CN108486268A - Based on plant ITS2 karyogenes DNA bar code to the identification method of opium poppy - Google Patents
Based on plant ITS2 karyogenes DNA bar code to the identification method of opium poppy Download PDFInfo
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Abstract
The present invention provides the identification method to Papaveraceae papaver opium poppy (opium) based on plant nucleus gene ITS2.This method collects the sample in multiple places by the distributed area for the poppy that takes a broad survey, and builds the database of the ITS2 sequences of the platymiscium, it is found that the distinctive informative site of opium poppy by comparing, in the ITS2 sequences of overall length 653bp, 47bp is C, 55th 56bp is GG, and 114bp is A, and 131bp is G, 153rd is G, 219bp is A, and 248bp is T, and 494bp is C, 513rd 516bp is TAAA, and 552bp is G.Creamcups can be identified using the difference of above-mentioned site and other species.
Description
Technical field
The invention belongs to molecular identification technical fields, and in particular to the data based on karyogene ITS2DNA bar codes, and profit
The method for carrying out opium poppy (opium) identification with the data.
Background technology
Opium poppy (Papaver somnferium) is Papaveraceae (Papaveraceae) papaver (Papaver) plant.It should
There are about 7 kinds in China for platymiscium：Opium poppy (Papaver somniferum), corn poppy (P.rhoeas), black ring opium poppy
(P.pavoninum), long pod opium poppy (P.dubium), wild poppy (P.nudicaule), Papaver canescens (P.canescens) and
Changbai Mountain opium poppy (P.radicatum).The opium or opium that wherein opium poppy is namely commonly called as, originate in Europe, are found in earliest
German Rhine river valley, the period Tang Dynasty bring China by Arabic businessman.《Full Tang poetry》In have it is numerous about chanting cry of certain animals poppy flower
Poem, but at this time opium poppy only as a kind of ornamental flower cultivate.It is all on the books in Song, member, bright, clear medical book afterwards, it can be a small amount of
It is used as medicine, there is analgesic, antibechic, calmness, antidiarrheal and other effects.To 1830s, foreign opium largely smuggles China, opium poppy
Plantation also by south to northern, once spread all over China.After in 1945, Chinese Government it is severe forbid under, opium poppy (crow
Piece) just mark is looked for from this noise elimination.But also there is one's share of expenses for a joint undertaking of breaking laws and commit crime on a small quantity still to ignore state's laws, plant opium poppy (opium) privately,
Huge injury is brought to the country and people, causes inevitably to lose.Therefore, firmly prevent opium poppy (opium) is
The responsibility that each citizen should use up.However, poppy appearance is more similar, flower, fruit are even more to be difficult to differentiate between, and have no way of differentiating.
Especially the plant corn poppy (P.rhoeas) of the category be the common ornamental flower of afforestation, because with opium poppy morphological feature without bright
Aobvious difference, is often looked at as drugs opium poppy and causes civic panic, bring unnecessary loss.
DNA bar code (DNA barcoding) technology is directly to carry out species identification using molecular DNA sequences, is had only
One without two repeatability；It is comparable between different plant species, or even global species identification can form unified standard；
DNA bar code technology only needs a small number of universal primers, you can identification species；DNA molecular sequence is opposite for each species
Fixed, variation is smaller, has opposite stability.Therefore, DNA bar code technology be now widely used for door, guiding principle, mesh, section, category,
It plants, the identification of mutation.
For a long time, ITS2 is the bar code sequence for being used as DNA of plants, as most important in Study on Molecular Phylogeny and Evolution
One of label.Meanwhile used also as the standard bar code sequence of medicinal plant identification.It is convenient for conserved region at its sequence both ends
Universal primer designs；Sequence amplification is relatively easy to；Sequence has enough variable regions, distinguishable different species etc. excellent
Gesture.
Currently, in poppy, the research of species is identified using Molecular tools, it is also relatively fewer, because of species
Identification library structure sample it is insufficient, the extremely difficult acquisition of some samples causes appraisal to stay in traditional method all the time：
Sample or online search pictures are checked in specimen museum, cause appraisal there are certain error and error, reliability and standard
True property is low.
