CN106434988A - Wu-he dipsacus asper ISSR-PCR molecule marking method - Google Patents
Wu-he dipsacus asper ISSR-PCR molecule marking method Download PDFInfo
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- CN106434988A CN106434988A CN201611074613.5A CN201611074613A CN106434988A CN 106434988 A CN106434988 A CN 106434988A CN 201611074613 A CN201611074613 A CN 201611074613A CN 106434988 A CN106434988 A CN 106434988A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention relates to a wu-he dipsacus asper ISSR-PCR molecule marking method. The method is characterized by comprising the following steps: 1, extracting the genome of the wu-he dipsacus asper, 2, conducting 1% agarose electrophoresis for 20 minutes, testing the integrity of the genome, at the same time using the DNA bar code ITS2 primer to conduct the PCR amplification of the extracted wu-he dipsacus asper DNA, verifying the purity of the extracted DNA, 3, based on the initial screened primer conducting the ISSR-PCR. Compared with the prior art, the reaction system created by the method has a simple optimization method. The amplified bands is both clear and stable with good polymorphism. The method compensates the shortcomings in the study of the genetic diversity of the wu-he dipsacus asper. The method can be used in the analysis of the genetic relationship and the verification of authenticity of the teasle, thus having good application value.
Description
Technical field
The present invention relates to biology field, especially relate to a kind of pentanucleotide repeats ISSR-PCR molecule labelling method.
Background technology
Radix Dipsaci is a kind of conventional Chinese medicine, is Radix Dipsaci（Dipsacus asper Wall.）Dry root.Have nourishing the liver and kidney,
The curative effects such as strong muscles and bones, arresting bleeding and miscarriage prevention；And originate in Enshi State of Hubei Province Hefeng County and the Radix Dipsaci in Wu Feng county of Yichang City, Yin Qigen
The excellent quality such as portion is sturdy and even thickness, quality are relatively soft, is assigned " pentanucleotide repeats ", is the important genuine medicine in Enshi
One of material.Existing research shows that the quality of Radix Dipsaci medical material is not only variant with place of production difference, and the life between different populations
Thing characteristic etc. there is also more apparent difference, points out Radix Dipsaci to there is abundant bio-diversity.
Current research focuses primarily upon historical Textual Research and DEVELOPMENT PROSPECT and karyotyping, component analyses, Planting characteristic etc.,
And the research of the basic research such as Physiology and biochemistry and genetic diversity is very few.In recent years, ISSR technology obtains in various plants
Arrive application it was demonstrated that its result is reliable and stable, be the effective means of research Genetic Diversity and germplasm identification.
Therefore, set up a relatively scientific and reasonable effective ISSR-PCR reaction system, seek each component of optimal PCR and use
Amount, can analyze for follow-up development pentanucleotide repeats Germplasm Resources Diversity and breeding provides good theoretical direction.
Content of the invention
The problem to be solved in the present invention is to provide a kind of pentanucleotide repeats ISSR-PCR molecule labelling method, simplifies molecular marker
Reaction system optimization step.
A kind of pentanucleotide repeats ISSR-PCR molecule labelling method is it is characterised in that comprise the steps：
1）Extract pentanucleotide repeats genomic DNA；
2）1% sepharose electrophoresis 20min detection genomic DNA integrity, adopts DNA bar code ITS2 primer pair to extract simultaneously
Pentanucleotide repeats DNA enters performing PCR amplification, the DNA purity that checking is extracted；
3）ISSR-PCR is carried out according to primary dcreening operation primer.
A kind of present invention pentanucleotide repeats ISSR-PCR molecule labelling method it is characterised in that：Described ISSR-PCR amplification
Reaction system is：2 × Taq Master Mix 10.44 μ l, template DNA 45ng, primer is contained in the reaction system of 18 μ L
0.35μM.
A kind of present invention pentanucleotide repeats ISSR-PCR molecule labelling method it is characterised in that：Described primer sequence is as follows
Shown.
Primer sequence（5’-3’）
UBC809 AGAGAGAGAGAGAGAGG
UBC815 CTCTCTCTCTCTCTCTG
UBC818 CACACACA CACACACAG
UBC840 GAGAGAGAGAGAGAGAYT
UBC842 GAGAGAGAGAGAGAGAYG
A kind of present invention pentanucleotide repeats ISSR-PCR molecule labelling method it is characterised in that：The expansion of described ISSR-PCR reaction
Increasing condition is：94 DEG C of annealing 5min, then 94 DEG C of 30s, 52 DEG C of 1min, 40 circulations of 72 DEG C of 1.5min, last 72 DEG C are prolonged
Stretch 10min, 4 DEG C of preservations.
