CN111363845B

CN111363845B - Special primer for specific identification of Chinese Huangguo ginseng and PCR method

Info

Publication number: CN111363845B
Application number: CN202010384567.9A
Authority: CN
Inventors: 王洪涛; 李桂生; 王英平; 王戎博; 陈玉春
Original assignee: Yantai University
Current assignee: Yantai University
Priority date: 2020-05-08
Filing date: 2020-05-08
Publication date: 2022-11-11
Anticipated expiration: 2040-05-08
Also published as: CN111363845A

Abstract

The invention relates to a short single copy region of ginseng chloroplastccsANon-synonymous mutation SNP locus of Chinese Huangguo ginseng is explored in the gene, and a conventional and fluorescent quantitative PCR method for specifically identifying the Chinese Huangguo ginseng is provided according to the locus. The conventional PCR method established by the invention can complete detection in a general molecular biology laboratory, and the established fluorescent quantitative PCR method does not need to mark a primer, can analyze a large number of samples in a short time, and can effectively realize large-scale rapid identification and screening in the field of the radix polygonati officinalis. Therefore, the invention provides an effective DNA molecular marker technical means for variety identification of the yellow fruit ginseng, and has important significance for germplasm resource protection, variety management and ginseng product quality optimization of the yellow fruit ginseng in China.

Description

Special primer for specific identification of Chinese Huangguo ginseng and PCR method

Technical Field

The invention relates to the technical field of molecular identification of traditional Chinese medicinal materials, in particular to a special primer and a PCR (polymerase chain reaction) method for specifically identifying Chinese yellow fruit ginseng.

Background

Ginseng (Panax ginseng c.a. meyer) is a perennial herb of araliaceae, is a traditional famous and precious Chinese medicinal material, has rich medicinal and edible values, and has the reputation of "king of herbaceous plant". The main effective component of Ginseng radix is ginsenoside, and has anti-aging, anti-fatigue and anti-inflammatory effects. After the artificially planted ginseng is officially approved as a new resource food by the Ministry of health, the development of the ginseng industry is greatly promoted. The cultivated ginseng has more than two thousand years of ginseng cultivation history, and is continuously subjected to genetic variation and differentiation in the artificial and natural selection processes, and the cultivated ginseng mainly comprises farmer varieties such as big horse teeth, two horse teeth, long neck, round winged reed rhizome, yellow fruit ginseng and the like. The Huangguo ginseng is a new ginseng cultivation variety bred by a systematic breeding method in China, and has the characteristic of high content of active ingredients such as ginsenoside, volatile oil, amino acid and the like. The successful breeding of the Huangguo ginseng increases new precious germplasm resources for the cultivation and breeding of the ginseng in China. However, the morphological characteristics of different ginseng varieties are very similar in the early growth and development stages, and in addition, a rapid and accurate ginseng variety identification method is lacked in China, so that different ginseng varieties are mixed and distributed in cultivation groups in various regions, and the varieties are disordered and the production seeds are mixed. The hybrid distribution of the cultivated population seriously affects the germplasm purity and the yield of the xanthoceras sorbifolia bunge, so that the establishment of a molecular identification method of the xanthoceras sorbifolia bunge is urgently needed to protect the germplasm resources of the xanthoceras sorbifolia bunge and maintain the quality stability of the xanthoceras sorbifolia bunge.

Since the morphological markers of ginseng are easily affected by the growth and development stages and the environment, various DNA molecular marker techniques have been widely used in recent years for genetic diversity analysis, germplasm resource identification and evaluation in ginseng species. At present, researches on the xanthoceras sorbifolia bunge mainly focus on aspects of genetic diversity, genetic structure analysis and the like, for example, RAPD (Random Amplified Polymorphic DNA) and a cluster analysis technology are utilized by any one of the Elliparis spinosa and the like to research the genetic relationship between the xanthoceras sorbifolia bunge and the American ginseng, and molecular markers of RAMP (Random Amplified microscopic Polymorphic) and ISSR (Inter-simple Sequence Repeat) are utilized by Wangzqing and the like to research the genetic difference between the "Formica Fuscai 01" ginseng and the farmhouse ginseng such as the xanthoceras sorbifolia bunge, but a molecular marker method for quickly and accurately identifying the xanthoceras sorbifolia bunge is not seen so far.

