KR100673067B1 - A method for discriminating genetical identification of aristolochia and akebia species - Google Patents

Abstract

The present invention relates to a method for differentiating genes between species of a tree by Pyrosequencing method, and specifically, genotype analysis of stalk and shrub barrels by Pyro sequencing method, and the species specific sequencing primer of the tree The present invention relates to a differential method for evaluating species associations and genetic variation between Akebia and Aristolochia species. Through the discrimination method of the present invention, by determining the species of the barrel, it is possible to supply the exact raw material of the barrel, and can be utilized as a quality control method of the barrel.

Crushed Vine, Shrub Barrel, Pyrosequencing, Differentiation

Description

A method for discriminating genetical identification of aristolochia and akebia species}             

1 is a diagram showing the results of performing a pyrosequencing method for shrub barrel ( Aristolochia manshuriensis Kom.),

2 is a diagram showing the results of performing a pyro sequencing method on Akebia quinata Decaisne .

The present invention relates to a method for discriminating genes between species of a neck by Pyrosequencing.

A tree trunk ( Akebia quinata Decaisne ) is native to the central and southern regions of Korea, and crosses the stems of vines, eight leaf vines, and similar plants called tongcho, jungong (丁 翁), and late spring. It is cut and has pharmacological effects such as diuretic effect, gastric ulcer prevention effect, suppression of elevated serum cholesterol, phospholipid and triglyceride content, anti-inflammatory and menstrual pain, and low back pain. The taste is bitter. The main ingredients are Akebosides, Kalium salt and Aristolochic acid (Information Interpretation, Shin Mingyo, Korean Medicine Dictionary, Younglimsa, pp521, 1998).

In Japan, as a plant of origin for the neck, only Akebia species produced in various parts of Japan have been used for a long time. The main ingredient of wood barrel is Akebosides, which is not a relatively widely used herbal medicine. Since ancient times, it was able to supply enough with Japanese herbal medicine, but as the demand for herbal medicine increased rapidly, the domestic herbal medicine was scarce and foreign imports Promoted.

The first thing to consider at this time was the identity of the plant of origin, and the only thing that could be dealt with immediately by the same species of origin was the Korean tree, which belongs to the creeper family. Silver was mainly used for pore rather than medicinal purpose and could not be used for medicinal purpose. Against this background, it is assumed that Chinese wooden barrels are used.

In fact, although there are several species of genus Akevia in China, the ancient name in China is called Aristolochia mandschuriensis Kom., Which belongs to the Rataceae family produced in northeastern China, which is different from the Japanese species. The main ingredient was Aristolochic acid, which was different in the family and genus according to the plant taxonomy. Shrub barrels have been used for a long time in China for wood barrels, but they are also believed to have regional specificities.

In the case of conventional neck differential gene analysis, the gel-based method, which may have subjective factors, may have different results depending on the reader.

In this regard, the present inventors have used the Pyrosequencing method, a DNA sequencing technique used to evaluate genetic variation, which has been developed recently, from the Akebia and Aristolochia species. The present invention was completed by identifying the variation of the single base form from the extracted DNA.

An object of the present invention is to provide a differential method for discriminating species of a throat by analyzing genetic variations between species by pyrosequencing, an automatic genetic analysis method.

In order to achieve the above object, the present invention provides a differentiation method for discriminating the species of the neck by analyzing the genetic variation between the species of the neck by Pyrosequencing (Pyrosequencing) method.

Differentiation method of the present invention comprises the first step of extracting DNA from the neck sample; A second step of preparing a reaction solution of the extracted DNA and a primer; A third step of amplifying the prepared reaction solution; A fourth step of analyzing the entire nucleotide sequence of the amplified DNA; And a fifth step of discriminating the species of the neck through genotyping obtained by combining the amplified DNA and performing the pyrosequencing method.

The neck samples for the differentiation method include Akebia species and Aristolochia species, such as, for example, barrels ( Akebia quinata Decaisne ) or shrub barrels ( Aristolochia mandschuriensis Kom.). Outposts, roots, stems, or leaves are possible.

The primer used in the second step of the differentiation method is a biotinylated primer described in SEQ ID NO: 1 as a sense primer, and the general primer described in SEQ ID NO: 2 as an antisense primer. By amplifying the DNA.

Biotin binding primer described in SEQ ID NO: 1 is characterized in that it is designed based on the base sequence as 19 base sequence (mer).

In addition, the primer used in the pyro sequencing method of the fifth step is characterized in that it is used for genotyping as a species specific sequencing primer of the neck sample described in SEQ ID NO: 3.

Primer described in SEQ ID NO: 3 is characterized in that the 18 base sequence (mer) is designed to add a little away from the mutation site (mutation site) so that the secondary structure (secondary structure) is not formed.

When the pyro sequencing method of the fifth step is performed, light is generated when the nucleotide is synthesized from the elongated DNA strand, and polymorphism of the neck is automatically determined through this process. do.

The fifth step of the pyro sequencing method is carried out with an automated microtiter based microtiter-based pyrosequencer instrument capable of simultaneous analysis of samples in 15 minutes, and the preparation of each nucleotide is approximately one minute. As such, it can be used in a fast way to determine the exact sequence of genetic variation.

Differentiation method of the present invention can be discriminated in a short time by using a small amount of sample, a non-gel based method (non-gel based method) can be more accurate diagnosis in which subjective elements are excluded.

In addition, the differentiation method of the present invention can discriminate the species of the throat, preferably creepy vines or shrub barrels, using species specific primers in the throat difficult to discriminate with the naked eye.

Hereinafter, the present invention will be described in detail by reference examples, examples and experimental examples.