The present invention has substantive distinguishing features following outstanding and advantage：The pentanucleotide repeats ISSR-PCR that the present invention is set up is excellent
The band changing reaction system amplification is steady and audible, has preferable polymorphism feature.Operating procedure is less it is easy to quick analysis is examined
Survey.Compensate for the disappearance of the Study on Diversity of local genuine medicinal materials Radix Dipsaci, achievement of the present invention can be used for pentanucleotide repeats genetic diversity
Property analysis, the research of genuine medicinal materials Molecular Identification and biogeography aspect.
Brief description
The amplification that accompanying drawing 1 optimized for the ISSR-PCR Uniform Design first round, 1-12 is the process combination number of table 3, M
For marker, primer is UBC840；
Accompanying drawing 2 is ISSR-PCR Uniform Design second wheel fine setting amplification, and 1-12 is the process combination number of table 3, and M is
Marker, primer is UBC840；
Accompanying drawing 3 is the ISSR-PCR amplification figure of reaction system after nine producing region Radix Dipsaci sample checkings optimize, and 1-9 is 9 places of production
The electrophoretic band of Radix Dipsaci sample ISSR-PCR product；M is marker, and primer is UBC809；
Accompanying drawing 4 is the determination of primer UBC840 optimum annealing temperature, and M is marker, and primer is UBC840；From right to left temperature according to
Secondary it is：65℃、63.8℃、62℃、59.1℃、55.5℃、52.9℃、51.0℃、50℃.
Specific embodiment
With reference to embodiment, the present invention is described in further detail, but the embodiment not limited to this of invention.
Embodiment 1
A kind of present invention pentanucleotide repeats ISSR-PCR molecule labelling method, comprises the following steps：
（1）Sampling and extracting genome DNA
The mortar clear water of grind away is cleaned and dries, light after the dehydrated alcohol adding 5ml, add liquid nitrogen to carry out after fire extinguishes
Pre-cooling, chooses the seedling 0.5g of indoor germination 15 ~ 20d, puts into rapidly in the mortar of pre-cooling after removing seed hull, adds appropriate
Liquid nitrogen, 2 ~ 3 centimetres of sample was not had with liquid nitrogen, ground 3 ~ 5min, and then adopted RNA isolation kit to extract pentanucleotide repeats genome
DNA.
（2）The uniform Design scheme of first round ISSR-PCR reaction system
Uniformly optimization design screening each component optimum dose of ISSR reaction system, using U12(62× 4) mix homogeneously design table,
The factor level of PCR amplification system is shown in Table 2.With UBC840 as primer, using 12 systems in table 3 and table 4 to ISSR-PCR
Reaction system carries out two-wheeled optimal screening.Reaction system 18 μ l, concrete consumption is shown in Table 3 and table 4.ISSR-PCR amplification program is：
94 DEG C of annealing 5min, then 94 DEG C of 30s, 52 DEG C of 1min, 40 circulations of 72 DEG C of 1.5min, last 72 DEG C of extension 10min, 4
DEG C preserve.
The amplimer of table 1 ISSR screening and sequence
| Primer | Sequence 5 ' -3 ' |
| UBC809 | AGAGAGAGAGAGAGAGG |
| UBC815 | CTCTCTCTCTCTCTCTG |
| UBC818 | CACACACA CACACACAG |
| UBC840 | GAGAGAGAGAGAGAGAYT |
| UBC842 | GAGAGAGAGAGAGAGAYG |
The each factor level table of table 2（Unit：μl）
Table 3 ISSR-PCR first round mix homogeneously designs table [U12 (62 × 4)] unit：μl12 2
| Process numbering | Template DNA | Primer | 2×Taq Master Mix | ddH2O |
| 1 | 0.22 | 1.1 | 7.2 | 9.5 |
| 2 | 0.22 | 1.8 | 9 | 7.0 |
| 3 | 0.65 | 3.2 | 5.4 | 8.7 |
| 4 | 0.65 | 4.0 | 10.4 | 3.0 |
| 5 | 1.1 | 0.4 | 7.2 | 9.4 |
| 6 | 1.1 | 1.8 | 5.4 | 9.7 |
| 7 | 1.8 | 2.5 | 10.4 | 3.2 |
| 8 | 1.8 | 4.0 | 9 | 3.2 |
| 9 | 2.5 | 0.4 | 5.4 | 9.7 |
| 10 | 2.5 | 1.1 | 10.4 | 4.0 |
| 11 | 3.2 | 2.5 | 9 | 3.2 |
| 12 | 3.2 | 3.2 | 7.2 | 4.3 |
First round mix homogeneously design experiment result is shown in accompanying drawing 1, and in addition to 9, other several process all have an amplified band, wherein 1,
2nd, 3,5 and 6 all have faint amplified band, and but 10, No. 12 have band not clear, and 4,7,8 and 11 this 4 process bands are clear
Clear stable and amplified band quantity is more, it is more satisfactory process combination.On the basis of this 4 consumption, progressively reduce each
The Concentraton gradient of individual component carries out the second wheel fine tuning Uniform Design.