Disclosure of Invention

The invention aims to solve the technical problem of providing a special primer and a PCR method for specifically identifying Chinese wampee ginseng. The first problem to be solved is: provides a PCR special primer for specifically identifying the yellow fruit ginseng. The second technical problem to be solved by the invention is: providing a conventional PCR method for specifically identifying Chinese Huangguo ginseng; the third technical problem to be solved by the invention is: provides a fluorescent quantitative PCR method for specifically identifying Chinese Huangguo ginseng.

The PCR special primer for specifically identifying the Panaxacum xanthioides comprises 1 forward primer HgF (SEQ ID No:1 in a sequence table) and 1 reverse primer HgR (SEQ ID No:2 in the sequence table).

The sequence of the chloroplast ccsA gene of the yellow fruit ginseng is shown as SEQ ID No:3, the chloroplast ccsA gene sequences of other ginseng varieties are shown as SEQ ID No:4, respectively.

The base of the chloroplast ccsA gene sequence of the yellow fruit ginseng is G at 635bp, the base of other ginseng varieties at the position is A, and as shown in figure 1, a PCR special primer HgR for specifically identifying the yellow fruit ginseng is designed according to the SNP site of the yellow fruit at 635 bp. The derivative sequence of the primer sequence special for the yellow fruit ginseng also belongs to the invention. The derivative sequence is obtained by adding and reducing one or more bases to the 5 'end and/or 3' end of the sequence on the basis of the primer of the invention.

The conventional PCR method for specifically identifying Chinese Huangguo ginseng provided by the invention is to carry out agarose gel electrophoresis analysis on a PCR amplification product after carrying out PCR amplification on the genome DNA of a ginseng sample to be detected by using the primers. Because the primers HgF and HgR only have specificity to the xanthophylls sorbifolia bunge, only the xanthophylls sorbifolia bunge can amplify a specific strip (SEQ ID No:5 in a sequence table) with the length of 217bp, and other ginseng varieties cannot detect a PCR amplification product.

The total volume of the conventional PCR reaction was 20 μ Ι _: includes 1. Mu.L (10 ng) of genomic DNA of a sample to be tested, 2. Mu.L (0.5. Mu.M) of primer HgF, 2. Mu.L (0.5. Mu.M) of primer HgR, 5. Mu.L of sterilized ultrapure water, and 10. Mu.L of 2 Xeasy Taq Supermix DNA polymerase.

The reaction conditions of the conventional PCR are as follows: pre-denaturation at 94 ℃ for 4min; the 33 thermal cycle parameters included 94 ℃ denaturation 30s, 62 ℃ annealing 30s and 72 ℃ extension 30s, and 72 ℃ extension was continued for 5min after the cycle was completed.

The invention provides a fluorescent quantitative PCR method for specifically identifying Chinese Huangguo ginseng, which is characterized in that after the primer is used for carrying out fluorescent quantitative PCR amplification on the genome DNA of a ginseng sample to be detected, endPoint end point analysis is carried out on a PCR amplification product. Because the primers HgF and HgR only have specificity to the yellow fruit ginseng, only the yellow fruit ginseng generates a fluorescent signal in the amplification process, and other ginseng varieties cannot be amplified by the primers and cannot detect the fluorescent signal, so that the yellow fruit ginseng can be distinguished from other varieties by the level of the fluorescent signal.