However, the following Reference Examples, Examples, and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Reference Examples, Examples, and Experimental Examples.

Reference Example 1. Preparation of a Neck Sample

The vine sample was used to receive the native plants that were found in Korea in the Department of Herbology, Kyung Hee University (Seoul, Korea). Shrub barrel samples were purchased from Kyung Hee University herbal medicine (Suwon, Gyeonggi, South Korea) was used.

Example 1 DNA Purification of Neck

3g of the neck of the entire root portion is finely chopped with a sterile knife, pulverized with a sterile grinder and powdered. DNA separation kit (DNeasy, No. 69104, Qiagen Inc., Valencia, CA, USA) was used slightly modified according to the manufacturer's instructions. 300 mg of the powdered barrel sample was used after washing. Prior to elution of the sample, the column was dried at 37 ° C. for 5 minutes to evaporate the remaining ethanol. Samples were eluted in 200 ul total volume of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

Experimental Example 1. DNA amplification using PCR

Amplification of the extracted DNA was performed by PCR. The internal transcribed spacer regions (ITS) regions of each neck sample were amplified using 25 ng DNA, 5 pmol of each primer (forward: SEQ ID NO. 1, reverse: SEQ ID NO. 2). PCR amplification was performed using 0.5 unit Taq polymerase (HT Biotechnology Ltd., Cambridge, United Kingdom). 30 ul of the PCR reaction mixture is 10 mM Tris-HCl, pH 9.0, 1.5 mM magnesium chloride, 50 mM potassium chloride, 0.1% Triton-X 100, 0.01% [v / v] stabilizer, deoxynucleotide triphosphate (dNTP) 0.25 mM each, oligonucleotide primer (oligonucleotide primer) 0.1 M each. PCR process is denatured for 5 minutes at 95 ℃, 30 times at 95 ℃, 30 seconds at 60 ℃, 30 times at 30 ℃ 72 ℃ PCR reaction using a PCR system (PCR System, Astec, Japan) Was performed. Reverse primer is a biotin binding capable of single strand DNA combination. PCR products were identified by 1.5% agarose gel electrophoresis.

Experimental Example 2. Genotyping by Pyrosequencing

DNA preparation for pyrosequencing (Pyrosequencing AB, Uppsala, Sweden) was performed following the manufacturer's standard protocol. Streptavidin sepharose beads, Streptavidin Sepharose HP, Amersham Pharmacia Biotech, Uppsala, Sweden were immobilized on the PCR product. The sequencing primer of the neck sample was the sequence set forth in SEQ ID NO: 3, which was produced by terminal residue hybridization immediately adjacent to the base of the A / C mutation from Pyrosequencing AB (http: // www). .pyrosequencing.com). After standing at room temperature for 10 minutes, 20 ul of biotin binding PCR product was immobilized in streptavidin coated Sepharose beads, and the immobilized PCR product was transferred to a 96-well filter (Millipore, Bedford, MA, USA). . Beads were left in the wells and vacuum was used to remove other solutions and reagents and to obtain pure single stranded DNA. The beads immobilized with template were re-immersed in 55 ul of 4M acetic acid containing 0.35 uM of sequencing primers, and 45 ul of the suspension was transferred to PSQ 96 plates (Pyrosequencing AB, Uppsala, Sweden). Using the PSQ 96 Sample Prep Thermoplate, heat the PSQ 96 plate containing the sample for 5 minutes at 90 ° C. to anneal the sequencing primer and at room temperature for 10 minutes. After transfer, the PSQ 96 plates were transferred to a process chamber of the PSQ 96 instrument (Pyrosequencing AB, Uppsala, Sweden). Enzymes, substrates, and nucleotides were prepared from reagent cassettes in wells using the PSQ 96 SNP Reagent Kit (Pyrosequencing AB, Uppsala, Sweden). Light is generated when nucleotides are synthesized on elongated DNA strands. From this process, polymorphism in the neck was automatically genotyped.

In Experimental Example 2 was identified by the pyro sequencing method in order to distinguish between the two types of barrels of creeper and shrub barrel, as a result, the creeper showed a different shape compared to the shrub barrel showed a clear difference. The peak of shrub barrels was very high in the second A nucleotide (see FIG. 1), but little scab was seen (see FIG. 2). In addition, we designed a species-specific sequencing primer of the keg to compare the creeper and shrub barrels. In the sequence, the shrub has A nucleotide base but was not present in the creeper. Therefore, it was confirmed that the species-specific sequencing primer of the neck of the present invention was suitably designed.

The present invention analyzes the genotype of the neck using pyro sequencing method, and devised the species specific sequencing primer of the neck, and the genetic association between Akebia species and Aristolochia species By establishing the species identification test method for differentiating the variation of the barrel, it is possible to supply accurate raw material of the barrel and use it as a quality control method of the barrel. Can be used.

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Claims (4)

A first step of extracting DNA from a neck sample; A second step of preparing a reaction solution of the extracted DNA and the primers set forth in SEQ ID NO: 1 and SEQ ID NO: 2; A third step of amplifying the prepared reaction solution; A fourth step of analyzing the entire nucleotide sequence of the amplified DNA; Genes obtained by combining the amplified DNAs and carrying out the pyro sequencing method using SEQ ID NO: 3 are compared to the sequences shown in FIGS. And a fifth step of discriminating between shrubs representing peaks and clumps representing c peaks at third nucleotide bases. delete The method of claim 1, wherein the primer set forth in SEQ ID NO: 1 is a sense primer as a biotin binding primer, and the primer set forth in SEQ ID NO: 2 is an antisense primer. delete

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