CN107619876A - A kind of method for identifying molecules of Epimedium sagittatum and Sky-High Hill barrenwort - Google Patents
A kind of method for identifying molecules of Epimedium sagittatum and Sky-High Hill barrenwort Download PDFInfo
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- CN107619876A CN107619876A CN201711054789.9A CN201711054789A CN107619876A CN 107619876 A CN107619876 A CN 107619876A CN 201711054789 A CN201711054789 A CN 201711054789A CN 107619876 A CN107619876 A CN 107619876A
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- barrenwort
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Abstract
The invention discloses a kind of Epimedium sagittatum and the method for identifying molecules of Sky-High Hill barrenwort, it comprises the following steps：1) testing sample DNA is extracted；2) using testing sample DNA as template, fragment of the PCR amplifications containing chloroplaset psbA trnH sequences；3) amplified production is spliced, obtains complete excision psbA trnH sequences；4) K2P models are based on, the phylogenetic tree of spliced psbA trnH sequences is sequenced in structure.The inventive method strong applicability, it is simple to operate, the quick and precisely identification of Epimedium sagittatum and Sky-High Hill barrenwort can be realized, identifies great amount of samples in a short time.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to point of a kind of Epimedium sagittatum and Sky-High Hill barrenwort
Sub- authentication method.
Background technology
Epimedium Epimedium L. plants are perennial herb, are ancient original dicotyledon monoids, are planting
It is in special sort status in thing systematic evolution tree, and one of traditional important medicinal plant in China, there is kidney-replenishing, strengthening tendons
The effect of bone, wind-damp dispelling, and there is obvious effect helping pregnant, anti-osteoporosis, improving immunity and suppressing tumour etc..
《Chinese Pharmacopoeia》Version in 2015 has included 5 kinds of barrenwort：Barrenwort E.breviconu Maxim., Epimedium sagittatum
E.sagittatum (Sieb.et Zucc.) Maxim., E. Pubescens E.pubescens Maxim., korean epimedium herb
E.koreanum Nakai and E. wushanense T. S. Ying E.wushanense T.S.Ying.Barrenwort form is extremely similar, greatly
The big difficulty for adding Morphological Identification.Epimedium sagittatum and its nearly edge species Sky-High Hill barrenwort E.myrianthum
Stearn classification problem is extensively disputed on always, is easily caused simply by virtue of the fine difference on ammonia configuration to carry out discriminating to it
Differentiate the generation of the problems such as inaccurate.The base of medicinal material is accurately the premise of clinical accurate medication, it is therefore desirable to which one kind can be to arrow
The method that leaf barrenwort and its nearly edge species Sky-High Hill barrenwort are quick and precisely identified.
DNA bar code technology completes the identification to species according to species DNA difference, has broken away from traditional form identification side
Method relies on the constraint of protracted experience, is not influenceed by the features such as individual morphology, size and integrality and experience limits, can be directly
There is provided abundant discriminating foundation from gene level, operating procedure is easy, has the advantages that stable, efficient, accurate, is traditional mirror
Determine effective supplement of method.The present invention uses DNA bar code technology, can realize and Epimedium sagittatum and Sky-High Hill barrenwort are entered
Row is quick and precisely identified.
The content of the invention
It is an object of the invention to provide differentiate Epimedium sagittatum and the excessive sheep in Sky-High Hill using chloroplaset psbA-trnH
The method of the leaves of pulse plants.
Specifically include following steps：Epimedium sagittatum and the fresh blade of Sky-High Hill barrenwort are taken, in extracting genome DNA
Before, with 75% ethanol surface.
The authentication method of Epimedium sagittatum provided by the invention and Sky-High Hill barrenwort, including PCR amplification chloroplasets psbA-
TrnH sequences.Specifically comprise the following steps：
1) testing sample DNA is extracted；
2) PCR expands the fragment containing chloroplaset psbA-trnH sequence；
3) two-way sequencing is carried out to amplified production, splicing, removes primer, obtains complete excision psbA-trnH sequence.
