CN106350518A - Primer pairs and application thereof in germplasm identification of morinda officinalis - Google Patents
Primer pairs and application thereof in germplasm identification of morinda officinalis Download PDFInfo
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- CN106350518A CN106350518A CN201611062263.0A CN201611062263A CN106350518A CN 106350518 A CN106350518 A CN 106350518A CN 201611062263 A CN201611062263 A CN 201611062263A CN 106350518 A CN106350518 A CN 106350518A
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- 125000003729 nucleotide group Chemical group 0.000 claims description 36
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- 238000012360 testing method Methods 0.000 claims description 18
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- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 238000013095 identification testing Methods 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 241000157491 Morinda Species 0.000 abstract description 15
- 235000017524 noni Nutrition 0.000 abstract description 14
- 239000003814 drug Substances 0.000 abstract description 8
- 210000003763 chloroplast Anatomy 0.000 abstract description 6
- 229940079593 drug Drugs 0.000 abstract description 6
- 241000894007 species Species 0.000 abstract description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 2
- 238000003752 polymerase chain reaction Methods 0.000 abstract 2
- 108020004414 DNA Proteins 0.000 description 39
- 108700031407 Chloroplast Genes Proteins 0.000 description 11
- 241000196324 Embryophyta Species 0.000 description 8
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- 241001365511 Gynochthodes nanlingensis Species 0.000 description 1
- 241000607292 Maguireothamnus speciosus Species 0.000 description 1
- 208000019255 Menstrual disease Diseases 0.000 description 1
- 244000131360 Morinda citrifolia Species 0.000 description 1
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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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Abstract
The invention relates to the technical field of biology, and particularly relates to primer pairs and application thereof in germplasm identification of morinda officinalis. The invention provides the primer pairs with nucleotide sequences represented by SEQ ID NO: 1-2 and SEQ ID NO: 3-4, and provides application of the primer pairs in the germplasm identification of the morinda officinalis. By adopting the primer pairs provided by the invention, the morinda officinalis can be identified through only a PCR (Polymerase Chain Reaction) manner, and accurate and rapid identification on the morinda officinalis can be achieved by using specific sequences in a chloroplast genome of the morinda officinalis. Accordingly, the identification on several morphologically close species of morinda can be achieved from the aspect of molecular biology. Then, the introduction of morinda officinalis counterfeits in market circulation is avoided, and the quality and medication safety of finished drugs are improved.
Description
Technical field
The present invention relates to biological technical field, more particularly, to primer pair and its application in Radix Morindae Officinaliss Idioplasm identification.
Background technology
Radix Morindae Officinaliss (morinda officinalis how) be Maguireothamnus speciosus, another name Radix Morindae Officinalis, Herba Morindae Parvifoliae, Caulis Gneti bore,
Rabbit son's intestinal, triangle vine, bran rattan, main product is in Guangdong, Fujian, Guangxi and Hainan.Radix Morindae Officinaliss are used as medicine with root, and property is sweet, and acrid in the mouth is micro-
Temperature；Return kidney, Liver Channel；There is kidney-replenishing, bone and muscle strengthening, the effect of wind-damp dispelling.For impotence and seminal emission, uterus cold infertile, menoxenia, lack
Abdomen cold type of pain, rheumatic arthralgia, muscles and bones impotence.
Radix Morindae Officinaliss are loaded in Shennong's Herbal as one of conventional Chinese medicine beginning and are classified as top grade, and hereafter history tree all has note
Carry.Beam TAO Hong-Jing has simple description in Collective Notes to the Canon of Materia medica to this crude drug: " shape such as Paeonia suffruticosa and thin, outer red interior black, use it
Beat and remove the heart ".
Radix Morindae Officinaliss are one of China's famous " four great Nan medicines ", and its wild resource is closely exhausted, in " Chinese Plants Folium Styracis Suberifoliae
Book rare extinction plants " it is listed in three-level protective plant in a book.It is grown in wild Radix Morindae Officinaliss in the dense woods more, cover
Degree ratio is larger, and cultivates and plant in the gradient less hillside on Radix Morindae Officinaliss more, has dungarunga or shrub to shelter from heat or light more.Wild Radix Morindae Officinaliss
Due to being chronically at wild state, it is subjected to the natural selection of various disasters and poor environment, often save Common Cultivation kind
Do not have or disappearance some key properties, and cultivate Radix Morindae Officinaliss and define multiple Farm Races in the course of cultivation.Separately
Outward, also Morinda Citifolia (morinda citrifolia l.), the Herba Morindae Parvifoliae (morinda with Radix Morindae Officinaliss plesiomorphism
Parvifolia bartl.), Nanling Herba Morindae Parvifoliae (morinda nanlingensis y.z.ruan) etc., these plants belong to bar together
Halberd sky belongs to, and morphological characteristic is also very much like with Radix Morindae Officinaliss, but medical value but has larger difference with Radix Morindae Officinaliss.Only lean on naked eyes
It is difficult to distinguish Radix Morindae Officinaliss with Morinda Citifolia, Herba Morindae Parvifoliae, Nanling Herba Morindae Parvifoliae, therefore Radix Morindae Officinaliss germ plasm resource and its sibling specieses are ground
Study carefully and become the task of top priority.