The volume of the fluorescent quantitative PCR reaction is 10 mu L: includes 1. Mu.L (10 ng) of the genomic DNA of the sample to be tested, 1. Mu.L (0.5. Mu.M) of the primer HgF, 1. Mu.L (0.5. Mu.M) of the primer HgR, 2. Mu.L of sterilized ultrapure water, 5. Mu.L of 2 XSSYBR Green I Mastermix.

The fluorescent quantitative PCR reaction conditions are as follows: pre-denaturing at 95 ℃ for 10min; the 40 thermal cycle parameters included denaturation at 95 ℃ for 10s, annealing at 62 ℃ for 15s and extension at 72 ℃ for 20s, and the melting curve conditions increased from 72 ℃ to 95 ℃ after the cycle ended, with an increase of 1 ℃ per step. And analyzing the experimental result by using an EndPoint end point analysis method after the melting step is finished.

The invention discovers an SNP locus of the yellow fruit ginseng from a cccA (c-type cytochrome synthesis gene) gene of a chloroplast of the ginseng, designs a variety specific primer of the yellow fruit ginseng according to the locus, and establishes a PCR (polymerase chain reaction) method for specifically identifying the Chinese yellow fruit ginseng.

The invention discovers a nonsynonymous mutation SNP locus of Chinese wampee ginseng from a ginseng chloroplast short single copy region ccsA gene, and provides a conventional and fluorescent quantitative PCR method for specifically identifying Chinese wampee ginseng. The conventional PCR method can complete detection in a general molecular biology laboratory, fluorescent quantitative PCR removes bottlenecks in the rapid identification process of two ginseng varieties, namely gel preparation and electrophoretic analysis in the conventional PCR method, improves the detection speed and efficiency, can rapidly identify and analyze a large number of samples in a short time, and has stable and reliable identification results. Therefore, the invention provides an effective DNA molecular marker technical means for variety identification of the yellow fruit ginseng, and has important significance for germplasm resource protection, variety management and ginseng product quality optimization of the yellow fruit ginseng in China.

Drawings

FIG. 1 is a diagram showing the SNP sites and relative positions of primers of Huanggushen in the ccsA gene sequence.

FIG. 2 is a diagram showing the results of gel electrophoresis identification of conventional PCR products of Cucumis metuliferus.

FIG. 3 is a diagram of the result of fluorescent quantitative PCR identification of Huanggugen ginseng.

Detailed Description

The present invention will be described in detail with reference to specific examples.

The invention is made based on the discovery of specific SNP sites of the yellow fruit ginseng in the CCsA gene of the chloroplast short single copy region of the ginseng by the inventor. The identification of Huangguo ginseng using the fluorescent quantitative PCR technique is described in detail in the following examples.

The methods used in the examples are conventional methods unless otherwise specified, and the various biomaterials described in the examples are obtained by routes that provide experimental access for the specific purpose disclosed and are not intended to limit the source of the biomaterials of the present invention. All primer synthesis and sequencing work was done by Saimei Feishale (China) science and technology Limited.

The embodiments are provided in order to provide detailed embodiments and specific procedures, which will help understanding of the present invention, but the scope of the present invention is not limited to the following embodiments.

Example 1 PCR Special primer designed for specific identification of Panaxanthus gracilis

1. Amplification and comparison of different ginseng variety chloroplast short single copy region ccsA gene sequences

1.1 extraction of genomic DNA of different Ginseng species

Collecting multiple samples of different ginseng varieties from different producing areas, cleaning collected ginseng roots or leaves with deionized water, placing a proper amount of the samples in a mortar, adding liquid nitrogen and grinding into fine powder, taking about 20mg of the fine powder in a centrifuge tube, extracting ginseng DNA according to the operation instruction of the DNA extraction kit, and storing the extracted DNA in a refrigerator at-20 ℃ for later use.