Amplimer sequence is used in the present invention：
Forward primer PA：5′-GTTATGCATGAACGTAATGCTC-3′；
Reverse primer TH：5′-CGCGCATGGTGGATTCACAATCC-3′.
The system of the PCR amplifications is every 25 μ L：
2 × Taq PCR Mix 12.5 μ L, each 1 μ L of 2.5 μm of ol/L primers, DNA profiling 2 μ L (about 30ng), ddH2O 8.5
μL。
The condition of the PCR amplifications is 94 DEG C of denaturation 1min, and 55 DEG C of annealing 1min, 72 DEG C extend 1.5min, 30 circulations.
4) K2P models are based on, the phylogenetic tree of spliced psbA-trnH sequence is sequenced in structure.
Compared with the methods of traditional Morphological Identification, physics and chemistry identification, the present invention is simple to operate, as a result accurately, is obtained
PsbA-trnH sequence help to realize the Rapid identification of Epimedium sagittatum and Sky-High Hill barrenwort, shorten qualification time.
Brief description of the drawings
Fig. 1 show the inter-species variant sites figure of Epimedium sagittatum and Sky-High Hill barrenwort；
The adjoining (NJ) that Fig. 2 show Epimedium sagittatum and Sky-High Hill barrenwort based on psbA-trnH sequence structure is set.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
Embodiment 1
1. collect 3 parts of Epimedium sagittatum original plant from Anhui Province and Hubei Province；Sky-High Hill is collected from Guizhou Province different regions
4 parts of barrenwort original plant.Details are shown in Table 1：
The Epimedium sagittatum of table 1 and Sky-High Hill barrenwort sample message table
| Sequence number | Sample number into spectrum | Sample ID | Collect ground |
| 1 | JY01 | Epimedium sagittatum | Anhui Province |
| 2 | JY02 | Epimedium sagittatum | Anhui Province |
| 3 | JY03 | Epimedium sagittatum | Hubei Province |
| 4 | TPS01 | Sky-High Hill barrenwort | Guizhou Province |
| 5 | TPS02 | Sky-High Hill barrenwort | Guizhou Province |
| 6 | TPS03 | Sky-High Hill barrenwort | Guizhou Province |
| 7 | TPS04 | Sky-High Hill barrenwort | Guizhou Province |
2. the extraction of Epimedium sagittatum and Sky-High Hill barrenwort DNA
Epimedium sagittatum and the fresh blade of Sky-High Hill barrenwort are taken, with 75% ethanol surface, takes about 30mg samples, is used
After DNA extraction bevellers (Retsch MM400, Germany) grinding 2min (30 time/second), extracted and tried with plant genome DNA
Agent box (Tiangen Biotech Co., China) extracts STb gene, 65 DEG C of water-bath 40min, and remaining step illustrates according to kit
Carry out.DNA be stored in -20 DEG C it is standby.
3.PCR is expanded
PsbA-trnH sequence primer is：
Forward primer PA：5′-GTTATGCATGAACGTAATGCTC-3′；
Reverse primer TH：5′-CGCGCATGGTGGATTCACAATCC-3′.
Synthesized by Co., Ltd of Sheng Gong bio-engineering corporations (Beijing).Primer sterilizing distilled water dissolves and is diluted to 2.5 μ
mol/L。
PCR reaction volumes are 25 μ L, and system includes 2 × Taq PCR Mix 12.5 μ L, each 1 μ L of 2.5 μm of ol/L primers,
DNA profiling 2 μ L (about 30ng), ddH2O 8.5μL。
PCR response procedures：PsbA-trnH primer sequences carry out amplified reaction in PCR instrument according to following procedure：
PCR primer short-term preservation should be stored in -20 DEG C for a long time in 4 DEG C.
4. sequencing
PCR primer is directly sent Chinese Academy of Agricultural Sciences's Important Project laboratory be sequenced.The same PCR primer of sequencing primer.
5. sequence assembly
Sequence assembly is carried out to two-way sequencing peak figure using CodonCode Aligner V5.2.0 softwares, removes guiding region
And low mass region.