Chloroplast is that plant carries out the distinctive organelle of photosynthesis, provides the main source of energy, chloroplast for plant
Have the genome of oneself.Complete Chloroplast gene generally comprises four parts, single greatly copy area (lsc), little single copy
Area (ssr) and Liang Duan inverted repeat area (ir).The evolutionary rate of Chloroplast gene is in Matrix attachment region and mitochondrial genome
Between evolutionary rate.The Chloroplast gene sequencing of early stage is more complicated, with the development of new-generation sequencing technology, is chloroplast
The research of genome provides great convenience.Chloroplast gene is the important information reviewing chloroplast origin and Evolution History
Source.Analyzed it may be determined that in chloroplast evolutionary process, which gene there occurs loss by comparing evolution genomics,
Which gene transfer has arrived Matrix attachment region, infers the root that gene is lost and shifted.Chloroplast gene group analysis are to solve plant
The important channel of a phylogeny difficult problem.Chloroplast gene can provide genome structure and the data of two levels of dna sequence
For Phylogenetic Analysis, the solution to some difficulty phylogeny problems provides key evidence.Chloroplast gene also may be used
To be used for providing Important Theoretic Foundation for species identification.But at present, not yet have and planted for Radix Morindae Officinaliss Chloroplast gene
The relevant report of identification.
Content of the invention
In view of this, the technical problem to be solved in the present invention is to provide primer pair and its in Radix Morindae Officinaliss Idioplasm identification
Application.The primer pair that the present invention provides is capable of quick, precise Identification to Radix Morindae Officinaliss germplasm.
The invention provides as shown in seq id no:1～2 nucleotide sequence primer pair.
The invention provides as shown in seq id no:3～4 nucleotide sequence primer pair.
The primer pair that the present invention provides is according to the design of Radix Morindae Officinaliss Chloroplast gene specific regions, this region and close thing
Kind Chloroplast gene to compare variability higher, this region is detected, is capable of quick, accurate to Radix Morindae Officinaliss germplasm
Really identify.
As shown in seq id no:1～2, the annealing temperature of the primer pair of nucleotide sequence is 56 DEG C.
As shown in seq id no:3～4, the annealing temperature of the primer pair of nucleotide sequence is 56 DEG C.
Application in Radix Morindae Officinaliss Idioplasm identification for the primer pair that the present invention provides.
The primer pair that the present invention provides can differentiate Radix Morindae Officinaliss, particularly can be from the close multiple Morinda offcinalis Hows of morphological feature
Radix Morindae Officinaliss are identified in its platymiscium.Close Morinda plant includes described morphological feature: Radix Morindae Officinaliss, Herba Morindae Parvifoliae, sea
Morinda offcinalis How and Nanling Herba Morindae Parvifoliae.
Present invention also offers a kind of Radix Morindae Officinaliss Idioplasm identification test kit, the primer pair providing including the present invention.
Pcr reagent is also included in this test kit that the present invention provides.
Described pcr reagent can be dntp, taq dna polymerase, the mg of subpackage2+, amplification buffer.
Preferably, pcr reagent is taq enzyme premixed liquid.
Electrophoresis reagents are also included in the test kit that the present invention provides.
Described electrophoresis reagents include agar and dyestuff.
Described dyestuff is bromophenol blue and dimethylbenzene is blue or green.
Dna molecular marker is also included in the test kit that the present invention provides.
Preferably, dna molecular marker includes the dna molecule of different molecular weight.
In the embodiment of the present invention, in dna molecular marker, including molecular weight be respectively 4500bp, 3000bp, 2000bp,
The dna molecule of 1200bp, 800bp, 500bp and 200bp.
Pcr reaction tube is also included in the test kit that the present invention provides.
Present invention also offers a kind of Radix Morindae Officinaliss germplasm identification method, with the genome dna of testing sample as template, respectively
With the primer pair of nucleotide sequence shown in seq id no:1～2, the primer pair of nucleotide sequence shown in seq id no:3～4 it is
Primer carries out pcr amplification；
Amplification obtains the length of fragment or sequence meets one of following condition, then this sample is Radix Morindae Officinaliss；
Obtain the length of fragment with the primer pair amplifies of nucleotide sequence shown in seq id no:1～2 as 1349bp；
Obtain the sequence such as seq id no:5 of fragment with the primer pair amplifies of nucleotide sequence shown in seq id no:1～2
Shown；
Obtain the length of fragment with the primer pair amplifies of nucleotide sequence shown in seq id no:3～4 as 905bp；
Obtain the sequence such as seq id no:6 of fragment with the primer pair amplifies of nucleotide sequence shown in seq id no:3～4
Shown.
