CN107557486A - EST SSR primer sets and its acquisition methods based on the exploitation of root of bidentate achyranthes transcript profile - Google Patents
EST SSR primer sets and its acquisition methods based on the exploitation of root of bidentate achyranthes transcript profile Download PDFInfo
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Abstract
The invention discloses a kind of EST SSR primer sets based on the exploitation of root of bidentate achyranthes transcript profile, totally 23 pairs of primers, its nucleotide sequence is as shown in sequence table.The invention further relates to the acquisition methods for the EST SSR primer sets developed based on root of bidentate achyranthes transcript profile.The present invention utilizes the est sequence of root of bidentate achyranthes transcript profile, develops the EST SSR sequences of the root of bidentate achyranthes in batches first, and the SSR repetitive sequence information obtained is directed to mRNA, detection be gene expression difference between different germplasm materials information, directly contacted with phenotype；The research such as structure for the accurate evaluation of root of bidentate achyranthes germ plasm resource, the positioning of specific traits gene, genetic map provides important information platform；By the optimization of PCR reaction systems and its program, a kind of PCR amplification system of suitable identification root of bidentate achyranthes EST SSR markers is established, the EST SSR markers to identify new from the root of bidentate achyranthes provide strong instrument.
Description
Technical field
The present invention relates to technical field of molecular biology, and in particular to a kind of EST-SSR based on the exploitation of root of bidentate achyranthes transcript profile
Primer sets and its acquisition methods.
Background technology
The root of bidentate achyranthes is the plant of Amaranthaceae Achyranthes, has ignite descending, filling liver kidney, invigorate blood circulation, inducing diuresis for treating strangurtia, strengthening the bones and muscles etc.
Effect.It is clinically used for treating the illnesss such as irregular menstruation, dysmenorrhoea, difficult urination, Urethra astringent pain.
The root of bidentate achyranthes is various in style, and genetic background obscures, and variet complexity phenomenon is serious, and homonym problem occurs often, market
On also often sell alternative, the mixed product for having the root of bidentate achyranthes, specification and drug safety to Chinese Medicinal Materials Markets bring certain unfavorable factor；
Simultaneously in addition with miscarriage, Confounding Factor between the limitation of planting area and kind, the seed selection of root of bidentate achyranthes kind drastically influence
And market circulation.
The simple repeated sequence that the principle of EST-SSR molecular labelings is made up of 1-6 nucleotides, is distributed widely in eucaryon
In biological genome；The usually relatively conservative single-copy sequence in SSR sequences both sides, can design a pair of special primers to expand
The site, through polyacrylamide gel electrophoresis, know Different Individual on this site because elementary cell number of repetition is different
And the polymorphism formed.Traditional genome SSR marker development process is relatively slow, it is necessary to carry out the structure of genomic library, repeat
The links such as sequence clone, screening, sequencing, design of primers, time-consuming effort, cost are high, efficiency is low.With the hair of functional genomics
Exhibition, substantial amounts of plant EST have become the valuable source of New molecular marker of the exploitation based on PCR
EST-SSR is as a kind of New molecular marker, and because repetitive sequence information is directed to mRNA, therefore what is detected is not
With the information of gene expression difference between germplasm materials, this species diversity can directly contact with phenotype.EST-SSR is as gene
A part, its usual flanking sequence conservative is higher, thus, the SSR marker based on est sequence exploitation has between different plant species
Good versatility, the research of other species is can be used for from the EST-SSR marks of a species exploitation, utilizes mark heredity to make
The conversion for making linkage information between species faster, can serve as calibration marker by figure, realize that multiple collection of illustrative plates are integrated, and then to compare base
Because group and homologous gene clone provide new approach.The clone during genome SSR primer developments and survey are eliminated simultaneously
Sequence step, existing sequencing data is taken full advantage of, reduces development cost.Due to above-mentioned advantage so that EST-SSR marks are being lost
It is numerous to pass map construction, Relationship iden- tification, functional gene positioning and comparative map and molecular marker assisted selection breeding etc.
Research field has higher application value.
With the fast development of Modern Molecular Biotechnology, EST-SSR polymorphisms are excavated on a large scale using computer approach
Site drastically increases SSR marker development efficiency.What is more important, the application of EST-SSR labelling techniques are believed for genome
The research of fruit germplasm resource for ceasing deficient species provides strong instrument.EST-SSR is developed using transcript profile, will be carried forward vigorously
The research and improved Varieties seed selection of Medicinal Plant Germplasm Resources.