Invention content
In view of the foregoing, in order to overcome the shortcomings of the prior art, present invention aims to point
The sequencing of sub- DNA bar code, same base position by finding out opium poppy (opium) base and the category other species difference reflect
It is fixed.The method being sequenced using molecule, can improve the accuracy, reliability and safety of appraisal.
In order to realize the above-mentioned purpose of the present invention, the present invention provides the following technical solutions：
Based on plant nucleus gene ITS2 to the identification method of Papaveraceae papaver opium poppy, this method uses poppy
ITS2 karyogenes detect opium poppy (opium), and find the peculiar base of opium poppy as the sequence difference of it and poppy
Informative site distinguishes plant similar with opium poppy in papaver by these characteristic information sites.
According to it is described based on plant nucleus gene ITS2 to the identification method of Papaveraceae papaver opium poppy, wherein the small-mouthed jar
The ITS2 karyogene overall lengths of grain be 653bp, 47bp be C, 55-56bp be GG, 114bp be A, 131bp be G, the 153rd
It is A for G, 219bp, 248bp is T, and 494bp is C, and 513-516bp is TAAA, and 552bp is G.
According to it is described based on plant nucleus gene ITS2 to the identification method of Papaveraceae papaver opium poppy, wherein the opium poppy
Karyogene ITS2 sequence, as shown in ITS2.
The present invention also provides a kind of based on plant nucleus gene ITS2 to the identification method of Papaveraceae papaver opium poppy, the party
Method includes the following steps：
(1) extraction sample to be tested DNA；
(2) PCR amplification is carried out using DNA bar code primer, obtains amplified production, the DNA bar code primer sequence
For ITS2；
(3) amplified production is sequenced, the species identification of creamcups, small-mouthed jar is carried out according to following specific position
The feature site of grain is：Overall length is 653bp, and 47bp is C, and 55-56bp is GG, and 114bp is A, and 131bp is G, the
153 be G, and 219bp is A, and 248bp is T, and 494bp is C, and 513-516bp is TAAA, and 552bp is G.
(4) papaverous plant species amplified production of doubting to be measured is sequenced, if meeting the condition of above-mentioned (3),
Then the sample to be tested is opium poppy (opium)；If not meeting the condition of above-mentioned (3), which is not opium poppy.
According to it is described based on plant nucleus gene ITS2 to the identification method of Papaveraceae papaver opium poppy, wherein the PCR
Amplification system be 25 μ L, system include Mg Cl22.0 μ L, d NTP, 2.0 μ L, 10 × PCR buffer solutions, 2.5 μ L, primer are each
1.0 μ L, ex Taq enzyme 1.25U, 2.5 μ L of total DNA；The amplification condition of the PCR is：94 DEG C of denaturation 5min, 1 cycle；94℃
It is denaturalized 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 45s carry out 30 cycles；72 DEG C of extension 10min.
According to it is described based on plant nucleus gene ITS2 to the identification method of Papaveraceae papaver opium poppy, wherein the sequencing
For bidirectional sequencing, the sequencing primer is shown in ITS2；The reaction system of the sequencing is 5 μ L：Wherein ddH2O 3 μ L, 10 μ
0.5 μ L of mol/L sequencing primers, 0.5 μ L, PCR purified product of sequencing reaction mixture, 1 μ L；The reaction condition of the sequencing is：94
DEG C pre-degeneration 10s；94 DEG C of denaturation 30s, 50 DEG C of annealing 5s, 60 DEG C of extension 4min carry out 30 cycles.
The opium poppy DNA bar code based on molecular data that present invention further provides a kind of, the DNA bar code primer are
ITS2, overall length 653bp, 47bp are C, and 55-56bp is GG, and 114bp is A, and 131bp is G, and the 153rd is G, the
219bp is A, and 248bp is T, and 494bp is C, and 513-516bp is TAAA, and 552bp is G.
Compared with prior art, the present invention has the following advantages：
The present invention is collected into 7 kinds of whole by 7 kinds of progress Sampling Surveys to the papaver in distribution in China, and
It is acquired as far as possible in different places, constructs the poppy ITS2 karyogene DNA bar code databases of distribution in China.
By the comparison of the series to all plants of the category, the opium poppy informative site different from other species bases of the category is found out,
Further obtain its distinctive DNA bar code.