In the present invention, testing sample is Radix Morindae Officinaliss, Herba Morindae Parvifoliae, Morinda Citifolia or Nanling Herba Morindae Parvifoliae.
Amplification obtains the length of fragment or sequence meets one of following condition, then this sample is Herba Morindae Parvifoliae；
Obtain the length of fragment with the primer pair amplifies of nucleotide sequence shown in seq id no:1～2 as 1366bp；
Obtain the sequence such as seq id no:7 of fragment with the primer pair amplifies of nucleotide sequence shown in seq id no:1～2
Shown；
Obtain the length of fragment with the primer pair amplifies of nucleotide sequence shown in seq id no:3～4 as 818bp；
Obtain the sequence such as seq id no:8 of fragment with the primer pair amplifies of nucleotide sequence shown in seq id no:3～4
Shown.
Amplification obtains the length of fragment or sequence meets one of following condition, then this sample is Morinda Citifolia；
Obtain the length of fragment with the primer pair amplifies of nucleotide sequence shown in seq id no:1～2 as 779bp；
Obtain the sequence such as seq id no:9 of fragment with the primer pair amplifies of nucleotide sequence shown in seq id no:1～2
Shown；
Obtain the length of fragment with the primer pair amplifies of nucleotide sequence shown in seq id no:3～4 as 892bp；
Sequence such as seq id no with the primer pair amplifies acquisition fragment of nucleotide sequence shown in seq id no:3～4:
Shown in 10.
Amplification obtains the length of fragment or sequence meets one of following condition, then this sample is Nanling Herba Morindae Parvifoliae；
Obtain the length of fragment with the primer pair amplifies of nucleotide sequence shown in seq id no:1～2 as 1282bp；
Sequence such as seq id no with the primer pair amplifies acquisition fragment of nucleotide sequence shown in seq id no:1～2:
Shown in 11；
Obtain the length of fragment with the primer pair amplifies of nucleotide sequence shown in seq id no:3～4 as 836bp；
Sequence such as seq id no with the primer pair amplifies acquisition fragment of nucleotide sequence shown in seq id no:3～4:
Shown in 12.
In practical operation, in order to save appraisal cost, by the way of electrophoresis, result is identified.It is preferably agarose
Gel electrophoresiss.
In one embodiment of the invention, Radix Morindae Officinaliss germplasm identification method is, with the genome dna of testing sample as template,
First with the primer pair of nucleotide sequence shown in seq id no:1～2, the genome dna of testing sample can be expanded, if institute
Obtain clip size and be then accredited as Morinda Citifolia for 779bp, if gained clip size is 1282bp, be accredited as Nanling Herba Morindae Parvifoliae；If institute
Obtaining clip size is 1349bp～1366bp it is difficult to be differentiated to agarose gel electrophoresiies result by naked eyes, then further
With the primer pair of nucleotide sequence shown in seq id no:3～4, the genome dna of testing sample is expanded, such as obtained sheet
Duan great little is then accredited as Radix Morindae Officinaliss for 905bp, if gained clip size is 818bp, is accredited as Herba Morindae Parvifoliae.
Testing sample dna is genome dna.It extracts and adopts Plant Genome dna extracts kit.
Described testing sample is fresh stem, leaf；Or the stem of drying, leaf；Or the stem through processing, leaf.
It is preferably fresh blade.
Through liquid nitrogen grinding before testing sample dna extraction.
In the present invention, the reaction system of pcr amplification is:
In the present invention, the program of pcr amplification is:
Can be by carrying out to the electrophoresis of amplified production to the judgement of testing result, also can be by entering to the sequencing of amplified production
OK.In order to shorten qualification time, by the way of electrophoresis, described electrophoresis is agarose gel electrophoresiies or polypropylene for the judgement of result
Acrylamide gel electrophoresis.Preferably, electrophoresis is agarose gel electrophoresiies.
The invention provides nucleotides sequence is classified as the primer pair shown in seq id no:1～2 and seq id no:3～4, and
Provide application in Radix Morindae Officinaliss Idioplasm identification for these primer pairs.The primer pair being provided using the present invention can only pass through pcr's
Mode is identified to Radix Morindae Officinaliss, using the specific sequence in Radix Morindae Officinaliss Chloroplast gene, is capable of to Radix Morindae Officinaliss
Accurately Rapid identification.So as to the aspect from molecular biology, realize the mirror to the close species of several morphology of Morinda
Not.And then avoid being mixed into of Radix Morindae Officinaliss counterfeit merchandise in market circulation, improve quality and the drug safety of finished medicines.