Because root of bidentate achyranthes hereditary basis is deficient, genomic information not yet discloses, and at present, the correlative study of root of bidentate achyranthes molecular labeling is equal
It is that the research of root of bidentate achyranthes germ plasm resource is carried out according to the universal primer in other plant molecular labeling site, can not finds special in the root of bidentate achyranthes
Different molecular labeling, because these marks used are without specificity, it is impossible to the heredity back of the body of accurate evaluation root of bidentate achyranthes germ plasm resource
Scape.It would therefore be highly desirable to establish effective EST-SSR molecular labelings, and then build fast and efficiently kind（System）Identification system, use
In the Phylogenetic Analysis of analysis root of bidentate achyranthes germplasm, its genetic fingerprint collection of illustrative plates is built, base is established for the seed selection of root of bidentate achyranthes improved Varieties
Plinth.
The content of the invention
The technical problem to be solved in the present invention is to provide it is a kind of based on the root of bidentate achyranthes transcript profile exploitation EST-SSR primer sets and its
Acquisition methods.
In order to solve the above technical problems, the technical scheme specifically used is as follows：
A kind of EST-SSR primer sets based on the exploitation of root of bidentate achyranthes transcript profile are designed, the primer sets include following 23 pairs of primers, and each pair is drawn
Thing is made up of sense primer and anti-sense primer, the nucleotide sequence such as sequence table SEQ ID NO of each pair primer:1 to SEQ ID
NO:Shown in 48.
A kind of method for obtaining the EST-SSR primer sets based on the exploitation of root of bidentate achyranthes transcript profile is designed, is comprised the following steps：
（1）Root of bidentate achyranthes transcript profile library is built, transcript profile sequencing data in library is spliced, de-redundancy obtains Unigenes；
（2）EST-SSR site screenings are carried out to Unigenes using MISA softwares；
（3）The EST-SSR primers that design of primers is designed are carried out to EST-SSR sites with the softwares of Primer 3；
（4）Root of bidentate achyranthes genomic DNA is extracted using CTAB methods, entering performing PCR with EST-SSR primer pair roots of bidentate achyranthes DNA expands, and then carries out
Agarose gel electrophoresis, EST-SSR molecular labeling primers are verified according to electrophoresis result.
The acquisition methods step of EST-SSR primer sets based on the exploitation of root of bidentate achyranthes transcript profile（2）In, what SSR can be detected
The parameter of repetitive sequence is arranged to：Mononucleotide is at least repeated 10 times, and dinucleotides is at least repeated 6 times, and trinucleotide is at least heavy
Multiple 5 times, tetranucleotide is at least repeated 5 times, and pentanucleotide is at least repeated 5 times, and Hexanucleotide is at least repeated 5 times.
The acquisition methods step of EST-SSR primer sets based on the exploitation of root of bidentate achyranthes transcript profile（4）In, the reactant of PCR amplifications
It is to be：Overall reaction system is 20 μ L, and 2~24 mg/ L DNA is included in system；0.05~0.6 μm of ol/L primer；50~
400 μm of ol/L dNTPs and 5~80 U/L archaeal dna polymerase, react 25~35 at being 57~60 DEG C in annealing temperature
PCR cycle.
Preferable pcr amplification reaction system be 20 μ L, included in system 6 mg/ L DNA, 0.2 μm of ol/L primer,
200 μm of ol/L dNTPs and 60 U/L archaeal dna polymerase, react 35 PCR cycles at being 57 DEG C in annealing temperature.
The advantageous effects of the present invention are：
1. the present invention utilizes the est sequence of root of bidentate achyranthes transcript profile, the EST-SSR sequences of the root of bidentate achyranthes are developed in batches first, due to obtaining
SSR repetitive sequence information be directed to mRNA, rather than genome, therefore the gene table between being different germplasm materials detected
Up to the information of difference so that this species diversity can directly contact with phenotype.
It is accurate evaluation, the specific traits of root of bidentate achyranthes germ plasm resource 2. the present invention develops the distinctive EST-SSR marks of the root of bidentate achyranthes
The researchs such as the positioning of gene, the structure of genetic map provide important information platform.
3. the present invention on the basis of economizing on resources, by the optimization of PCR reaction systems and its program, establishes a kind of suitable
The PCR amplification system of identification root of bidentate achyranthes EST-SSR marks is closed, the EST-SSR marks to identify new from the root of bidentate achyranthes have provided
The instrument of power.
