CN104152564B - A kind of nucleotide sequence, molecular probe and method differentiating purple striae pocket orchid - Google Patents
A kind of nucleotide sequence, molecular probe and method differentiating purple striae pocket orchid Download PDFInfo
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- 206010040925 Skin striae Diseases 0.000 title claims abstract description 50
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- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 241000519406 Paphiopedilum Species 0.000 description 2
- 241000519419 Paphiopedilum armeniacum Species 0.000 description 2
- 241000519045 Paphiopedilum micranthum Species 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
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Abstract
The present invention relates to a kind of nucleotide sequence, molecular probe and the method for differentiating purple striae pocket orchid.Does the present invention differentiate that the nucleotide sequence of purple striae pocket orchid is as SEQ? ID? shown in NO.1.For differentiating the nucleic acid molecule probe upstream ZWF of purple striae pocket orchid: as SEQ? ID? shown in NO.2; Downstream ZWR: as SEQ? ID? shown in NO.3.Utilize nucleic acid molecule probe ZWF/ZWR, differentiate that purple striae pocket is blue by conventional PCR method.Inventive samples consumption is few, only needs a small amount of sample just can complete whole operation; Accurately, highly sensitive, ZWF/ZWR is the blue specificity molecular probe of purple striae pocket, if the blue kinds of other pockets, is negative reaction; Method is simple, and adopt round pcr to detect, the time is short, within 1st, can complete.
Description
Technical field
The invention belongs to the technical field utilizing molecular biology method to differentiate purple striae pocket orchid, relate to a kind of nucleotide sequence, molecular probe and the method for differentiating purple striae pocket orchid.
Background technology
Purple striae pocket orchid (
paphiopedilumpurpuratum) be the orchid family (Orchidaceae) Paphiopedilum (
paphiopedilum) plant, be mainly distributed in the provinces and regions such as China Guangdong, Guangxi, Yunnan and Hong Kong, also there is distribution in Vietnam.Purple striae pocket orchid not only spends U.S., and blade is also very beautiful, and the grid spot that blade face has light green alternate with sap green, has very high ornamental value, thus has the refined title of " Hong Kong girl ".In recent years; owing to artificially excessively gathering, smuggling abroad wildness and the reason such as Habitat destruction is serious; the distribution range atrophy gradually of wild purple striae pocket orchid; quantity sharply declines; wild resource has arrived the edge of extinction, is classified as first class of protection plant by " Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) ".Therefore, in order to the blue wild population of purple striae pocket that available protecting and searching are new, the method setting up quick, accurate discriminating purple striae pocket orchid is very necessary.
Along with the fast development of Protocols in Molecular Biology, in recent years, molecular markers for identification technology achieves fast development, and this also makes plant Rapid identification become possibility.Polymorphic (the startcodontargetedPolymorphism of target initiator codon, SCoT) mark is a kind of goal gene based on translation initiation site mark new technology, primer is designed according to ATG translation initiation site flank conserved sequence, amplification candidate's functional gene region and then produce dominant polymorphism mark, has the features such as simple to operate, reproducible.This experimental applications SCoT labeling technique carries out analysis of genetic diversity to cypripedium; obtain the blue SCoT finger printing of pocket; therefrom filter out the specific sequence fragment of purple striae pocket orchid; by clone, order-checking etc.; design and develop the specific probe for the identification of purple striae pocket orchid, differentiated effective and protect the blue germ plasm resource of purple striae pocket to have very important significance.
Summary of the invention
First object of the present invention there is provided one and utilizes molecular biology method, obtains the nucleotide sequence differentiating purple striae pocket orchid.
The present invention is for differentiating that the characteristic nucleotide sequence of purple striae pocket orchid derives from SCoT universal primer 19(5 '-ACCATGGCTACCACCGGC-3 ') the blue specific nucleotide sequences of purple striae pocket of amplification gained, concrete sequence is:
ACCATGGCTACCACCGGCATGTTAAGTTCCTCTATAAATCCACCCTATTCTTTGGAAGATAGAGGGCCTAGTGAATTCATAGTCCCCATCAACTCACCTGGATGCCCCATAAATCTGCCTTCTTCCCTAAGAAGCTTTAGTGAATGATCAAGTCAAAATTAGTCGGCACCCCCAATGTCGGCCTGGGTAACATCCCCGTATCCCTGTAATTATTATTTTTGGCATGACTTCTATTCAAGTATGACGTACTACTTGCTTTCGCAATAATTTTATTTGTTTTTGCACTTTATGCATGGGGCACTATTCCAGTCCTAGTTTAATAATATAATTTGCAGCTATTTAAATCTTTGCAGAGACTCTTTGAAATTACTTAGTGTCATGAGAATAATTCCGAACAACTCAATGTATGGGAAACCCACTTAACCTACTCAATTGACACTCTTATCATCAGTCTTGCCCACGCACAACTGTGATAGCTGAGTTCAAACTAGAGCGGTCGAAGTTAGGGTCGGAATCATTTACCTTGTCCTATTCAATTATCTCTCAGCACACTGATGATTCCAAAGAGTAGCCCAGATATACGAAGCAAAATCATCAAGTTGCATAAAAAAGCTAACATAATTTATAATATTATGCTGTACAAATAAGTTCGACACCCCCATGGTCAGTATGAAGGCTGACAATCCCATGGTCAGTATAGCTGGCACCCCTATGGCCGGTGGTAGCCATGGT, such as SEQIDNO. 1.
