CN107254528B - Detection of sibicose A in Polygala tenuifolia5With sibicose A6Method of content - Google Patents
Detection of sibicose A in Polygala tenuifolia5With sibicose A6Method of content Download PDFInfo
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Abstract
The invention discloses a method for detecting sibicose A in polygala tenuifolia medicinal material5With sibicose A6The PCR primer and the method comprise the following steps: an upstream primer: 5'-AGATGGTGAGCGTGATT-3', respectively; a downstream primer: 5'-CAAGCCCTCTTTCAATA-3' are provided. The method comprises the following steps: extracting genome DNA of polygala tenuifolia samples; taking genome DNA as a template, and carrying out PCR amplification by using the specific primer; detection of the contained compound, namely, the sibiricose A, by PCR amplification products5With sibicose A6Can amplify polygala tenuifolia samples with a strip length of 629bp, and contains the compound of sibiricose A5With sibicose A6The content of (A) is low. The PCR primer and the method have the advantages of convenience, rapidness and high accuracy, and can be applied to fine variety breeding of polygala tenuifolia medicinal materials, so that the breeding time can be greatly shortened, and the breeding efficiency can be improved.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a method for detecting compound sibicoseA in polygala tenuifolia medicinal materials5With sibicose A6High-low PCR primer and method.
Background
Polygala tenuifolia Willd, which is listed as the top grade and is a commonly used Chinese medicine, was originally recorded in Shen nong's herbal Jing. Warm in nature, bitter and pungent in flavor, and has the effects of soothing nerves, promoting intelligence, eliminating phlegm and reducing swelling. Modern researches show that the main effective components of the polygala tenuifolia medicinal material comprise saponins, sugar esters, oroxanthones, alkaloids and the like, and have pharmacological effects of inhibiting bacteria, resisting inflammation, improving cognition, improving memory, resisting senile dementia, depression, tumors, aging and the like.
The strength of the drug effect of the traditional Chinese medicinal materials is closely related to the quality of the medicinal materials, and the quality of the medicinal materials is closely related to the content of main chemical components contained in the medicinal materials. The research shows that the sugar ester compound sibiricose A5With sibicoseA6Has strong anti-dementia and anti-depression activity. Currently on the compound sibiricose A5With sibicose A6The qualitative and quantitative analysis of (a) mainly focuses on methods such as extraction and separation, hplc (uplc) content determination, fingerprint, one-test-multiple-evaluation or metabonomics, but these methods have many disadvantages: the extraction and separation have great blindness and long time consumption, and simultaneously, a great deal of repetitive work is inevitably involved; HPLC (UPLC) content determination, fingerprint spectrum, one-test-multiple-evaluation or metabonomics and other methods, the used instruments are expensive, the technical requirements on experimental operators are high, and the popularization and the application are difficult. The accumulation degree and content change of secondary metabolites in the traditional Chinese medicinal materials are influenced by growth years, the growth years of the traditional Chinese medicinal materials, particularly rhizome traditional Chinese medicinal materials are long, generally 2-3 years are taken as main raw materials, and long time is needed for determining the content of compounds contained in the traditional Chinese medicinal materials.
The compound content is a complex character controlled by multiple genes, and the content difference of the compounds among different polygala tenuifolia samples is large. sibicose A5With sibicose A6Has the same mother nucleus structure, and the content of the mother nucleus structure can be controlled by the same type or the same gene. Therefore, the invention screens out the peptide-Sibiricose A5With sibicose A6Content-related specific DNA molecular bands, aiming at designing detectable sibicose A aiming at the specific bands5With sibicose A6High content of specific PCR primers and hopeful to be applied practically.
Disclosure of Invention
The invention aims to provide a method for detecting sibicose A in polygala tenuifolia medicinal materials5With sibicose A6PCR primers with high and low content; and detecting the sibicose A in the polygala tenuifolia medicinal material by using the PCR primer5With sibicose A6The method has high content. The method has the advantages of convenience and rapidness,The method has the advantage of good accuracy, can be applied to fine variety breeding of polygala tenuifolia medicinal materials, can greatly shorten breeding time and improve breeding efficiency.