The present invention carries out the identification of molecular core gene DNA bar code to opium poppy (opium) for the first time, using ITS2 karyogenes to this
Belong to 7 kinds of plants to be expanded and be sequenced, obtain base informative site, find the difference of opium poppy and other species, quickly judges true
Puppet, as a result accuracy is high.It is direct using this method, accurate, quick, safe and efficient to obtain judging result.
Description of the drawings
Fig. 1 is the pcr amplification product collection of illustrative plates of papaver Activities of Some Plants sample, wherein right " 1 " is GeneRuler 100bp
Plus DNA Ladder；
Fig. 2 is the sample segment aligned sequences of poppy as a result, being part poppy ITS2 sequences.
Specific implementation mode
Below in conjunction with the accompanying drawings, the essentiality content further illustrated the present invention with the embodiment of the present invention, but not with
This limits the present invention.
Embodiment 1
The present invention has carried out the poppy of extensive acquisition distribution in China, of acquisition different location as much as possible
Body, the karyogene DNA bar code database for building the platymiscium obtain the base ratio pair in the DNA bar code of opium poppy (opium)
Obtain its distinctive DNA bar code informative site.
The opium poppy DNA bar code of the present invention is to expand to obtain by ITS2 karyogenes, overall length 653bp.Wherein there are 10
Variant sites are different from other species of papaver, and the individual in several places of opium poppy (opium) is all identical.In comparison,
The specific recognition site of opium poppy is respectively：47bp is C, and 55-56bp is GG, and 114bp is A, and 131bp is G, the
153 be G, and 219bp is A, and 248bp is T, and 494bp is C, and 513-516bp is TAAA, and 552bp is G.The present invention's
Amplimer using ITS2 sequences universal primer, in the sequence of the 653bp expanded, according to above-mentioned specific position
Base type can identify whether be opium poppy (opium).
Since the opium poppy DNA bar code of the present invention is based on the poppy materials obtained as far as possible more, it is contemplated that its
Morphological feature is more similar, the smaller problem of inevitable DNA sequence dna difference, therefore can greatly be differentiated using ITS2 karyogenes
Poppy.Using the above-mentioned opium poppy DNA bar code of the present invention can quick, accurate, safe efficient identification opium poppy (opium),
Avoid the problem that congener morphological feature is similar, identification is low for a long time.
The present invention is extracted by the DNA to sample to be tested, is expanded and is sequenced using Molecular tools, by the sequence of amplified production
Row be compared with the specific recognition site in opium poppy DNA bar code of the present invention, can directly obtain identify the true and false as a result, examine
The process of survey is fast, efficient, safe, accurate, is with the relatively simple method with identification opium poppy.
In the present invention, the DNA extraction method of sample to be tested uses method well-known to those skilled in the art.In the present invention
It is embodied in case, the total DNA of sample to be tested is extracted using 2 × CTAB methods of improvement.The present invention carrys out DNA in sample to be tested
Source does not have particular/special requirement, can use leaf, root, the stem of sample to be tested.
PCR amplification and amplimer in the present invention sequence used when being sequenced are universal sequence, specially：
ITS2F：5’-ATGCGATACTTGGTGTGAAT-3’
ITS2R：5’-GACGCTTCTCCAGACTACAAT-3’
In the present invention, PCR amplification method and the method that amplified production is sequenced are ripe using those skilled in the art
The method known.
Present invention preferably employs the PCR amplification system of 25 μ L, system proportioning is as follows：MgCl22.0 μ L (25mmol/L),
(it is limited that work is given birth in Shanghai by dNTP2.0 μ L (2.5mmol/L), PCR buffer solutions 2.5 μ L (10 ×), each 1.0 μ L of primer (2.5 μm of ol/L)
Company), exTaq enzyme 1.25U, 2.5 μ L (about 50ng) of total DNA.It is special that the present invention does not have the reagent source in amplification system
It limits, Commercial reagents well-known to those skilled in the art can be used in reagent used in amplification system.
The amplification condition of PCR is：94 DEG C of denaturation 5min (1 cycle)；94 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C are prolonged
Stretch 45s (carrying out 30 cycles)；72 DEG C of extension 10min.All alternative sample DNAs are equal under above-mentioned amplification system and amplification condition
The sequence using primer can be amplified.Those skilled in the art can be based on the above technical solution to amplification system condition
Carry out appropriate rational adjustment.It is timely such as to change the volume of amplification system, the concentration of system constituent, the temperature of adjustment amplification
Between etc. conditions, all belong to the scope of protection of the present invention.The present invention preferably purifies amplified production.Using people in the art
Purification process known to member carries out.If ITS2 primer amplifications obtain 653bp, then sample to be tested is compared specifically by being sequenced
Property recognition site can be determined whether as opium poppy (opium).