Second object of the present invention is to provide the nucleic acid molecule probe (ZWF/ZWR) for differentiating purple striae pocket orchid that the nucleotide sequence (SEQIDNO.1) based on above-mentioned discriminating purple striae pocket orchid designs, and this nucleic acid molecular probe sequence is as follows:
Upstream primer ZWF:5 '-CCCATAAATCTGCCTTCT-3 ', as shown in SEQIDNO.2;
Downstream primer ZWR:5 '-GGGTGCCAGCTATACTGA-3 ', as shown in SEQIDNO.3.
Above-mentioned nucleic acid molecule probe (ZWF/ZWR), has high specificity, only reacts with purple striae pocket orchid, and does not react with other pockets orchid kinds.Utilize this probe can be differentiated purple striae pocket orchid fast and accurately by standard PCR amplification.
3rd object of the present invention is to provide a kind of method differentiating purple striae pocket orchid.
The present invention adopts above-mentioned nucleic acid molecular probe (ZWF/ZWR) as specificity amplification primer, and with the blue genomic dna of purple striae pocket for template, through pcr amplification, electrophoresis detection, can obtain the blue signature band of purple striae pocket clearly, realize the discriminating to purple striae pocket orchid.
Inventive samples consumption is few, only needs a small amount of sample just can complete whole operation; Accurately, highly sensitive, ZWF/ZWR is the blue specificity molecular probe of purple striae pocket, if the blue kinds of other pockets, is negative reaction; Method is simple, and adopt round pcr to detect, the time is shorter, within 1st, just can complete.
Accompanying drawing explanation
Fig. 1 is the characteristic nucleotide sequence of purple striae pocket orchid of the present invention and the site plan to the blue specificity nucleic acid molecular probe of purple striae pocket ZWF and ZWR, left side is 5 ' end, right side is 3 ' end, and wherein black part is divided into the sequence fragment size and location that specificity nucleic acid molecule probe ZWF and ZWR increases;
Fig. 2 be adopt SCoT primer 19 conveniently PCR method the reacted electrophorogram of PCR is carried out to the blue sample of different pocket (band of arrow indication is the blue distinctive band of purple striae pocket, molecular weight is 732bp), wherein, 1, Malipo pocket orchid (Malipo, Yunnan), 2, Malipo pocket orchid (Zhejiang Hangzhou), 3, paphiopedilum armeniacum (Lijiang, yunnan), 4, Paphiopedilum micranthum (Yunnan mountain of papers), 5, volume calyx pocket orchid (Nanning), 6, Henry pocket orchid (Malipo, Yunnan), 7, band leaf pocket orchid (Longzhou), 8, purple striae pocket orchid (Guangzhou Guangdong), 9, bright cloud pocket orchid (Yunnan mountain of papers), M is DNA molecular amount standard markerDL2000.
Fig. 3 is the PCR primer electrophorogram utilizing nucleic acid molecular probe ZWF/ZWR of the present invention to detect the blue sample of different pocket, 1, Malipo pocket orchid (Malipo, Yunnan), 2, Malipo pocket orchid (Zhejiang Hangzhou), 3, paphiopedilum armeniacum (Lijiang, yunnan), 4, Paphiopedilum micranthum (Yunnan mountain of papers), 5, calyx pocket orchid (Nanning) is rolled up, 6, Henry pocket orchid (Malipo, Yunnan), 7, leaf pocket orchid (Longzhou) is with, 8, purple striae pocket orchid (Guangzhou Guangdong), 9, bright cloud pocket orchid (Yunnan mountain of papers), M is DNA molecular amount standard markerDL2000.
Embodiment
The present invention can be comparatively objective and accurate from molecular level discriminating purple striae pocket blue, by following examples, the present invention will be further described:
Embodiment 1: the preparation of the blue characteristic nucleotide sequence of purple striae pocket
1. the extraction of genomic dna
The blue blade 0.2g of pocket that clip-80 DEG C is preserved puts into mortar, add liquid nitrogen grinding immediately to powder, then the UNIQ-10 pillar plant genome DNA extraction agent box of Shanghai Sheng Gong biotechnology company limited is used to extract cypripedium genomic dna, with 1% agarose gel, electrophoresis detection is carried out to gained DNA, and by UV spectrophotometer measuring DNA concentration, be diluted to 50ng/ μ l.