Brief description
Fig. 1 show Radix Morindae Officinaliss identification pcr after through agarose gel electrophoresiies detection after result；Wherein,
Swimming lane 1 shows the Radix Morindae Officinaliss genome dna through primer pair amplifies shown in seq id no:1～2；
Swimming lane 2 shows the Morinda Citifolia genome dna through primer pair amplifies shown in seq id no:1～2；
Swimming lane 3 shows the Herba Morindae Parvifoliae genome dna through primer pair amplifies shown in seq id no:1～2；
Swimming lane 4 shows the Nanling Herba Morindae Parvifoliae genome dna through primer pair amplifies shown in seq id no:1～2；
Swimming lane 5 shows the Radix Morindae Officinaliss genome dna through primer pair amplifies shown in seq id no:3～4；
Swimming lane 6 shows the Herba Morindae Parvifoliae genome dna through primer pair amplifies shown in seq id no:3～4；
Swimming lane m shows dmarker iii, and marker is made up of 7 dna fragments, respectively 4,500bp, 3,000bp, 2,
000bp, 1,200bp, 800bp, 500bp and 200bp.
Specific embodiment
The invention provides primer pair and its application in Radix Morindae Officinaliss Idioplasm identification, those skilled in the art can use for reference
Present disclosure, is suitably modified technological parameter and realizes.Specifically, all similar replacements and change are to this area skill
For art personnel it is it will be apparent that they are considered as including in the present invention.The method of the present invention and application have been passed through relatively
Good embodiment is described, and related personnel substantially can be to methods herein in without departing from present invention, spirit and scope
It is modified with application or suitably changes and combine, to realize and to apply the technology of the present invention.
The sample that the present invention adopts, instrument are all common commercially available product, all can buy in market.
With reference to embodiment, the present invention be expanded on further:
Embodiment 1
100 parts of Radix Morindae Officinaliss product is buied in market, identifies through morphology and other prior arts (mode such as finger printing), its
70 parts of middle Radix Morindae Officinaliss, 12 parts of Herba Morindae Parvifoliae, 9 parts of Morinda Citifolia and 9 parts of Nanling Herba Morindae Parvifoliae.
Each sample is carried out with genome dna extraction, is fully ground fresh blade to be checked first by liquid nitrogen, then uses
(Plant Genome dna extracts kit, buy from health is the limited public affairs of century biotechnology to Plant Genome dna extracts kit
Department, catalog number (Cat.No.): cw0553) extracted, extraction step reference reagent box description.
Each dna sample is expanded.Amplification is in Suzhou Dongsheng Xingye Scientific Instrument Co., Ltd. etc811 pcr amplification
Carry out on instrument；
First, with the primer pair of nucleotide sequence shown in seq id no:1～2, the genome dna of testing sample is carried out
Amplification, reaction system is characterized as: adds the material of following amount: 2 × taq mastermix 10 μ l, primer in 200 μ lpcr pipes
The each 0.75 μ l of 10 μm of upstream and downstream, dna 1 the μ l, ddh of extraction2O 7.5 μ l, total system is 20 μ l；Amplification program condition is: 94
DEG C denaturation 3 minutes, 94 DEG C of degeneration 30 seconds, anneal 30 seconds for 56 DEG C, 72 DEG C extend 1 point 30 seconds, degeneration, annealing, extend three and walk
Rapid circulation 30 times, finally again 72 DEG C fully extend 10 minutes.After amplification, enter row agarose gel electrophoresis, amplified production is carried out
Detection.
If gained clip size is 779bp, it is accredited as Morinda Citifolia, if gained clip size is 1282bp, be accredited as south
Ridge Herba Morindae Parvifoliae；If gained clip size is 1349bp～1366bp it is difficult to be carried out to agarose gel electrophoresiies result by naked eyes
Differentiate, then with the primer pair of nucleotide sequence shown in seq id no:3～4, the genome dna of testing sample is carried out further
Amplification,
With the primer pair of nucleotide sequence shown in seq id no:3～4, the genome dna of testing sample is expanded,
Reaction system is characterized as: adds the material of following amount: 2 × taq mastermix 10 μ l, 10 μm of primer in 200 μ lpcr pipes
The each 0.75 μ l of upstream and downstream, dna 1 the μ l, ddh of the extraction of extraction2O 7.5 μ l, total system is 20 μ l；Amplification program condition
For 94 DEG C of denaturations 3 minutes, 94 DEG C of degeneration 30 seconds, anneal 30 seconds for 56 DEG C, 72 DEG C extend 1 point 30 seconds, degeneration, annealing, extension three
Individual step cycle 30 times, finally again 72 DEG C fully extend 10 minutes.After amplification, enter row agarose gel electrophoresis, to amplified production
Detected.