The present invention does not have the mode of sequencing special restriction, using sequencing approach well-known to those skilled in the art,
Use unidirectional sequencing or bidirectional sequencing.The present invention, with rigorous attitude, takes two-way survey in specific implementation process
The method of sequence.
Preferably use the amplification system of PCR for the sequencing reaction system of 5 μ L in the present invention：ddH2O 3 μ L, 10 μm of ol/L
0.5 μ L of sequencing primer, 0.5 μ L, PCR purified product of sequencing reaction mixture, 1 μ L.Reagent used in experimentation can be adopted
With Commercial reagents well known in the art.The reaction condition of sequencing is preferably：96 DEG C of pre-degeneration 10s, 50 DEG C of annealing 5s, 60 DEG C
Extend 4min, 33 cycles.Those skilled in the art can be based on the above technical solution to sequencing reaction system condition
Appropriate rational adjustment is carried out, such as changes volume, the concentration of component, the time of sequencing and the temperature condition of reaction system,
It belongs to the scope of protection of the present invention.After the completion of reaction, product is settled, is purified, after thermal denaturation, upper machine is sequenced.This
Invention is not particularly limited sedimentation, purifying, thermal denaturation and sequencing procedure, using the method known to those skilled in the art.
The sequencing result of sample to be tested is compared with the opium poppy DNA bar code of the present invention, if meeting following condition
Sample to be tested is opium poppy (opium)：47bp is C, and 55-56bp is GG, and 114bp is A, and 131bp is G, and the 153rd is G,
219bp is A, and 248bp is T, and 494bp is C, and 513-516bp is TAAA, and 552bp is G.It is on the contrary then be not opium poppy.
The present invention can be to it using universal primer and the operable PCR amplification of those skilled in the art and sequencing approach
Rapid identification is carried out, the process of identification is time saving, quick, accuracy is higher.
To make the objectives, technical solutions, and advantages of the present invention clearer, with reference to examples of implementation to the present invention
It is described in detail, but they cannot be interpreted as the restriction of the scope of the present invention.
Embodiment 2：
1. the collection of specimens and protection of poppy
The holding items of consulting literatures and domestic specimen museum formulate the side of sampling according to the sampling request of DNA bar code
Case, each species acquire the individual of separate sources in its distributed area as far as possible, and the blade of all individuals is preserved with silica dehydrator.
7 kinds of poppy, total number of individuals are blade materials of 25 (contain mutation) individual, the sequence information of sample segment from
NCBI gene pools download (total 32 sequences).Wherein, the sample of opium poppy (opium) comes from the wild fragmentary growth in Xinjiang Yili of China field
In woods side, each place respectively acquires 3 parts of leaf samples.The bill of materials and sample source are shown in Table 1,2.
1 poppy sample of table and source
The primer of 2 poppy of table and No. Genebank
2. Genome DNA extraction
The total DNA that above-mentioned poppy blade is extracted using 2 × CTAB methods of improvement, is as follows：
(1) using clean mortar, pestle high pressure sterilization, drying, cooling.
(2) it takes clean blade to be put into mortar, firmly be ground after stiffness becomes fragile with liquid nitrogen coolant
Material is allowed to thin such as powder, is then transferred to ground material in the centrifuge tube of the 2ml of precooling；
(3) 2 × CTAB extracting solutions and 2 μ L beta -mercaptoethanols (2%V/V) of 1ml preheatings are added in the centrifuge tube of 2ml,
Material is put into extracting solution completely, and is mixed well.Warm bath about 1.5 hours in 65 DEG C of water-baths are put into, 4-6 is during which shaken up
It is secondary.
(4) after taking out warm bath material, isometric chloroform isoamyl alcohol (volume ratio 24 is added：1) solution shakes up 5-10 points
Then clock is centrifuged 5 minutes with 10000-12000 revs/min.
(5) supernatant (about 700~800 μ L) is transferred in a new centrifuge tube and (pays attention to avoiding in suction process miscellaneous
Matter).