6. sequence alignment
The psbA-trnH sequence of sequencing and spliced each sample is compared with MEGA6.0 softwares, compares postorder
Row length is 518bp.As a result show, the larger (figure of psbA-trnH sequence intraspecific variablity of Epimedium sagittatum and Sky-High Hill barrenwort
1), sequence is respectively as shown in SEQ ID No.1-3 and SEQ ID No.4-7.
7. being based on K2P models, the phylogenetic tree of spliced psbA-trnH sequence is sequenced in structure, can be excessive by arrow leaf
The sheep leaves of pulse plants effectively separates (Fig. 2) with Sky-High Hill barrenwort.
Claims (7)
1. a kind of method for identifying molecules for Epimedium sagittatum and Sky-High Hill barrenwort, including PCR amplification chloroplasets psbA-
TrnH sequences.
2. according to the method for claim 1, comprise the following steps：
1) sample DNA is extracted；
2) PCR expands the fragment of chloroplaset psbA-trnH sequence；
3) amplified production is spliced, obtains chloroplaset psbA-trnH sequence；
4) K2P models are based on, the phylogenetic tree of spliced psbA-trnH sequence is sequenced in structure.
3. according to the method for claim 2, it is characterised in that the sample is Epimedium sagittatum and Sky-High Hill barrenwort.
4. method according to claim 1 or 2, it is characterised in that the primer of PCR amplification is：
Forward primer PA：5′-GTTATGCATGAACGTAATGCTC-3′；
Reverse primer TH：5′-CGCGCATGGTGGATTCACAATCC-3′.
5. method according to claim 1 or 2, it is characterised in that the system of the PCR amplifications is every 25 μ L：2×Taq
PCR Mix 12.5 μ L, each 1 μ L of 2.5 μm of ol/L primers, DNA profiling 2 μ L (about 30ng), ddH2O 8.5μL。
6. method according to claim 1 or 2, it is characterised in that the condition of the PCR amplifications is 94 DEG C of denaturation 1min,
55 DEG C of annealing 1min, 72 DEG C of extension 1.5min, 30 circulate.
7. any one of claim 1-6 methods described is differentiating the application of Epimedium sagittatum and Sky-High Hill barrenwort.
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| CN201711054789.9A CN107619876A (en) | 2017-11-01 | 2017-11-01 | A kind of method for identifying molecules of Epimedium sagittatum and Sky-High Hill barrenwort |
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| CN201711054789.9A CN107619876A (en) | 2017-11-01 | 2017-11-01 | A kind of method for identifying molecules of Epimedium sagittatum and Sky-High Hill barrenwort |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114085920A (en) * | 2021-10-18 | 2022-02-25 | 贵州贵基生物医药有限公司 | DNA bar code and primer pair of epimedium herb and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1190894A (en) * | 1995-06-07 | 1998-08-19 | 路德维格癌症研究所 | Method for identifying or isolating a molecule and molecules indentified thereby |
| CN103805522A (en) * | 2014-02-25 | 2014-05-21 | 福建农林大学 | Pleurotus eryngii var. tuoliensis and molecular marker identification method thereof |
-
2017
- 2017-11-01 CN CN201711054789.9A patent/CN107619876A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1190894A (en) * | 1995-06-07 | 1998-08-19 | 路德维格癌症研究所 | Method for identifying or isolating a molecule and molecules indentified thereby |
| CN103805522A (en) * | 2014-02-25 | 2014-05-21 | 福建农林大学 | Pleurotus eryngii var. tuoliensis and molecular marker identification method thereof |
Non-Patent Citations (1)
| Title |
|---|
| YU JIANG ET AL: "Identification of the genus Epimedium with DNA barcodes", 《JOURNAL OF MEDICINAL PLANTS RESEARCH》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114085920A (en) * | 2021-10-18 | 2022-02-25 | 贵州贵基生物医药有限公司 | DNA bar code and primer pair of epimedium herb and application thereof |
| CN114085920B (en) * | 2021-10-18 | 2024-01-26 | 贵州贵基生物医药有限公司 | DNA bar code of epimedium, primer pair and application thereof |
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Application publication date: 20180123 |