As gained clip size is then accredited as Radix Morindae Officinaliss for 905bp, if gained clip size is 818bp, it is accredited as clavuss
Rattan.
Amplification such as table 1, Fig. 1 to each sample:
Table 1: amplified production fragment length (bp)
As can be seen from Table 1,100 parts of Morinda samples are identified, its result is consistent completely with expection, this is described
The primer pair that invention provides has good accuracy, and it is to the accuracy rate of Radix Morindae Officinaliss Idioplasm identification up to 100%.
The above is only the preferred embodiment of the present invention it is noted that coming for those skilled in the art
Say, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should be regarded as
Protection scope of the present invention.
sequence listing
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tatacgcccc aatctagact cttttcttcg atagatagat tggtaaatat ttttcggaag 180
aagttttagc aagaactctt aaatttttta attttggaat tggaatctgt atcttcacgt 240
atcaaagatt cgacttaatt tctaaggaat tattaaatat aatgaaaaag gtgttgctat 300
cattatttga agaaaacagt aagggccata tttgatcatc atagaaaaaa gataagtata 360
agtccccttt tttgtaatat taattattaa tattaattga ataataaact cgatacaaca 420
aaaagtagga aaaagagttg ttttcctatc tatatctatt aaattgaatg aacaactacc 480
ctgtcaccat tctaaatagt ctatattcca aatttcggat cacaaacctt tttcacaaaa 540
taaaaagact tttgttttgt tattctattg aatgttattc tattgaaata gaacaagaaa 600
tgtatatatg ataaactttt actcactttc ttttctataa caaatgatat tcaaagaaac 660
tacatcagtt agatataata taacactgga tttttgttac atttttggta atgtaattcg 720
ggcagacgcg gatattttaa tattaattaa tactttcgat taatgattaa ctcttatctg 780
gtcacctagc ctgtgggact aatgggacat aataaaaatg aaaagggcga ttccggtcct 840
ggtccagacg cttactccta ttaagtttga gtgaatttta ttagtaccaa aacaaaatag 900
tttgaattca gaaaaattca taaataatta tactacccca tttacttacg atataaaaga 960
gtaatagata agattatgga aaattcgatt tttaataaaa tcgaaaagaa aaagaatagt 1020
tacaagaggt ttttctaaat aactaacttc cctaatccta aatagaaaga gtttgaacat 1080
atgatgaaaa ggccaaccga cttttttttg aagtcaaact ctcagaatcc aggttcctac 1140
cttacctgaa tcctaaatac atgaaatgac acctgcgtgt catttgaact agattaggtt 1200
tagtttgtga aaaagagtat gattcataac agataaaaat cagaagaaat taaaggcatt 1260
ccgccgaaaa tgcgacttta aattaaaaaa aaaaacgtcc ttttttttct attctaagat 1320
aatggatatc ctctgggacg gaaggattc 1349
<210> 6
<211> 905
<212> dna
<213>artificial sequence
<400> 6
attcgaccta attgtccccc gtactatttt aaaaagtgtt ctgagtgagt tagttgggtt 60
ccatgctttt cttcctttga atccaaattc aatagcgaac taagttaaat tgtttgttat 120
ttgtttttta gcaaatgagt agttagttta tcgaaataaa agtaactaag aagggaagaa 180
gggaattttt gttggtttcg taagaactaa cttagaaagt atcaaaaggt tcggataatt 240
cttttttttt tttttaccta tattgtcgat tactaattaa gaagtcttta tcaaaaaaag 300
ggtgaattct tcaaattagg aaaacaaaac gaatttcttc ttatcgtacg gatataatta 360
aaataaaaaa tgtaaataga gagttttttc tttattctct atttacattt tttattctct 420
atttacattt atcgaaaata ctgttttagc taccaatccc agttggttcg gattctaatg 480
aatatttcac taatgtttaa gaaattcttt cgttattagg attaaattag aagaaacgaa 540
aaagacaaaa aaatcaagaa aaaagcacat tcgcctgcat atctttcgtg tcgaaagatg 600
actaagttca tttagttagt tctacatttc ttgcgtttat tctatatatt aacttcgata 660
tttaactatc tattctagat agttagctct atactaagaa tttcaaattg aattaataaa 720
aaaattcata agaatgcaaa ttcttaatta taattagtat aaaatagaaa atccttttat 780
ttataaataa ataatataca ggtacaaata ataaattgag gcacccattt tatgacaact 840
ttcagctttc cttctatttt tgtgccttta gtaggcctag tatttccggc aattgcaatg 900
gcttc 905
<210> 7
<211> 1366
<212> dna
<213>artificial sequence
<400> 7
cgttgctttc taccacatcg tttcaaacga agttttagca tcacattcct cttaatttgg 60
aattggtatg gaattgattc aattgtgaaa tcatgcatag tcattggttt aattagtgtg 120