(6) step (4), (5) are repeated twice.
(7) supernatant (about 450~600 μ L) is transferred to the isoamyl alcohol that 70% volume is added in new centrifuge tube, settled
DNA is gently overturned 2~3 times, it is seen that white flock precipitate stands 30 minutes or more in 4 DEG C of refrigerators, then 12000 leaves the heart 5
~10 minutes, abandon supernatant.
(8) it is respectively washed 2 times with 76% ethyl alcohol of 200 μ L and absolute ethyl alcohol, then 12000 leaves the heart 5~10 minutes, abandon
Clear liquid.Centrifuge tube is put into 37 DEG C of baking ovens (or at room temperature) dry, after ethyl alcohol volatilization, 30~50 μ L TE solution and 1 are added
~2 μ L RNase A, and be put into 37 DEG C of baking oven and digested 2~3 hours with ribalgilase (RNase A), be finally putting into-
It is spare in 20 DEG C of refrigerator.
3.PCR amplified reactions
DNA concentration is detected with ultraviolet specrophotometer (UV-VIS spectrophotometer (TU-1800)), last dilute
It releases spare to 50~100ng/ μ L.
Using following primer amplification rDNA (ITS2) sequence：
ITS2F：5’-ATGCGATACTTGGTGTGAAT-3’
ITS2R：5’-GACGCTTCTCCAGACTACAAT-3’
PCR reacts amplification system, and system proportioning is as follows：Mg Cl22.0 μ L of 2.0 μ L (25mmol/L), d NTP
(2.5mmol/L), PCR buffer solutions 2.5 μ L (10 ×), each 1.0 μ L of primer (2.5 μm of ol/L) (Shanghai Sheng Gong Co., Ltds),
ExTaq enzyme 1.25U, 2.5 μ L (about 50ng) of total DNA.
The primer of all standby screenings can Successful amplification under following amplification condition：94 DEG C of denaturation 5min (1 cycle)；94
DEG C denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C extend 45s (carry out 30 cycle)；72 DEG C of extension 10min.
Amplified production is purified, method is illustrated to operate by kit (giving birth to work Sangon in Shanghai).Use 1.5% fine jade
Sepharose electrophoresis detection PCR product, the results show that the papaver sample primer of all standby screenings is equal under amplification condition of the present invention
It can Successful amplification.Sample segment electrophoresis pattern is referring to Fig. 1.
4. amplified production is sequenced
Sequencing reaction reagent uses the PRISM Dye Terminator Cycle Sequencing of ABI companies
Reaction Kit, bidirectional sequencing.
Sequencing reaction system is 5 μ L：Wherein ddH23 μ L of O, 0.5 μ L of sequencing primer (10 μm of ol/L), sequencing reaction mixing
1 μ L of object (mix, Big Dye v3.1) 0.5 μ L, PCR purified product.
The sequence that ITS2 primers are sequenced uses amplimer sequence in step 3.
Sequencing reaction condition is：96 DEG C of pre-degeneration 10s, 50 DEG C of annealing 5s, 60 DEG C of extension 4min, 33 recycle；After
Add 20 μ L sedimentation agent (absolute ethyl alcohols：Sodium acetate=20 3M：1), 4 DEG C of sedimentations are stayed overnight.
Sedimentation products add 20 μ L ddH after purification through 70% alcohol2Loading after O is denaturalized 2 minutes under the conditions of 95 DEG C, in ABI
It is sequenced on 3730 automatic sequencers.
5. data analysis
Sequencing result is spliced and is compared respectively with Sequencher4.1.4 and BioEdit (ver 7.0.0),
Insertion and deletion in matrix is replaced with "-".The matrix of the chloroplast DNA bar code of poppy is built, DNA bar code is obtained
Database.
6. sequence alignment and the sequence signature of opium poppy
The result of sequence alignment is：It is 653bp that overall length, which is sequenced, in ITS2, by 25 Sequence compositions.Wherein, 32 are downloaded from NCBI
Item, the present invention obtain 25, and sequence part belongs in papaver 7 and (contain mutation) 25 individuals.Opium poppy all samples in a matrix
The information recognition sites of karyogene ITS2DNA bar codes be：47bp is C, and 55-56bp is GG, and 114bp is A, the
131bp is G, and the 153rd is G, and 219bp is A, and 248bp is T, and 494bp is C, and 513-516bp is TAAA, 552bp
For G.If only seeing the sequence of single species, the opium poppy extension increasing sequence of all separate sources is almost the same, is expanded with primer I TS2
Obtained sequence product informative site is completely the same.