The invention provides a method for detecting sibicose A in polygala tenuifolia medicinal material5With sibicose A6The content of PCR primers, the specific PCR primers are shown as follows:
an upstream primer: 5'-AGATGGTGAGCGTGATT-3' (SEQ ID No. 1);
a downstream primer: 5'-CAAGCCCTCTTTCAATA-3' (SEQ ID No. 2).
The invention provides a method for detecting sibicose A in polygala tenuifolia medicinal material5With sibicose A6The content method comprises the following steps:
(1) extracting genome DNA of polygala tenuifolia samples;
specifically, a CTAB method is adopted to extract genome DNA; because the DNA of individuals, tissues and organs and even cells in each development period is consistent, is not influenced by environment and is stable in heredity, the polygala tenuifolia samples in any growth period and any tissue part can be selected to extract the DNA.
(2) Performing PCR amplification by using the PCR primer by using the genome DNA as a template;
wherein, when the specific primer is adopted for PCR amplification, the preferable PCR amplification reaction system is 25 mu L, and the method comprises the following steps: mu.L of template DNA, 1. mu.L of each of 10. mu.M upstream and downstream primers, 12.5. mu.L of 2 Xeasy Taq PCR Supermix, and the reaction volume was made up with sterilized ultrapure water;
the PCR amplification procedure is preferably: 5min at 93 ℃; 35 cycles of 93 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 30 s; 5min at 72 ℃ and 10min at 10 ℃.
(3) Detection of Compound sibiricose A by PCR amplification product5With sibicose A6The content of (A);
electrophoresis is carried out by using 1.0-2.0% agarose gel, PCR amplification products are detected, the agarose gel is observed under ultraviolet rays, polygala tenuifolia samples with 629bp bands can be amplified, and a compound, namely, the sibiricose A contained in the polygala tenuifolia samples5With sibicose A6The content of (A) is low.
The agarose gel concentration is preferably 1.5%.
Compared with the prior art, the invention has the beneficial effects that:
(1) the primer of the invention can be used for accurately and efficiently detecting the sibicose A in polygala tenuifolia medicinal materials5With sibicose A6The quality of the polygala tenuifolia medicinal material is preliminarily identified;
(2) the primer of the invention can be used for detecting polygala seeds or young leaves of polygala medicinal materials in seedling stage, and greatly improves the compound sibiricose A5With sibicose A6The screening efficiency of the polygala tenuifolia fine variety with higher content accelerates the breeding process of polygala tenuifolia medicinal materials and shortens the breeding period.
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FIG. 1 is an agarose gel electrophoresis of 7 polygala tenuifolia samples amplified by specific primers, wherein 1-7 are 7 polygala tenuifolia samples, and the PCR amplification products of No.1 and No. 3-6 polygala tenuifolia samples are 629bp in length; m is DL 2000DNA Marker, which is 2000bp, 1000bp, 750bp and 500bp from top to bottom.
Detailed Description
The following examples are intended to illustrate the process but are not intended to limit the scope of the invention.
Unless otherwise indicated, the following examples follow conventional experimental conditions, such as the protocols described in the molecular cloning Laboratory Manual (New York: Gold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations; all reagents used are commercially available.
The specific method for designing the specificity identifying primer (upstream primer: 5'-AGATGGTGAGCGTGATT-3'; downstream primer: 5'-CAAGCCCTCTTTCAATA-3') provided by the invention comprises the following steps: firstly, the compound sibiricose A in polygala root medicinal material5With sibicose A6Performing content determination, combining ISSR (inter-simple sequence repeat) molecular marker technology, and screening out the compound, namely the siRNA A, by using SPSS 16.0 software5And sibicosse A6Content-related specific bands, performing gel recovery and clone sequencing on the specific bands, and aiming at a target by using Primer Premier5.0 softwareDesigning specific PCR primer.