Brief description of the drawings
Fig. 1 is the root of bidentate achyranthes DNA agarose electrophoresis testing result figures of extraction；
Fig. 2 is influence figure of the different DNA concentrations to EST-SSR pcr amplification product；
Fig. 3 is influence figure of the different primer concentrations to EST-SSR pcr amplification product；
Fig. 4 is influence figure of the different dNTPs concentration to EST-SSR pcr amplification product；
Fig. 5 is influence figure of the different DNA polymerase concentrations to EST-SSR pcr amplification product；
Fig. 6 is influence figure of the different PCR response procedures to EST-SSR pcr amplification product；
Fig. 7 is the EST-SSR sequence pcr amplification product electrophoresis result figures of identification.
Embodiment
Illustrate the embodiment of the present invention with reference to the accompanying drawings and examples, but following examples are used only in detail
Describe the bright present invention in detail, the scope not limiting the invention in any way.Involved instrument and equipment is such as in the examples below
It is routine instrument device without special instruction；Involved reagent is commercially available conventional reagent unless otherwise instructed.It is involved
Experimental method, be normal experiment method unless otherwise instructed.
Embodiment 1：
（1）The root of bidentate achyranthes transcript profile library of structure,
Plantation 1 year is collected respectively（R1）With continuous plantation 2 years（R2）" radix achyranthis bidentatae 1 " kind seed forms the root system of phase（Repeat 3
It is secondary, totally 6 samples）, after extracting total serum IgE with Trizol methods, with Oligo（dT）Enrichment with magnetic bead mRNA.Add
MRNA is broken into short-movie section by fragmentation buffer, using mRNA as template, with 6 base random primers（random
hexamers）Reverse transcription synthesizes first cDNA chain, then adds buffer solution, dNTPs, RNase H and DNA polymerase
I synthetic double chain cDNA chains, then purify double-strand cDNA, adjunction head with AMPure XP beads, cDNA first carries out end reparation plus A
Tail simultaneously connects sequence measuring joints, then carries out clip size selection with AMPure XP beads.Finally enter performing PCR amplification, be used in combination
AMPure XP beads purified pcr products, 6 sequenators of sequencing library Illumina HiSeq 4000 built up are surveyed
Sequence.Original reads caused by 6 sequencing storehouses are common, original reads are after removing joint, remove low-quality reads, 6 storehouse common properties lifes
268,190,758 clean reads sequences, sequence assembly is carried out using Trinity softwares, the final average length that obtains is
The 87 of 1060 bp, 256 Unigenes sequences, detailed data is as shown in table 1, wherein wherein, R1-1, R1-2, R1-3 difference table
Show that three biology of R1 samples repeat；R2-1, R2-2, R2-3 represent that three biology of R2 samples repeat respectively；Q20、
Q30 represents that base of the Phred numerical value more than 20,30 accounts for the percentage of overall base respectively.
Reads quality and quantities caused by 6 sequencing storehouses of the root of bidentate achyranthes root of table 1 collect
（2）Transcript profile library is sequenced, sequencing total length is 92,453,545bp, using MISA softwares to the root of bidentate achyranthes 87,256
Individual Unigenes carries out EST-SSR site screenings, and the parameter setting for the repetitive sequence that each SSR can be detected is respectively：
Mononucleotide is at least repeated 10 times, and dinucleotides is at least repeated 6 times, and trinucleotide is at least repeated 5 times, and tetranucleotide at least repeats
5 times, pentanucleotide is at least repeated 5 times, and Hexanucleotide is at least repeated 5 times.SSR of the length of identification more than 18bp has 23,844
It is individual；Unigenes comprising sequence contained by SSR has 19,045；Unigenes wherein more than 1 SSR has 3,822；SSR
The Unigenes quantity for being repetitively appearing in more than 2 is 1,371.
（3）According to SSR flanking sequence conservatisms, with the Software for Design SSR sites both sides primers of Primer 3, design of primers
Parameter be arranged to default parameters, randomly selected 23 EST-SSR sequences and its primer are as shown in table 2.
The EST-SSR primer sets that table 2 is identified based on root of bidentate achyranthes transcript profile
（4）The genomic DNA of " radix achyranthis bidentatae 1 " treating to plant extensively using the extraction of CTAB methods, with 1% agarose gel electrophoresis
DNA integralities and RNA pollution conditions are detected, DNA concentration and purity are detected with NanDrop1000 detectors.As shown in figure 1, carry
Bands of the DNA taken through electrophoresis detection is bright, clear, the de- band of nothing.The amount of DNA of 1~4 swimming lane is respectively 2 μ g, 1.5 μ g, 1 μ
g、0.5 µg。
Enter performing PCR to root of bidentate achyranthes DNA with the EST-SSR primers Cluster-6082.39339 of the design to expand, Ran Houjin
Row agarose gel electrophoresis, EST-SSR molecular labeling primers are verified according to electrophoresis result.