tatacgcccc aatctagact cttttcttcg atagatagat tggtaaatat ttttcggaag 180
aagttttagc aagaactctt aaatttttta attttggaat tggaatttgt atcttcacgt 240
atcaaagatt cgacttaatt tctaaggaat tattaaatat aatgaaaaag gtgttgctat 300
cattatttga agaaaacagt aagggccata tttgatcatc atagaaaaaa gataagtata 360
agtccccttt tttgtaatat taattattaa tattaattga ataataaact cgatacaaca 420
aaaagtagga aaacgagttg ttttcctatc tatatctatt aaattgaatg aacaactacc 480
ctgtcaccat tctaaatagt ctatattcca aatttcggat cacaaacctt tttcacaaaa 540
taaaaagact tttgttttgt tattctattg aatgttattc tattgaaata gaacaagaaa 600
tgtatatatg ataaactttt actcactttc ttttctataa caaatgatat tcaaagaaac 660
tacatcagtt agatataata taacactgga tttttgttac atttttggta atgtaattcg 720
ggcagacgcg gatattttaa tattaattaa tactttcgat taatgattaa ctcttatctg 780
gtcacctagc ctgtgggact aatgggacat aataaaaatg aaaagggcga ttccggtcct 840
ggtccagacg cttactccta ttaagtttga gtgaatttta ttagtaccaa aacaaaatag 900
tttgaattca gaaaaattca taaataatta tactacccca tttacttacg atataaaaga 960
gtaatagata agattattga aaattccatt tttaataaaa tcgaaaagaa aaagaatagt 1020
tacaagaggt ttttctaaat aactaacttc cctaatccta aatagaaaga gtttgaacat 1080
atgatgaaaa gtccaaccga cttttttttg aagtcaaact ctcagaatcc aggttcctac 1140
cttacctgaa tcctaaatac atgaaatgac acctgcgtgt catttgaact agattaggtt 1200
tagtttgtga aaaagagtat gattcataac agataaaaat cagaagaaat taaaggcatt 1260
ccgccgaaaa tgcgacttta aattaaaaaa aaaacgtcct ttttttctat tctaagataa 1320
tttttctatt ctaagataat ggatatcctc tgggacggaa ggattc 1366
<210> 8
<211> 818
<212> dna
<213>artificial sequence
<400> 8
attcgaccta attgtcccac gtaatatttt aaaaagtgtt ctgagtgagt tagttgcgtt 60
ccatgctttt cttcctttga atccaaattc aatagcgaac taagttaaat tgtttgttat 120
ttgtttttta gcaaatgagt agttagttta tcgaaataaa agtaactaag aagggaagaa 180
gggaattttt gttggtttcg taagaactaa cttagaaagt atcaaaaggt tcggataatt 240
cttttttttt ttttacctat acggatataa ttaaaataaa aaatgtaaat agagagtttt 300
ttctttattc tctatttaca ttttttattc tctatttaca tttatcgaaa atactgtttt 360
agctaccaat cccagttggt tcggattcta atgaatattt cactaatgtt taagaaattc 420
tttcgttatt aggattaaat tagaagaaac gaaaaagaca aaaaaatcaa gaaaaaaggg 480
cattcgcctg catatctttc gtgtcgaaag atgactaagt tcatttagtt agttctacat 540
ttcttgcgtt tattctatat attaacttcg atatttaact atctattcta gatagttagc 600
tctatactaa gaatttcaaa ttgaattaat aaaaaaattc ataagaattc aaattcttaa 660
ttataattag tataaaatag aaaatccttt tatttataaa taaataatat acaggtacaa 720
ataataaatt gaggcaccca ttttatgaca actttcagct ttccttctat ttttgtgcct 780
ttagtaggcc tagtatttcc ggcaattgca atggcttc 818
<210> 9
<211> 779
<212> dna
<213>artificial sequence
<400> 9
cgttgctttc taccacatcg tttcaaacga agttttagca tcacattcct cttaatttgg 60
aattggtatg gaattgattc aattgtgaaa tcatgcatag tcattggttt aattagtgtg 120
tatacgcccc aatctagact cttttcttcg atagatagat tggtaaatat ttttcggaag 180
aagttttagc aagaactctt aaatttttga attttggaat tggaatctgg atcgtcacgt 240
atcaaagatt cgacttaatt tctaaggaat tattaaacat aatgaaaagg gtgttgctat 300
cattatttga agaaaacagt cagggctcta tttgatcctc atagaaaaaa gataagtata 360
agtccccctt tttcgttttt tgatattgta atattaattg aataagaaac tcgatacaac 420
aaaaagtagg caaaagagtt gttttcatat ctattaacta taagtttttc taaataacta 480
acttccctaa tcctaaatag aaagagtttg aacatatgat gaaaaggcca accaactttt 540
tttgaagtca aactttcaga atccgggttc ctaccttacc tgaatcctaa atagattaaa 600
ttacacctgt gtgtcatttg cactagattg ggtttagttt gtgaaaaaga gtatgattca 660
taacagataa aaatcagaag aaataagggc attccgccga aaatgcgact ttcaattttt 720
ttaaaaccgt ccttttttat attctaagat aatggatatc ctctgggacg gaaggattc 779
<210> 10
<211> 892
<212> dna
<213>artificial sequence
<400> 10
attcgaccta attgtcccac gtaatatttt aaaaagtgtt ctgagtgagt tatttgcgtt 60