Example 1 Sibiricose A in Polygala tenuifolia Willd5With sibicose A6Detection method for content
1. Sample source
The polygala root medicinal material adopted in the embodiment is dried roots of 7 batches of polygala root medicinal materials collected from different producing areas in Shanxi province, and the specific information of polygala root samples is shown in Table 1.
TABLE 1 Polygala tenuifolia sample information Table
2. Extraction of genomic DNA
About 60mg of each of the above samples was ground into powder with liquid nitrogen, and DNA was extracted by the CTAB method. The kit is purchased from Beijing Etdelley Biotechnology GmbH, and has the following model: CTAB plant genome DNA rapid extraction kit (cat # DN14), 50 times. The specific experimental method refers to the kit instruction.
PCR amplification
And (3) taking the genomic DNA of each polygala tenuifolia medicinal material obtained in the step (2) as a DNA template for PCR amplification, and performing conventional PCR amplification by taking 5'-AGATGGTGAGCGTGATT-3' as an upstream primer and 5'-CAAGCCCTCTTTCAATA-3' as a downstream primer. The PCR amplification reaction system is 25 mu L and comprises: mu.L of template DNA, 1. mu.L of each of 10. mu.M upstream and downstream primers, 12.5. mu.L of 2 × EasyTaq PCR SuperMix, and the reaction volume was made up with sterilized ultrapure water. The PCR amplification procedure was: 5min at 93 ℃; 35 cycles of 93 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 30 s; 5min at 72 ℃; PCR amplification products of polygala root medicinal materials are obtained at 10 ℃ for 10 min.
4. Sibiricose A in polygala tenuifolia5With sibicose A6Detection of high or low levels
Loading the amplified fragment of each polygala tenuifolia medicinal material into a sample hole of agarose gel with the concentration of 1.5 percent, and carrying out agarose gel electrophoresis; the voltage was 138V, the electrophoresis time was 30min, DL 2000DNA Marker was used as a standard for DNA molecular size, and the electrophoresis result was photographed in a gel imaging system to obtain an agarose gel electrophoresis image as shown in FIG. 1.
As can be seen from FIG. 1, no bands were amplified in the polygala tenuifolia samples No.2 and No. 7, and the compound, sibiricose A, was found in these 2 polygala tenuifolia samples5With sibicose A6The contents are higher and are all higher than 1.900mg/g, and the polygala tenuifolia hybrid strain can be used as a good variety for breeding polygala tenuifolia medicinal materials; the 5 polygala tenuifolia samples except No.2 and No. 7 all amplified a strip with the length of 629bp, and the 5 polygala tenuifolia samples contain the compound of the sibiricose A5With sibicose A6Low content of sibicose A5The contents are all lower than 1.650mg/g, sibicose A6The content is less than 1.400 mg/g.
Example 2PCR products with sibicose A in Polygala tenuifolia Willd5With sibicose A6Correlation study of content
1. With sibiricose A in Polygala tenuifolia Willd5With sibicose A6Detection of content-related Gene sequences
Taking 5 parts (No. 1, No.3, No. 4, No. 5 and No. 6) of PCR amplified fragments of polygala tenuifolia medicinal materials obtained in the step 3 of the embodiment 1, delivering the fragments to the biological engineering (Shanghai) GmbH and Shanghai Sangni Biotech Co, respectively, performing secondary bidirectional sequencing, and analyzing by bioinformatics software such as DNAMAN and the like, wherein the DNA sequences of the 5 parts of polygala tenuifolia medicinal materials are completely the same, the length of the PCR fragment is 629bp, and the DNA sequence is shown as SEQ ID No. 3.
2. Sibiricose A in polygala tenuifolia5With sibicose A6Determination of the content
(1) Taking 7 parts of dried root of polygala tenuifolia medical material obtained in the step 1 of the embodiment 1, beating by using a stick or rubbing by hands, removing the wood core to obtain a polygala tenuifolia cylinder, crushing, and sieving by a third sieve of pharmacopeia for later use.