Embodiment 2
Analysis is to the influence situation of EST-SSR pcr amplification product under different PCR treatment conditions, and experiment and analysis result are such as
Under：
（1）Pcr amplification reaction system is 20 μ L, Taq DNA polymerase concentrations（60 U/L）, positive and negative primer concentration（0.2μ
mol/L）, dNTP concentration（200 μmol/L）, template DNA set 8 dosage gradients, respectively 2,4,6,8,10,12,
14th, 16,18,20,22,24 mg/ L, carried out in the type PCR amplification instruments of American AB I 2720.Response procedures are：94 DEG C of pre-degenerations
2 minutes；94 DEG C of denaturation are annealed 30 seconds, 72 DEG C for 30 seconds, 57 DEG C to be extended 40 seconds, 30 circulations；72 DEG C extend 5 minutes；With 1% fine jade
Sepharose electrophoresis detection.
As a result as shown in Fig. 2 M swimming lanes are DL2000 Marker, root of bidentate achyranthes DNA concentration equal energy in the range of 2~24 mg/ L
Special clear band is obtained, wherein being 6mg/ L in DNA concentration（3rd swimming lane）When the band that obtains it is the brightest.
（2）In other experiment conditions and step（1）In the case of middle pcr amplification reaction system identical, primer concentration is set
12 gradients（Positive anti-primer is identical）, i.e., 0.05,0.1,0.15,0.2,0.25,0.3,0.35,0.4,0.45,0.5,0.55,
0.6 μmol/L.Detected with 1% agarose gel electrophoresis.
As a result as shown in figure 3, primer concentration is 0.2 μm of ol/L（4th swimming lane）When the band that obtains it is the most special, bright,
And without traction phenomenon.
（3）In other experiment conditions and step（1）In the case of middle pcr amplification reaction system identical, dNTPs concentration is set
Put 8 gradients, respectively 50,100,150,200,250,300,350,400 μm of ol/L.Examined with 1% agarose gel electrophoresis
Survey.
As a result as shown in figure 4, NTPs concentration is 200 μm of ol/L（4th swimming lane）When the band that obtains it is the most special, clear.
（4）In other experiment conditions and step（1）In the case of middle pcr amplification reaction system identical, DNA polymerases are dense
Degree set 9 gradients, respectively 5,10,20,30,40,50,60,70,80 U/L.Examined with 1% agarose gel electrophoresis
Survey.
As a result as shown in figure 5, DNA polymerase concentrations are 60 U/L（7th swimming lane）With 80 U/L（9th swimming lane）Shi Junneng
Obtain special, clearly band；In view of economic and practical, the effect being optimal with minimum enzyme amount, therefore DNA polymerases
Concentration should be when being 60 U/L the present invention PCR reaction systems in optimum amount.
（5）In other experiment conditions and step（1）In the case of middle pcr amplification reaction system identical, response procedures are set：
57 DEG C of annealing temperature, 25 circulations, 57 DEG C, 30 circulations, 57 DEG C, 35 circulations, 58 DEG C, 25 circulations, 58 DEG C, 30 are followed
Ring, 58 DEG C, 35 circulations, 59 DEG C, 25 circulations, 59 DEG C, 30 circulations, 59 DEG C, 35 circulations, 60 DEG C, 25 circulations, 60
DEG C, 30 circulation, 60 DEG C, 35 circulation.
As a result as shown in fig. 6, in 55 DEG C, 35 circulations of annealing temperature（3rd swimming lane）Reaction condition under, the band of acquisition
It is the most special, bright.
In summary, the optimal pcr amplification reaction system of the present invention is DNA, 0.2 μ for including 6 mg/ L in 20 μ L systems
Mol/L primer, 200 μm of ol/L dNTPs and 60 U/L archaeal dna polymerase, react 35 PCR at being 57 DEG C in annealing temperature
Circulation.