TABLE 1 Ginseng samples of different varieties

1.2 Chloroplast ccsA gene sequence of different ginseng varieties amplified by PCR

The 20 μ L PCR reaction system was: mu.L (10 ng) of genomic DNA of Panax ginseng, 2. Mu.L (0.5. Mu.M) of primer ccsF (5 '-AGACACGCCTGCTCTTAGGAAG-3'), 2. Mu.L (0.5. Mu.M) of primer ccsR (5 '-ACCCGATTGTTGGTCTTTG-3'), 5. Mu.L of sterilized ultrapure water, 10. Mu.L of 2 × Easy Taq Supermix DNA polymerase (formulation: 2. Mu.L 10 × Easy Taq buffer, 1.60. Mu.L of 2.5mM dNTPs, 0.4. Mu.L Easy Taq DNA polymerase, 6. Mu.L H ₂ O)。

The PCR reaction conditions were: pre-denaturation at 94 ℃ for 4min; then, 33 cycles of reaction are carried out at 94 ℃ 30s,58 ℃ 30s and 72 ℃ 60 s; finally, the reaction is finished by extension at 72 ℃ for 7min and heat preservation at 4 ℃.

1.3 sequence alignment

And (3) directly sequencing the PCR amplification product, and splicing the DNA sequencing result by utilizing SeqMan software. Alignment of chloroplast ccsA gene sequences of yellow fruit and other ginseng varieties using ClustalW Omega software is shown in FIG. 1. At the 635 th base of the ccsA gene, the bases of the ginseng radix huang herein are G, while the other ginseng is a. The mutation site is a non-synonymous mutation, resulting in the corresponding amino acid changing from glutamine (Q) to arginine (R). The chloroplast ccsA gene sequence of the xanthophylls sorbifolia is shown as SEQ ID No:3, the chloroplast ccsA gene sequence of other ginseng varieties is shown as SEQ ID No:4, respectively.

2. Special primer for specific identification of Huangguo ginseng

Based on the specific SNP site of the yellow fruit ginseng found in the sequence alignment, a forward primer HgF is designed at the base positions 438-458 of the picture 1, and the sequence is 5' (SEQ ID No:1 in the sequence table). A reverse primer HgR is designed at the 635-654 th base, and the sequence is 5-. In order to ensure the absolute specificity of HgR to the Huanggushen, the penultimate base A at the 3' end is changed into G, so that the HgR has mismatch of two bases for ginseng varieties except the Huanggushen. The amplification interval of the positive primer and the negative primer is 438 bp-654 bp, so that the specific amplification fragment length of the yellow fruit ginseng is 217bp. The relative positions of the primers and the length of the fragment expected to be amplified are shown in FIG. 1.

Example 2 routine PCR identification of Panaxanthus gracilis

Genomic DNAs of different ginseng species shown in Table 1 were used as templates, and PCR was performed using primers HgF and HgR.

The total volume of the PCR reaction was 20. Mu.L: includes 1. Mu.L (10 ng) of genomic DNA of a sample to be tested, 2. Mu.L (0.5. Mu.M) of primer HgF, 2. Mu.L (0.5. Mu.M) of primer HgR, 5. Mu.L of sterilized ultrapure water, and 10. Mu.L of 2 Xeasy Taq Supermix DNA polymerase. The reaction conditions of PCR were: pre-denaturation at 94 ℃ for 4min; the 33 thermal cycle parameters comprise denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s and extension at 72 ℃ for 30s, extension at 72 ℃ is continued for 5min after the cycle is finished, and the reaction is finished by heat preservation at 4 ℃.

3 μ L of the PCR product was detected by electrophoresis in a 1% agarose gel containing ethidium bromide, and the results were observed using a gel imaging system. As shown in FIG. 2 (lanes 1-5 are Huangguo, 6-7 are Dama teeth, 8-9 are Era teeth, 10-11 are edgings, 12-13 are long necks, 14-15 are round shoulder reeds), only Huangguo ginseng produced specific bands amplified by HgF and HgR, but none of the other ginseng species produced bands. Recovering the specific band of the xanthoceras sorbifolia bunge and sequencing, wherein the sequence length is 217bp, and the sequences (SEQ ID No:5 in a sequence table) and SEQ ID No: and 3, the corresponding sequence results are completely consistent, and the reliability of the experimental result is verified. The gel electrophoresis detection result shown in FIG. 2 shows neat and bright target sequence bands, and no non-specific amplification band or sheet-shaped dragging occurs, so that the 217bp specific band generated by the yellow fruit ginseng can be used for accurately distinguishing the yellow fruit ginseng from other ginseng varieties.