The mirror of opium poppy can be used for as the DNA bar code of opium poppy according to the specific information site of the obtained opium poppy of the present invention
It is fixed.The identification method of the present invention passes through above-mentioned 10 merely with general versatility primer by PCR amplification and sequencing approach
Specific recognition site can carry out Rapid identification to opium poppy.
Embodiment 3
The opium poppy having no result without flower acquired using medicinal material growing area and field and other papaver plant leafs are adopted as material
DNA extractions, PCR amplification and sequencing are carried out with the method for embodiment 1, qualification result is shown, is obtained in known creamcups
The 47bp of DNA bar code sequence is C, and 55-56bp is GG, and 114bp is A, and 131bp is G, and the 153rd is G, 219bp
It is T for A, 248bp, 494bp is C, and 513-516bp is TAAA, and 552bp is G.And other poppy (nothings
The opium poppy and other papaver plant that flower is had no result) have no above-mentioned specific position, it is possible thereby to identify opium poppy sample.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Poppy is analyzed in the embodiment of the present invention has used 25 individuals altogether.Wherein opium poppy (Papaver
Somnferium) individual has 5, and the present invention will be put into this 5 individual ITS2 sequences in sequence table below.5 of opium poppy
The sequence of individual has the missing of 7 bases with other 20 kinds of comparisons.
Sequence table
<110>Kunming Inst. of Botany, Chinese Academy of Sciences
<120>Based on plant ITS2 karyogenes DNA bar code to the identification method of opium poppy
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3274
<212> DNA
<213>Opium poppy（Papaver somnferium L.）(2 Ambystoma laterale x Ambystoma
jeffersonianum)
<400> 1
aavrsmnrmt cgaaacctgc ccagcagaac gacccgtgaa cacgtgaatc caagtccagt 60
ggtggtgcaa gtggggagag atcccccttg ctccaccgct cggtcgggga gttggctaac 120
accctctctt tgtgccggaa aacgaaccca aggcgcggtg agcgccaagg aaaaaacaaa 180
tggatgctag cgggcctctt ctctttctcc tgcctcggtg ggaaaaatgc agcggtaggt 240
gtcgcgaaat cctatcttcg aacgactctc ggcaacggat atctcggctc tcgcatcgat 300
gaagaacgta gcgaaatgcg atacttggtg tgaattgcag aatcccgtga accatcgagt 360
ttttgaacgc aagttgcgcc ccaagccttc gggccgaggg cacgcttgcc tgggcgtcac 420
gcaccgagtc tccccctcca actcatgtcc ttggcgcctt ctggcgacat tggcattggg 480
cagtgaatgg ggaggacatt gaccccccgt gcctttaaag tgcggtcggt ctaaacacag 540
gccctgggag gccggcgtca cgattcgtgg tggtcgacac tcgttgtctc tcttcattcc 600
tgaatccgtg tctgctgtgc ttaccgtgaa ggaccataag gaacccatcg ggccaaavrs 660
mnrmtcgaaa cctgcccagc agaacgaccc gtgaacacgt gaatccaagt ccagtggtgg 720
tgcaagtggg gagagatccc ccttgctcca ccgctcggtc ggggagttgg ctaacaccct 780
ctctttgtgc cggaaaacga acccaaggcg cggtgagcgc caaggaaaaa acaaatggat 840
gctagcgggc ctcttctctt tctcctgcct cggtgggaaa aatgcagcgg taggtgtcgc 900
gaaatcctat cttcgaacga ctctcggcaa cggatatctc ggctctcgca tcgatgaaga 960
acgtagcgaa atgcgatact tggtgtgaat tgcagaatcc cgtgaaccat cgagtttttg 1020