Embodiment 3
All primers are designed using Primer3 softwares in selection table 1.Based on the root of bidentate achyranthes EST- optimized in embodiment 2
SSR amplification systems, and randomly choose the validity that 22 EST-SSR verify established amplification system.As shown in fig. 7,21
Individual EST-SSR sequences are detected special purpose band, illustrate the PCR inspections for the identification EST-SSR sequences that the present invention is established
Survey method can be used for the subsequent experimental of identification root of bidentate achyranthes EST-SSR marker research.
The present invention is described in detail above in conjunction with drawings and examples, still, those of skill in the art
Member can also be carried out it is understood that on the premise of present inventive concept is not departed to each design parameter in above-described embodiment
Change, forms multiple specific embodiments, is the common excursion of the present invention, is no longer described in detail one by one herein.
SEQUENCE LISTING
<110>He'nan University of Technology
<120>EST-SSR primer sets and its acquisition methods based on the exploitation of root of bidentate achyranthes transcript profile
<130> 2017
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<170> PatentIn version 3.2
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Claims (5)
- A kind of 1. EST-SSR primer sets based on the exploitation of root of bidentate achyranthes transcript profile, it is characterised in that：The primer sets include following 23 pairs Primer, each pair primer are made up of sense primer and anti-sense primer, the nucleotide sequence such as sequence table SEQ ID NO of each pair primer:1 To SEQ ID NO:Shown in 46.
- 2. a kind of method for obtaining the EST-SSR primer sets based on the exploitation of root of bidentate achyranthes transcript profile described in claim 1, its feature exist In comprising the following steps：（1）Root of bidentate achyranthes transcript profile library is built, transcript profile sequencing data in the library is spliced, de-redundancy obtains Unigenes；（2）EST-SSR site screenings are carried out to the Unigenes using MISA softwares；（3）The EST-SSR primers that design of primers is designed are carried out to the EST-SSR sites with the softwares of Primer 3；（4）Root of bidentate achyranthes genomic DNA is extracted using CTAB methods, entering performing PCR with the EST-SSR primer pairs root of bidentate achyranthes DNA expands, then Enter row agarose gel electrophoresis, EST-SSR molecular labeling primers are verified according to electrophoresis result.
- 3. the acquisition methods of the EST-SSR primer sets according to claim 2 based on the exploitation of root of bidentate achyranthes transcript profile, its feature exist In：The step（2）In, the parameter for the repetitive sequence that SSR can be detected is arranged to：Mononucleotide is at least repeated 10 times, and two Nucleotides is at least repeated 6 times, and trinucleotide is at least repeated 5 times, and tetranucleotide is at least repeated 5 times, and pentanucleotide at least repeats 5 Secondary, Hexanucleotide is at least repeated 5 times.
- 4. the acquisition methods of the EST-SSR primer sets according to claim 2 based on the exploitation of root of bidentate achyranthes transcript profile, its feature exist In step（4）In, the reaction system of PCR amplifications is：Overall reaction system is 20 μ L, includes 2~24 mg/ L's in system DNA；0.05~0.6 μm of ol/L primer；50~400 μm of ol/L dNTPs and 5~80 U/L archaeal dna polymerase, are being moved back Fiery temperature is that 25~35 PCR cycles are reacted at 57~60 DEG C.
- 5. the acquisition side of the EST-SSR primer sets based on the exploitation of root of bidentate achyranthes transcript profile according to claim 2 or claim 4 Method, it is characterised in that the pcr amplification reaction system is 20 μ L, and the DNA comprising 6 mg/ L in system, 0.2 μm of ol/L draw Thing, 200 μm of ol/L dNTPs and 60 U/L archaeal dna polymerase, react 35 PCR cycles at being 57 DEG C in annealing temperature.
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| CN113981105A (en) * | 2021-10-22 | 2022-01-28 | 广东一方制药有限公司 | Primer for multiplex PCR identification of achyranthes and cyathula medicinal materials, standard decoction and traditional Chinese medicine formula granules and application thereof |
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| CN105274198A (en) * | 2015-05-26 | 2016-01-27 | 江苏省农业科学院 | Method for sequencing and development of Asplenium nidus L. EST-SSR primers based on transcriptome |
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| CN105274198A (en) * | 2015-05-26 | 2016-01-27 | 江苏省农业科学院 | Method for sequencing and development of Asplenium nidus L. EST-SSR primers based on transcriptome |
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| CN113981105A (en) * | 2021-10-22 | 2022-01-28 | 广东一方制药有限公司 | Primer for multiplex PCR identification of achyranthes and cyathula medicinal materials, standard decoction and traditional Chinese medicine formula granules and application thereof |
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