(2) Preparing a reference substance solution: weighing of sibiricose A5With sibicose A6And (3) precisely weighing a proper amount of reference substances, fixing the volume with methanol, and preparing reference substance stock solutions with mass concentrations of 0.247 and 0.153mg/mL respectively.
(3) Preparing a test solution: weighing about 0.2g of polygala tenuifolia medicinal powder which is sieved by pharmacopoeia No. three, precisely weighing, placing in a conical flask with a plug, precisely adding 20mL of 70% methanol, weighing, carrying out ultrasonic treatment for 30min (power 500W, frequency 40kHz), placing to room temperature, weighing again, complementing the weight loss by 70% methanol solution, shaking up, filtering, taking subsequent filtrate, and filtering by using a 0.22 mu m microporous membrane to obtain the medicinal powder.
(4) Analyzing the reference substance and the sample solution of the polygala tenuifolia sample by using a Waters ACQUITY UPLC H-Class UPLC chromatograph, and calculating the compound, namely, the sibiricose A in the polygala tenuifolia sample solution according to a linear equation constructed by the reference substance solution5With sibicose A6The content of (a).
(5) Chromatographic conditions are as follows: the chromatographic column is Phenomenex Kinetex C18(100X 2.10mm, 2.6 μm); the mobile phase is eluted by the gradient of acetonitrile (A) -0.1 percent formic acid solution (B), and the elution gradient is as follows: 0-3min, 5-10% of A; 3-6min, 10-12% of A; 6-16min, 12-22% A; 16-17min, 22% A; 17-22min, 22-27% A; 22-25min, 27-32% A; 25-27min, 32-35% A; 27-30min, 35% A; 30-36min, 35-40% A; 36-37min, 40-100% A; 37-39min, 100-5% A. Detection wavelength: 320 nm; sample introduction amount: 2 mu L of the solution; collecting time: 39 min; column temperature: 40 ℃; flow rate: 0.4 mL/min; the theoretical plate number is not less than 6000.
(6) Precision suction of sibiricose A5With sibicose A6Appropriate amount of control solution, gradually diluting with methanol for 1, 2, 4, 8, 16 and 32 times, and shaking. And (4) carrying out sample injection analysis according to the chromatographic condition in the item (5), wherein the sample injection amount is 2 mu L, and determining the peak area. And drawing a standard curve by taking the peak area as a vertical coordinate (Y) and the mass concentration as a horizontal coordinate (X) to obtain a linear regression equation. Compound sibiricose A5The linear regression equation of (1) is that (1.0) is multiplied by (10)7X+54664(R20.9995); compound sibicoseA6The linear regression equation of (1) is that (1.0) is multiplied by (10)7X+22829(R2＝0.9997)。
(7) And (3) sample content determination: measuring the compound sibiricose A in 7 batches of polygala tenuifolia samples according to the chromatographic conditions in the item (5)5With sibicose A6The results are shown in Table 2. As can be seen from Table 2, the sibicose A selected in example 15With sibicose A6Radix Polygalae with low content except No.2 and No. 7The 5 polygala samples all amplified a strip with the length of 629bp, and the compound of the 5 polygala samples is the sibiricose A5With sibicose A6Low content of sibicose A5The contents are all lower than 1.650mg/g, sibicose A6The content is less than 1.400 mg/g.
TABLE 27 Compound sibiricose A in Polygala tenuifolia samples5With sibicose A6Mass fraction (mg/g)
SEQUENCE LISTING
<110> university of Shanxi
<120> method for detecting content of sibicose A5 and sibicose A6 in polygala tenuifolia
<130>.
<160>3
<170>PatentIn version 3.5
<210>1
<211>17
<212>DNA
<213>Polygala tenuifolia Willd.
<400>1
agatggtgag cgtgatt 17
<210>2
<211>17
<212>DNA
<213>Polygala tenuifolia Willd.
<400>2
caagccctct ttcaata 17
<210>3
<211>629
<212>DNA
<213>Polygala tenuifolia Willd.