ccatggtttt cttcctttga atcaaaattc aatagcgaac taagttaaat tgtttgttat 120
ttgtttttta gcaaatgagt agttagttta tcgaaataaa agtaactaag aagggaattt 180
ttgttggttt cgtaagatca aacttagaaa gtatcaaaag gttcggataa ttcttttttt 240
tttacctata ttgtcgatta ctaattaaga agtctttatc aaaacaaaaa aggggtgaat 300
tcttcaaatt aggaaaacaa aaacgaattt cttcttatcg tacggatata attaaaataa 360
aaaaaatcaa aaaatagaga gttttttctt tattctctat ttacatttat cgaaaatact 420
gttttagcta acaatcccag ttggttcgga ttctaatgaa tctttcacta atgtttaaga 480
aattctttct ttatttcttt attaggatta aattagaaga aactaaaaag aataaaagac 540
aaaaaaatca agaaaaaagg gcattcgcct gcatatcttt cgtgtcgaaa gatgactaag 600
ttcatttagt tagttctaca tttcttgcgt ttattctata tattaacttc gatatttaac 660
tatctattct agatagttag ctatatacta agaatttcaa attgaattaa taaaaaaatt 720
cataagaatt caaattctta attataatta gtataaaatc tttttattta taaataaata 780
atatacaggt acaaataata aattgaggca cccattttat gacaactttc agctttcctt 840
ctatttttgt gcctttagta ggcctagtat ttccggcaat tgcaatggct tc 892
<210> 11
<211> 1282
<212> dna
<213>artificial sequence
<400> 11
cgttgctttc taccacatcg tttcaaacga agttttacca taacattcct ctaatttgga 60
accagtatgg aattgattca attatggaat catgaatagt catcggttcg tagacatcgt 120
aatctatacc cttttcttct ataattagaa tagagccata aatcgctaaa gggatttctg 180
aagaagtttt agcaagtcct cttaattaaa gattgaaaag aatttcaatt taataataga 240
ttagattaaa tctagatagg ttagggttaa atatttcaat ttcaaaattt caaatagagg 300
ggatcaaaaa cctttttcac aaaaatagaa tagaaggata catatacgtt aaacatttcc 360
tcatttttta tgtattttgt ttaagctgtg tagttcagca agatttcgtt aatcgaatct 420
tgccatacat aatagaatag aaagtgaata aaagatacta aaagatataa aacactccct 480
tctaaagtgt tacataaatt tacaattatt tattaggacc aaacacgaaa tacttaaaac 540
gcaagattca aagaaaatac attgggtaga tatataatta catatataat ttttaatgca 600
attcaggcag acacagaaat ctgaatacct tcgattaatg tattcgtccc ccgggtactc 660
cgggactaat ggagtataag agaaatcaaa atgggaattc tgatctggga tgcgggctgg 720
ctaggcacaa accccaacaa gtccaaatta agttgctcct atttttattt tagtctaacc 780
ccttaagatt aagagaatta atcctattga ctttgaatga atttcattag taccaaaatt 840
agtaacaaaa tagttagaat taagaaatct atatcaaaat tcataaaaaa attagtttag 900
aggataggga ataatagata ggatccttga aaattcaaat attgataaaa tagaaaatca 960
atatgggacg gaatgttttt ctaaataact aacttcccga aacagggtgg gattgggcat 1020
aggatgaaaa gaccgccctt tttttaattc aaactcttgg aacccgtgtt cccaatttac 1080
ctggattaga aatagagtca attacatctg cgtgtcattt gaactcgatt caattcagtt 1140
tgtgtaaaac tgatatgatt catgcataaa agagaaaaat cagaagcaag aaagatattc 1200
cagctaacat tagactttac ccattcaaca aaatctttat tctattctaa cataatgaat 1260
atgctctggg acggaaggat tc 1282
<210> 12
<211> 836
<212> dna
<213>artificial sequence
<400> 12
attcgaccta atcgtaccac gtaatctttt aaaaagtgtt ctgaatgagt tattcaagct 60
ccacgctttc tttcctttga ttaaaaattc aatcaaaacc aaatagagtg ctaagttcaa 120
ttatttgtag caaacgagta gttagtttat tcggaattaa aggaaatagg aattttgttt 180
ggtgacctac gatctaattg tagaaagaat caaaagctgc ggataacccc ttttacctat 240
atttctgatt actaatcaag aagtctctat caaaaaaagg gtgaattttt cctttcatga 300
aattaggcaa acaaaatgaa tttcttcttc tgatcttacg tatataattc aaatagaaaa 360
aatagaaaat aaggttttgt gtttttctcg atttctcgag aatccggttt agctaaaaat 420
acctccgggt tcggattcta acgaatcctt cgacaatctg gaagaaagtc tttctttagt 480
aaaaaggtaa aaagaataaa agaaaaagaa atcaagaaaa agaataaagt tgattatcat 540
acatatcttt catgtagaaa gatgaataag ttcatttatt tagctctaca ttccttgcac 600
ttattctata tcctcactta gatatataga tacttagatc tatactaaga attgaattaa 660
taattaaata ataatgaaaa ttcttaatta taattattat aagatatctt tatttataaa 720
taataataac aggtacgaac aataaatcga ggtacccatt ctatgacaac tttgaacctt 780
tcccctattt ttgtgccttt agtaggccta gtatttccgg caattgcaat ggcttc 836
Claims (10)
1. as shown in seq id no:1～2 nucleotide sequence primer pair.