Example 3 fluorescent quantitative PCR identification of Panaxanthus gracilis

And respectively taking genome DNA of different ginseng varieties as templates, and performing fluorescent quantitative PCR amplification by using primers HgF and HgR. Sample materials of different ginseng species are shown in table 1.

The 10 μ L fluorescent quantitative PCR reaction system is: 1. Mu.L (10 ng) of genomic DNA of the sample to be tested, 1. Mu.L (0.5. Mu.M) of the primer HgF, 1. Mu.L (0.5. Mu.M) of the primer HgR, 2. Mu.L of sterilized ultrapure water, 5. Mu.L of 2 XSSYBR Green I Mastermix.

The fluorescent quantitative PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 10min; the 40 thermal cycle parameters include denaturation at 95 ℃ for 10s, annealing at 62 ℃ for 15s and elongation at 72 ℃ for 20s, and the melting step after the cycle is increased from 72 ℃ to 95 ℃ by 1 ℃ in each step. And analyzing the experimental result by using an EndPoint end point analysis method after the melting step is finished.

The end point analysis results of EndPoint are shown in FIG. 3 (sample 1 is ultrapure water as a negative control, 2-6 are big horse teeth, 7-11 are two horse teeth, 12-16 are edgings, 17-21 are long necks, 22-26 are round winged reeds, and 27-50 are yellow fruits). SYBR Green I is a dye with a Green excitation wavelength that binds to all double-stranded DNA double-helix minor groove regions. At the beginning of PCR amplification, dye molecules not bound to the double-stranded DNA emit weak fluorescence, and the fluorescence signal intensity of SYBRGreen I is gradually increased along with the amplification of the double-stranded PCR product. Thus, the presence of a particular allele in a target can be determined by detection of a fluorescent signal. As can be seen from FIG. 3, all the yellow fruit ginseng samples were produced with amplification products, and the fluorescence Signal levels (Signal Level%) thereof were all 80 or more, while the negative control and other ginseng species were unable to perform PCR amplification due to the specificity of the primers for the yellow fruit ginseng species, and only weak fluorescence signals emitted from dye molecules not bound to the double-stranded DNA could be detected. Therefore, the primers and the fluorescent quantitative PCR technology can be used for easily distinguishing and identifying the Huangguo ginseng from other ginseng varieties. Compared with other non-gel electrophoresis analysis such as TaqMan and molecular beacon, the fluorescent quantitative PCR method developed by the research does not need to mark the primer, the result identification time does not exceed two hours, a large number of samples can be analyzed in a short time, and the field large-scale rapid identification and screening of the radix polygonati officinalis can be effectively realized.

Sequence listing

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atgatatttt caactttaga gcatatatta actcatattt ccttttcaat cgtttcaatt 60

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tcagaaaagg gaatgatagc tattttttta tgtataacag gattattagt cactcgttgg 180

atttattcga gacatttccc actaagtgat ttatatgaat cattaatctt tctttcatgg 240

agtttatcag ttattcatat agttccgtat ttcaaaaaaa agaaaaaaaa tttaagcaca 300

ataactgcgt caagtgttat ttttacccag ggctttgcta cttcaggtct tttaactgaa 360

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ctagtcatta catttcgaaa aaacagaaat attttttgta aaaggaatcc tttattaaat 540