<400>3
caagccctct ttcaatatgc aatctggcaa tggaggcaac ttgtaaagtg caatgtcaag 60
aagcaccttc acccctttga cagattcagg aagcaaatac tctgaccagc ttgagaagcc 120
aatcctctca aggcttgaat tggtaattga ttcagtcaaa gcagtgacag tattagttgc 180
caccatagaa tcaaccagct caggatacat ttcagccatc ttaaaaccaa ccattccacc 240
ataactaaaa ccaaccaaga tgcatttctc aactccgatc ttcttcaatc ctctcgctac 300
acactcagct tgaaaagcag gtgatctctc aagcttgtct gtgcttgagc tcccaaagaa 360
aatgaaatct gggacatata catcatatgt tcttgctaga cctaaaacct ggaactgcca 420
tgttaagata ccatcaaatg caaacccatg aagaaacact acagaaggct tagcaggacc 480
cttatatcta cttgatgacg ttggtttgat agggacccag aagttcatga cagttcctgg 540
ttcgatttca acaatctggg ttttcattcc taccaatttc aacagtaacc ccagtagaga 600
tttgtatgca gcaatcacgc tcaccatct 629
Claims (4)
1. Detection of sibiricose A in polygala tenuifolia medicinal material5With sibicose A6The content of the PCR primer is characterized in that the PCR primer is as follows:
an upstream primer: 5'-AGATGGTGAGCGTGATT-3', respectively;
a downstream primer: 5'-CAAGCCCTCTTTCAATA-3' are provided.
2. Detection of sibiricose A in polygala tenuifolia medicinal material5With sibicose A6The content method is characterized by comprising the following steps:
(1) extracting genome DNA of polygala tenuifolia samples;
(2) performing PCR amplification using the PCR primer according to claim 1 using the genomic DNA as a template;
(3) detection of the contained compound, namely, the sibiricose A, by PCR amplification products5With sibicose A6Can amplify polygala tenuifolia samples with a strip length of 629bp, and contains the compound of sibiricose A5The content of (A) is less than 1.650mg/g, sibicose A6The content of (B) is less than 1.400 mg/g.
3. The method according to claim 2, wherein in the step (1), the genomic DNA is extracted by CTAB method.
4. The method according to claim 2, wherein in step (1), the polygala tenuifolia is a polygala tenuifolia sample at any tissue site and at any growth stage.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101590065A (en) * | 2008-05-30 | 2009-12-02 | 中国人民解放军总医院 | Siberia Radix Polygalae sugar A1, Siberia Radix Polygalae sugar A5 and the tenuifoliside A application in preparation treatment depression product |
| CN104818337A (en) * | 2015-05-13 | 2015-08-05 | 山西大学 | Method for identifying Fenyuan No.2 Yuanzhi by using specific primers |
| CN106701921A (en) * | 2016-12-01 | 2017-05-24 | 辽宁师范大学 | Primer for real-time fluorescence PCR (Polymerase Chain Reaction) specific detection on polygala tenuifolia and detection method |
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- 2017-06-26 CN CN201710495176.2A patent/CN107254528B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101590065A (en) * | 2008-05-30 | 2009-12-02 | 中国人民解放军总医院 | Siberia Radix Polygalae sugar A1, Siberia Radix Polygalae sugar A5 and the tenuifoliside A application in preparation treatment depression product |
| CN104818337A (en) * | 2015-05-13 | 2015-08-05 | 山西大学 | Method for identifying Fenyuan No.2 Yuanzhi by using specific primers |
| CN106701921A (en) * | 2016-12-01 | 2017-05-24 | 辽宁师范大学 | Primer for real-time fluorescence PCR (Polymerase Chain Reaction) specific detection on polygala tenuifolia and detection method |
Non-Patent Citations (2)
| Title |
|---|
| Possible mechanism of the antidepressant effect of 3,6’-disinapoyl sucrose from Polygala tenuifolia Willd;Yuan Hu等;《Journal of Pharmacy and Pharmacology》;20110503;第63卷;第869-874页 * |
| 山西省西伯利亚远志种质资源的ISSR分析;刘超等;《热带农业科学》;20150415;第35卷(第4期);第45-50页 * |
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