2. as shown in seq id no:3～4 nucleotide sequence primer pair.
3. application in Radix Morindae Officinaliss Idioplasm identification for the primer pair described in claim 1～2.
4. a kind of Radix Morindae Officinaliss Idioplasm identification test kit is it is characterised in that include the primer pair described in claim 1～2.
5. Radix Morindae Officinaliss Idioplasm identification test kit according to claim 4 is it is characterised in that wherein also include pcr reagent.
6. the Radix Morindae Officinaliss Idioplasm identification test kit according to claim 4 or 5 is it is characterised in that wherein also include electrophoresis examination
Agent.
7. a kind of Radix Morindae Officinaliss germplasm identification method is it is characterised in that with the genome dna of testing sample as template, respectively with seq
The primer pair of nucleotide sequence shown in id no:1～2, the primer pair of nucleotide sequence shown in seq id no:3～4 are entered for primer
Row pcr expands；
Described amplification obtains the length of fragment or sequence meets one of following condition, then this sample is Radix Morindae Officinaliss；
Obtain the length of fragment with the primer pair amplifies of nucleotide sequence shown in seq id no:1～2 as 1349bp；
Obtain the sequence such as seq id no:5 institute of fragment with the primer pair amplifies of nucleotide sequence shown in seq id no:1～2
Show；
Obtain the length of fragment with the primer pair amplifies of nucleotide sequence shown in seq id no:3～4 as 905bp；
Obtain the sequence such as seq id no:6 institute of fragment with the primer pair amplifies of nucleotide sequence shown in seq id no:3～4
Show.
8. authentication method according to claim 7 it is characterised in that described testing sample be Radix Morindae Officinaliss, Herba Morindae Parvifoliae, Hai Ba
Halberd or Nanling Herba Morindae Parvifoliae.
9. authentication method according to claim 7 is it is characterised in that the reaction system of described pcr amplification is:
10. authentication method according to claim 6 is it is characterised in that the program of described pcr amplification is:
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|---|---|---|---|---|
| CN101701258A (en) * | 2009-11-13 | 2010-05-05 | 中国检验检疫科学研究院 | Primer for PCR identification of kidney bean and PCR identification method |
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| CN103468709A (en) * | 2013-07-16 | 2013-12-25 | 南京师范大学 | Chloroplast genome of dendrobium huoshanense and germplasm identification method |
| KR20160057258A (en) * | 2014-11-13 | 2016-05-23 | 서울대학교산학협력단 | Complete sequencing of Chloroplast genome and nrDNA of Panax ginseng-derived Marker, DNA primer set for discrimination of Panax ginseng cultivars and Panax species and uses thereof |
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2016
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| CN101701258A (en) * | 2009-11-13 | 2010-05-05 | 中国检验检疫科学研究院 | Primer for PCR identification of kidney bean and PCR identification method |
| CN102559908A (en) * | 2012-02-03 | 2012-07-11 | 中国检验检疫科学研究院 | Primer for PCR identification of Davidia involucrata Baill and PCR identification method |
| CN103468709A (en) * | 2013-07-16 | 2013-12-25 | 南京师范大学 | Chloroplast genome of dendrobium huoshanense and germplasm identification method |
| KR20160057258A (en) * | 2014-11-13 | 2016-05-23 | 서울대학교산학협력단 | Complete sequencing of Chloroplast genome and nrDNA of Panax ginseng-derived Marker, DNA primer set for discrimination of Panax ginseng cultivars and Panax species and uses thereof |
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