnaSP was used to calculate the nucleotide variation value (Pi) of all genes and intergenic regions of the chloroplast genomes of the three species, and to perform hypervariable region detection and sequence difference level analysis ( figure 2 ). The variation of the mean value of intergenic region Pi (0.004) in the chloroplast ge...

Embodiment 2

[0024] Example 2 Acquisition of three primitive species-specific DNA barcodes of rhubarb medicinal materials

[0025] 2.1 Experimental materials

[0026] A total of 42 samples were collected in this example, including the three basic species of rhubarb medicinal materials, Rheumtanguticum, medicinal rhubarb Rh.officinale and palm leaf rhubarb Rh.palmatum, as well as edible rhubarb Rh.rhaponticum and wave leaf rhubarb Rh.rhabarbarum Linnaeus, Rh.alexandrae Batal., Rh.nobile Hook.f.et Thoms. and Rumex crispus. See Table 2 for details.

[0027] Table 2 Sample information table of the three basic species of rhubarb medicinal materials and their counterfeit products

[0028]

[0029] 2.2 Genome Extraction

[0030] About 50-100 mg of the samples in Table 2 were taken, ground with liquid nitrogen, and the rest of the steps were carried out according to the standard operating method of the plant genomic DNA extraction kit.

[0031] 2.3 PCR amplification

[0032] Primers 1F / 1R, ...

Embodiment 3

[0039] Example 3 Identification and analysis of three primitive species-specific DNA barcodes of rhubarb medicinal materials

[0040] 3.1 K2P distance

[0041] The intra-species and inter-species K2P distances of the high-quality sequences obtained by sequencing were calculated according to MEGA7.0. In the present invention, the minimum genetic distance between species is greater than the maximum genetic distance within a species, and there is no overlap, indicating that the obtained sequences meet the requirements of DNA barcodes (Table 3).

[0042] 3.2 Phylogenetic tree of NJ

[0043]The NJ phylogenetic tree was constructed according to MEGA7.0, and the repetition value of the bootstrap was set to 1000. In the present invention, rhubarb tangutica is gathered into one branch alone, rhubarb palmata is gathered into one branch alone, and rhubarb palmata is gathered into one branch alone, indicating that rpsl6-trnQ, psaA-ycf3, psbE-petL, ndhF-rpl32 and trnT -trnL can be used ...

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Abstract

The invention relates to identification of three original species of a rhei radix et rhizoma medicinal material and an application of the identification. The identification specifically comprises the following steps: screening hypervariable regions based on chloroplast genomes, designing primers 1F / 1R, 2F / 2R, 3F / 3R, 4F / 4R and 5F / 5R by taking the screened hypervariable regions rps16-trnQ, psaA-ycf3, psbE-petL, ndhF-rpl32 and trnT-trnL sequences as templates, performing PCR amplification and Sanger sequencing on 42 samples, evaluating the species identification capability of the screened sequences by utilizing MEGA7.0, and determining specific DNA bar codes capable of being used for identifying dried roots and stems of rh. tanguticum maxim. ex balf., rh. officinale baill. and rheum palmatum L. The invention discloses five pairs of universal primers capable of effectively amplifying the hypervariable regions rps16-trnQ, psaA-ycf3, psbE-petL, ndhF-rpl32 and trnT-trnL, can be used for identifying original plants, seeds and medicinal materials of three basic original species of the rhubarb medicinal material, and provides a basis for classifying and identifying the three basic original species of the rhubarb medicinal material, ensuring the clinical medication safety of the rhei radix et rhizoma medicinal material and standardizing the rhei radix et rhizoma medicinal material market.

Description

technical field [0001] The invention belongs to the technical field of identification of source varieties of Chinese medicinal materials, and in particular relates to the development and identification of specific DNA barcodes for identifying three basic species of rhubarb medicinal materials based on the chloroplast genome. Background technique [0002] "Chinese Pharmacopoeia" records that the medicinal materials Rhei Radix et Rhizoma are the dried roots and rhizomes of the Polygonaceae plants Rheumpalmatum L., Rh.tanguticum Maxim.ex Balf. and Rh.officinaleBaill. The wide range of medicinal effects of rhubarb is mainly related to the large amount of active ingredients contained in rhubarb. The main chemical components of rhubarb are anthraquinones, anthrones, stilbenes, tannins and other compounds. There are differences in the chemical substances of rhubarb medicinal materials with different bases, and they show specialties in different effects. Research shows that rhubarb ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6895C12N15/11
CPCC12Q1/6806C12Q1/6895C12Q2531/113C12Q2563/185C12Q2535/101
Inventor 宋经元李冉郡辛天怡武立伟姚辉
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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