gagttatttt cctttggtga aatccaatac atgaatgaaa gaaacaatgt tttaagaact 600

actttttttt ctgctaagaa ttattacagg tcccggttga ttcaacagtt ggattattgg 660

agttatcgtg tcattagtct agggtttatc tttttaacca taggtattct ttcgggagca 720

gtatgggcta atgaagcatg gggatcatat tggaattggg acccaaagga aacttgggca 780

tttattactt ggatcgtatt tgcgatttat ttacacactc gaaccaatat aaaattacgg 840

ggtgcaaatt ccgcaattgt ggcgtctata ggctttctta taatttggat atgctatttt 900

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Claims

1. The PCR special primer for identifying the specific property of the yellow fruit ginseng is based on the short single copy area of the yellow fruit ginseng in chloroplastccsAThe 635 th base yellow fruit from the 5' end is G which is designed for the existence of single nucleotide polymorphism sites in the gene;

the nucleotide sequences of the PCR special primers of the ginseng huangguo are respectively shown as SEQ ID No:1 and SEQ ID No:2, respectively.

2. The PCR primer special for specifically identifying the ginseng radix linderae according to claim 1, wherein the primer comprises: the yellow fruit andchloroplast of other ginseng speciesccsAThe gene sequences are respectively shown as SEQ ID No:3 and SEQ ID No:4, respectively.

3. The method for identifying the specificity of the xanthophylls sorbifolia bunge by using the primers as claimed in claim 1 or 2, wherein the primers comprise: firstly, carrying out conventional PCR amplification on a sample to be detected, and then carrying out 1% agarose gel electrophoresis detection on a PCR amplification product, wherein if a single band with the length of 217bp appears, the sample source is xanthoceras sorbifolia bunge.

4. The method of authentication according to claim 3, wherein: the total volume of the PCR reaction was 20 μ L: the method comprises the steps of including 1 muL 10ng of genome DNA of a sample to be detected, 2 muL 0.5 muM primer HgF,2 muL 0.5 muM primer HgR,5 muL sterilization ultrapure water and 10 muL 2 Xeasy Taq Supermix DNA polymerase; the reaction conditions of PCR were: pre-denaturation at 94 ℃ for 4min; the 33 thermal cycle parameters comprise denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s and extension at 72 ℃ for 30s, extension at 72 ℃ is continued for 5min after the cycle is finished, and the reaction is finished by heat preservation at 4 ℃.

5. The method for identifying the specificity of the xanthophylls sorbifolia bunge by using the primers as claimed in claim 1 or 2, wherein the primers comprise: firstly, carrying out fluorescence quantitative PCR amplification on a sample to be detected, and after the amplification cycle is finished, heating the temperature from 72 ℃ to 95 ℃ to perform a melting step; analyzing an experimental result by using an EndPoint end point analysis method after the melting step is finished; the melting step after the circulation is finished is that the temperature is increased from 72 ℃ to 95 ℃, and the temperature is increased by 1 ℃ in each step; and (3) analyzing by using an EndPoint end point analysis method after the melting step is finished, wherein if the Level Signal Level% of the fluorescence Signal is more than 50, the sample source is the yellow fruit ginseng.

6. The method of authentication according to claim 5, wherein: the 10 mu L fluorescent quantitative PCR reaction system is as follows: 1. mu.L 10ng of genomic DNA of a sample to be tested, 1 mu.L 0.5 mu M primer HgF,1 mu.L 0.5 mu M primer HgR,2 mu.L of sterilized ultrapure water, and 5 mu.L 2 XSYBR Green I Mastermix;

the fluorescent quantitative PCR reaction conditions are as follows: pre-denaturing at 95 ℃ for 10min; the 40 thermal cycle parameters comprise denaturation at 95 ℃ for 10s, annealing at 62 ℃ for 15s and extension at 72 ℃ for 20s, and the melting step after the cycle is increased from 72 ℃ to 95 ℃ and is increased by 1 ℃ in each step; and analyzing the experimental result by using an EndPoint end point analysis method after the melting step is finished.