（3）The uniform Design scheme of the second wheel fine tuning ISSR-PCR reaction system
The uniform Design scheme of fine tuning ISSR-PCR reaction system is shown in Table 4, and result of the test is shown in accompanying drawing 2.Except processing 1 band
Beyond relatively weak, the band clearly becoming clear all has been run out of in remaining several process.4th, 6,7,8,11,12 amplifications appearance are more
Miscellaneous band, and the band that 2,3,5,9 and 10 process are run out of clearly becomes clear, and considers DNA profiling, primer and Taq Master
The consumption of Mix, processing 2 is optimization process, and its each amounts of components is respectively：2 × Taq Master Mix 10.44 μ l, template
DNA 45ng, 0.35 μM of primer.
Table 4 ISSR-PCR second takes turns mix homogeneously design table [U12 (62 × 4)] unit：μl
| Process numbering | Template DNA | Primer | 2×Taq Master Mix | ddH2O |
| 1 | 1.08 | 2.16 | 9.84 | 4.92 |
| 2 | 1.08 | 2.52 | 10.44 | 3.96 |
| 3 | 1.44 | 3.24 | 9.24 | 4.08 |
| 4 | 1.44 | 3.6 | 11.04 | 1.92 |
| 5 | 1.8 | 1.8 | 9.84 | 4.56 |
| 6 | 1.8 | 2.52 | 9.24 | 4.44 |
| 7 | 2.16 | 2.88 | 11.04 | 1.92 |
| 8 | 2.16 | 3.6 | 10.44 | 1.8 |
| 9 | 2.52 | 1.8 | 9.24 | 4.44 |
| 10 | 2.52 | 2.16 | 11.04 | 2.28 |
| 11 | 2.88 | 2.88 | 10.44 | 1.8 |
| 12 | 2.88 | 3.24 | 9.84 | 2.04 |
（4）The checking of the ISSR-PCR reaction system after optimization
Using the reaction system after optimizing, with nine producing region Radix Dipsaci samples as template, UBC809 is primer, is optimized system
PCR verifies, result shows, amplified band steady and audible as accompanying drawing 3, illustrate that this reaction system is suitable for the ISSR of pentanucleotide repeats and divides
Analysis.
（5）The determination of primer optimum annealing temperature
This experiment ISSR primer used comes from the primer sequence that Canadian Columbia University announces, and is responsible for by Shanghai English fine horse
Synthesis.
On the basis of two-wheeled uniform Design determines peak optimization reaction system, primer is carried out on Bole's PCR instrument and most preferably anneals
The determination of temperature, the setting of gradient is set with theoretical annealing temperature Tm-5 DEG C ~ Tm+5 DEG C, arranges 8 thermogrades altogether.Sieve
The primer optimum annealing temperature relevant information going out is shown in Table 5.
Table 5 primer optimum annealing temperature result
, from accompanying drawing 4 as can be seen that when annealing temperature is higher, almost there is no visible amplified band taking primer UBC840 as a example,
Illustrate that amplification efficiency is not high.When annealing temperature is gradually lowered it can be seen that amplified band gradually clearly becomes clear, band increases.
When annealing temperature is at 52.9 DEG C, amplified band number is more and clear, it is thus determined that the optimum annealing temperature of this primer is 52.9
℃.In the same manner, other primer optimum annealing temperatures are also followed this principle and are determined.
A kind of present invention pentanucleotide repeats ISSR-PCR molecule labelling method, with the pentanucleotide repeats DNA that gathers as sample, adopts
Mix homogeneously method for designing, to the template DNA consumption of impact ISSR reaction, primer concentration and 2 × Taq Master Mix this 3
Individual factor carries out gradient test, optimizes pentanucleotide repeats ISSR-PCR reaction system, and with the Radix Dipsaci DNA in 9 other producing regions as sample
Product, the ISSR-PCR reaction system after checking optimization.On the basis of optimization system is set up, temperature is carried out to each primer of screening
Degree grads PCR, determines primer optimum annealing temperature.Result shows, in 18 μ l reaction systems, when in system contain 2 × Taq
When Master Mix 10.44 μ l, template DNA 45ng, 0.35 μM of primer, and annealing temperature are optimal, expanding effect is best.