aacgcaagtt gcgccccaag ccttcgggcc gagggcacgc ttgcctgggc gtcacgcacc 1080
gagtctcccc ctccaactca tgtccttggc gccttctggc gacattggca ttgggcagtg 1140
aatggggagg acattgaccc cccgtgcctt taaagtgcgg tcggtctaaa cacaggccct 1200
gggaggccgg cgtcacgatt cgtggtggtc gacactcgtt gtctctcttc attcctgaat 1260
ccgtgtctgc tgtgcttacc gtgaaggacc ataaggaacc catcgggcca aavrsmnrmt 1320
cgaaacctgc ccagcagaac gacccgtgaa cacgtgaatc caagtccagt ggtggtgcaa 1380
gtggggagag atcccccttg ctccaccgct cggtcgggga gttggctaac accctctctt 1440
tgtgccggaa aacgaaccca aggcgcggtg agcgccaagg aaaaaacaaa tggatgctag 1500
cgggcctctt ctctttctcc tgcctcggtg ggaaaaatgc agcggtaggt gtcgcgaaat 1560
cctatcttcg aacgactctc ggcaacggat atctcggctc tcgcatcgat gaagaacgta 1620
gcgaaatgcg atacttggtg tgaattgcag aatcccgtga accatcgagt ttttgaacgc 1680
aagttgcgcc ccaagccttc gggccgaggg cacgcttgcc tgggcgtcac gcaccgagtc 1740
tccccctcca actcatgtcc ttggcgcctt ctggcgacat tggcattggg cagtgaatgg 1800
ggaggacatt gaccccccgt gcctttaaag tgcggtcggt ctaaacacag gccctgggag 1860
gccggcgtca cgattcgtgg tggtcgacac tcgttgtctc tcttcattcc tgaatccgtg 1920
tctgctgtgc ttaccgtgaa ggaccataag gaacccatcg ggccaavrsm nrmtcgaaac 1980
ctgcccagca gaacgacccg tgaacacgtg aatccaagtc cagtggtggt gcaagtgggg 2040
agagatcccc cttgctccac cgctcggtcg gggagttggc taacaccctc tctttgtgcc 2100
ggaaaacgaa cccaaggcgc ggtgagcgcc aaggaaaaaa caaatggatg ctagcgggcc 2160
tcttctcttt ctcctgcctc ggtgggaaaa atgcagcggt aggtgtcgcg aaatcctatc 2220
ttcgaacgac tctcggcaac ggatatctcg gctctcgcat cgatgaagaa cgtagcgaaa 2280
tgcgatactt ggtgtgaatt gcagaatccc gtgaaccatc gagtttttga acgcaagttg 2340
cgccccaagc cttcgggccg agggcacgct tgcctgggcg tcacgcaccg agtctccccc 2400
tccaactcat gtccttggcg ccttctggcg acattggcat tgggcagtga atggggagga 2460
cattgacccc ccgtgccttt aaagtgcggt cggtctaaac acaggccctg ggaggccggc 2520
gtcacgattc gtggtggtcg acactcgttg tctctcttca ttcctgaatc cgtgtctgct 2580
gtgcttaccg tgaaggacca taaggaaccc atcgggccaa avrsmnrmtc gaaacctgcc 2640
cagcagaacg acccgtgaac acgtgaatcc aagtccagtg gtggtgcaag tggggagaga 2700
tcccccttgc tccaccgctc ggtcggggag ttggctaaca ccctctcttt gtgccggaaa 2760
acgaacccaa ggcgcggtga gcgccaagga aaaaacaaat ggatgctagc gggcctcttc 2820
tctttctcct gcctcggtgg gaaaaatgca gcggtaggtg tcgcgaaatc ctatcttcga 2880
acgactctcg gcaacggata tctcggctct cgcatcgatg aagaacgtag cgaaatgcga 2940
tacttggtgt gaattgcaga atcccgtgaa ccatcgagtt tttgaacgca agttgcgccc 3000
caagccttcg ggccgagggc acgcttgcct gggcgtcacg caccgagtct ccccctccaa 3060
ctcatgtcct tggcgccttc tggcgacatt ggcattgggc agtgaatggg gaggacattg 3120
accccccgtg cctttaaagt gcggtcggtc taaacacagg ccctgggagg ccggcgtcac 3180
gattcgtggt ggtcgacact cgttgtctct cttcattcct gaatccgtgt ctgctgtgct 3240
taccgtgaag gaccataagg aacccatcgg gcca 3274
Claims (7)
1. based on plant nucleus gene ITS2 to the identification method of Papaveraceae papaver opium poppy, this method uses poppy
ITS2 karyogenes detect opium poppy, find letter of the peculiar base of opium poppy as the sequence difference of other plants in it and papaver
Site is ceased, plant similar with opium poppy in papaver is distinguished by these characteristic information sites.
2. it is according to claim 1 based on plant nucleus gene ITS2 to the identification method of Papaveraceae papaver opium poppy, it is special
Sign is that the ITS2 karyogene overall lengths of the opium poppy are 653bp, and 47bp is C, and 55-56bp is GG, and 114bp is A, the
131bp is G, and the 153rd is G, and 219bp is A, and 248bp is T, and 494bp is C, and 513-516bp is TAAA, 552bp
For G.
3. it is according to claim 1 based on plant nucleus gene ITS2 to the identification method of Papaveraceae papaver opium poppy, it is special
Sign is the sequence of the karyogene ITS2 of the opium poppy, as shown in ITS2.
4. it is a kind of based on plant nucleus gene ITS2 to the identification method of Papaveraceae papaver opium poppy, it is characterised in that this method includes
Following step：
(1) extraction sample to be tested DNA；
(2) PCR amplification is carried out using DNA bar code primer, obtains amplified production, the DNA bar code primer sequence is
ITS2；
(3) amplified production is sequenced, the species identification of similar creamcups, small-mouthed jar is carried out according to following specific position
The feature site of grain is：Overall length is 653bp, and 47bp is C, and 55-56bp is GG, and 114bp is A, and 131bp is G, the
153 be G, and 219bp is A, and 248bp is T, and 494bp is C, and 513-516bp is TAAA, and 552bp is G；
(4) the plant species amplified production of doubtful opium poppy to be measured is sequenced, if meeting the condition of above-mentioned (3), this is waited for
Test sample sheet is opium poppy, if not meeting the condition of above-mentioned (3), which is not opium poppy.
5. it is according to claim 4 based on plant nucleus gene ITS2 to the identification method of Papaveraceae papaver opium poppy, it is special
Sign is that the amplification system of the PCR is 25 μ L, and system includes Mg Cl22.0 μ L, d NTP, 2.0 μ L, 10 × PCR buffer solutions
2.5 μ L, primer each 1.0 μ L, ex Taq enzyme 1.25U, 2.5 μ L of total DNA；The amplification condition of the PCR is：94 DEG C of denaturation 5min,
1 cycle；94 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 45s carry out 30 cycles；72 DEG C of extension 10min.
6. it is according to claim 3 based on plant nucleus gene ITS2 to the identification method of Papaveraceae papaver opium poppy, it is special
Sign is that the sequencing is bidirectional sequencing, and the sequencing primer is shown in ITS2；The reaction system of the sequencing is 5 μ L：Its
Middle ddH2O 3 μ L, 10 μm of 0.5 μ L of ol/L sequencing primers, 0.5 μ L, PCR purified product of sequencing reaction mixture, 1 μ L；The sequencing
Reaction condition be：94 DEG C of pre-degeneration 10s；94 DEG C of denaturation 30s, 50 DEG C of annealing 5s, 60 DEG C of extension 4min carry out 30 cycles.
7. a kind of opium poppy DNA bar code based on molecular data, the DNA bar code primer is ITS2, overall length 653bp, the
47bp is C, and 55-56bp is GG, and 114bp is A, and 131bp is G, and the 153rd is G, and 219bp is A, and 248bp is T, the
494bp is C, and 513-516bp is TAAA, and 552bp is G.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111424076A (en) * | 2020-05-08 | 2020-07-17 | 中国科学院昆明植物研究所 | Method for identifying poppy by using chloroplast genome |
-
2018
- 2018-03-01 CN CN201810170228.3A patent/CN108486268A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| JIANGUO ZHOU: "Complete Chloroplast Genomes of Papaver rhoeas and Papaver orientale: Molecular Structures, Comparative Analysis, and Phylogenetic Analysis", 《MOLECULES》 * |
| 宋炳轲: "利用DNA ITS2 条形码序列鉴定植物大麻和罂粟", 《中国法医学杂质》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111424076A (en) * | 2020-05-08 | 2020-07-17 | 中国科学院昆明植物研究所 | Method for identifying poppy by using chloroplast genome |
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