SEQUENCE LISTING
<110>Research Institute of Traditional Chinese Medicine
<120>A kind of pentanucleotide repeats ISSR-PCR molecule labelling method
<130> 104
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 17
<212> DNA
<213>Artificial sequence
<400> 1
agagagagag agagagg 17
<210> 2
<211> 17
<212> DNA
<213>Artificial sequence
<400> 2
ctctctctct ctctctg 17
<210> 3
<211> 17
<212> DNA
<213>Artificial sequence
<400> 3
cacacacaca cacacag 17
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence
<400> 4
gagagagaga gagagayt 18
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence
<400> 5
gagagagaga gagagayg 18
Claims (4)
1. a kind of pentanucleotide repeats ISSR-PCR molecule labelling method is it is characterised in that comprise the steps：
1) extract pentanucleotide repeats genomic DNA；
2) 1% sepharose electrophoresis 20min detection genomic DNA integrity, adopts DNA bar code ITS2 primer pair to extract simultaneously
Pentanucleotide repeats DNA enters performing PCR amplification, the DNA purity that checking is extracted；
3) ISSR-PCR is carried out according to primary dcreening operation primer.
2. a kind of pentanucleotide repeats ISSR-PCR molecule labelling method according to claim 1 it is characterised in that：Described
The reaction system of ISSR-PCR amplification is：2 × Taq Master Mix 10.44 μ l, template is contained in the reaction system of 18 μ L
DNA 45ng, 0.35 μM of primer.
3. a kind of pentanucleotide repeats ISSR-PCR molecule labelling method according to claim 2 it is characterised in that：Described draws
Thing sequence is as follows.
4. a kind of pentanucleotide repeats ISSR-PCR molecule labelling method according to claim 1 it is characterised in that：Described
ISSR-PCR reaction amplification condition be：94 DEG C of annealing 5min, then 94 DEG C of 30s, 52 DEG C of 1min, 72 DEG C of 1.5min 40 follow
Ring, last 72 DEG C of extension 10min, 4 DEG C of preservations.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048592A (en) * | 2017-12-19 | 2018-05-18 | 广西壮族自治区林业科学研究院 | Hainan wind nanmu ISSR-PCR molecule labelling methods and its primer special |
| CN109055595A (en) * | 2018-09-07 | 2018-12-21 | 湖北省农业科学院中药材研究所 | A kind of Herba Epimedii ISSR-PCR molecule labelling method |
| CN109082476A (en) * | 2018-07-05 | 2018-12-25 | 湖北省农业科学院中药材研究所 | A kind of Emperor XianFeng's Rhizoma Atractylodis Macrocephalae ISSR molecular labeling optimization system construction method |
| CN112195262A (en) * | 2020-10-16 | 2021-01-08 | 湖北省农业科学院中药材研究所 | ISSR-PCR (inter-simple sequence repeat-polymerase chain reaction) amplification reaction system of Hubei fritillary bulb, ISSR-PCR molecular marking method and application |
-
2016
- 2016-11-30 CN CN201611074613.5A patent/CN106434988A/en active Pending
Non-Patent Citations (6)
| Title |
|---|
| D.X.CHEN ET AL: "Genetic diversity and population structure of wild Dipsacus asperoides in China as indicated by ISSR markers", 《GENETICS AND MOLECULAR RESEARCH》 * |
| 李晓玲等: "10份续断属植物亲缘关系的ISSR分析", 《植物研究》 * |
| 王瑜等: "紫花苜蓿ISSR-PCR反应体系的建立与优化", 《草地学报》 * |
| 罗洪斌等: "道地药材五鹤续断基因组脱氧核糖核酸提取方法的改进", 《时珍国医国药》 * |
| 赵志新等: "玄参ISSR-PCR反应体系的建立与优化", 《药物生物技术》 * |
| 雷美艳等: "基于ITS2条形码序列鉴定中药材续断及其混伪品", 《四川农业大学学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048592A (en) * | 2017-12-19 | 2018-05-18 | 广西壮族自治区林业科学研究院 | Hainan wind nanmu ISSR-PCR molecule labelling methods and its primer special |
| CN109082476A (en) * | 2018-07-05 | 2018-12-25 | 湖北省农业科学院中药材研究所 | A kind of Emperor XianFeng's Rhizoma Atractylodis Macrocephalae ISSR molecular labeling optimization system construction method |
| CN109055595A (en) * | 2018-09-07 | 2018-12-21 | 湖北省农业科学院中药材研究所 | A kind of Herba Epimedii ISSR-PCR molecule labelling method |
| CN112195262A (en) * | 2020-10-16 | 2021-01-08 | 湖北省农业科学院中药材研究所 | ISSR-PCR (inter-simple sequence repeat-polymerase chain reaction) amplification reaction system of Hubei fritillary bulb, ISSR-PCR molecular marking method and application |
| CN112195262B (en) * | 2020-10-16 | 2023-03-14 | 湖北省农业科学院中药材研究所 | ISSR-PCR (inter-simple sequence repeat-polymerase chain reaction) amplification reaction system of Hubei fritillary bulb, ISSR-PCR molecular marking method and application |
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