2.SCoT – PCR reacts, electrophoresis detection
Utilize SCoT universal primer 19(5 '-ACCATGGCTACCACCGGC-3 ') carry out PCR reaction, primer is synthesized by Shanghai Sheng Gong biotechnology company limited, and reaction system is: 2 μ l10 × Buffer, 2 μ lMgCl
2(25mM), 0.8 μ ldNTPs (10mM), 1 μ lSCoT primer 19 (10 μMs), 1 μ l template DNA (50ng/ μ l), 0.5 μ lTaq enzyme (2U/ μ l), 12.7 μ lddH
2o.Cumulative volume is 20 μ l.
PCR response procedures is: 94 DEG C of denaturation 4min; 35 circulations (72 DEG C extend 2min for 94 DEG C of sex change 45s, 55 DEG C of annealing 45s); Last in 72 DEG C of downward-extension 10min.
PCR primer electrophoresis result is shown in Fig. 2, arrow target band (molecular weight is 732bp), the blue signature band of the purple striae pocket filtered out when being and adopting SCoT primer 19 to carry out pcr amplification.As Fig. 2, (in figure, passage 1 ~ 9 is from 9 parts of blue samples of different pocket, specifically sees accompanying drawing explanation; Passage M is DNA molecular amount standard markerDL2000) shown in, only purple striae pocket orchid (passage 8) has band to occur in molecular weight 732bp position, and the blue kind of other pockets does not have band to occur in molecular weight 732bp position.Therefore, this band is the peculiar band of purple striae pocket orchid.
3. sequencing
SCoT universal primer 19 is utilized to carry out after pcr amplification terminates, PCR primer 1.5 ﹪ agarose gel electrophoresis, glue is cut to the special SCoT fragment only occurred in purple striae pocket orchid (in Fig. 2 arrow indication fragment), adopts PCR primer purification kit (the raw work in Shanghai) to reclaim the purifying section of section.Then purified DNA fragmentation is connected on PUCm-T carrier (the raw work in Shanghai), is transformed in competent escherichia coli cell, then send biotech firm to check order (the raw work in Shanghai), obtain the Nucleotide composition as shown in SEQIDNO.1 and arrangement.
Embodiment 2: the preparation of purple striae pocket blue specific nucle molecular probe ZWF/ZWR, PCR reacts, electrophoresis detection
On the basis obtaining the blue specific nucleotide sequences of purple striae pocket, utilize PrimerPrimer5.0 software design to draw the nucleotide sequence (being respectively shown in SEQIDNO.2, SEQIDNO.3) of ZWF/ZWR, primer is synthesized by Shanghai Sheng Gong biotechnology company limited.Then utilize the primer ZWF/ZWR of design and synthesis, augmentation detection is carried out to 9 parts of blue samples (specifically seeing that accompanying drawing illustrates) of different pocket.
PCR reaction system is 2 μ l10 × Buffer, 2 μ lMgCl
2(25mM), 0.8 μ ldNTPs (10mM), 1 μ l primer ZWF (10 μMs), 1 μ l primer ZWR (10 μMs), 1 μ l template DNA (50ng/ μ l), 0.5 μ lTaq enzyme (2U/ μ l), 11.7 μ lddH
2o.Cumulative volume is 20 μ l.
PCR response procedures is 94 DEG C of denaturation 4min; 35 circulations (72 DEG C extend 2min for 94 DEG C of sex change 45s, 58 DEG C of annealing 45s); Last in 72 DEG C of downward-extension 10min.
Detect PCR result with 1.5 ﹪ agarose gel electrophoresis, as shown in Figure 3, from Fig. 3, (figure, passage 1 ~ 9 is from 9 parts of different blue samples of pocket to electrophorogram, specifically sees accompanying drawing explanation; Passage M is DNA molecular amount standard markerDL2000) can find out, only purple striae pocket orchid (passage 8) can amplify specific fragment, and the blue kind of other pockets does not amplify any band, this shows that nucleic acid molecular probe of the present invention has high specificity, therefore may be used for differentiating that purple striae pocket is blue fast.The specific fragment of purple striae pocket orchid is sent to order-checking, and its sequence is as shown in 106 ~ 709 bit bases of SEQIDNO.1, and its length is 604bp, and concrete sequence information is shown in Fig. 1.
SEQUENCELISTING
<110> Hangzhou Pedagogic University
<120> mono-kind differentiates the nucleotide sequence of purple striae pocket orchid, molecular probe and method
<130>1
<160>3
<170>PatentInversion3.3
<210>1
<211>732
<212>DNA
<213> synthetic
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accatggctaccaccggcatgttaagttcctctataaatccaccctattctttggaagat60
agagggcctagtgaattcatagtccccatcaactcacctggatgccccataaatctgcct120
tcttccctaagaagctttagtgaatgatcaagtcaaaattagtcggcacccccaatgtcg180
gcctgggtaacatccccgtatccctgtaattattatttttggcatgacttctattcaagt240
atgacgtactacttgctttcgcaataattttatttgtttttgcactttatgcatggggca300
ctattccagtcctagtttaataatataatttgcagctatttaaatctttgcagagactct360
ttgaaattacttagtgtcatgagaataattccgaacaactcaatgtatgggaaacccact420
taacctactcaattgacactcttatcatcagtcttgcccacgcacaactgtgatagctga480
gttcaaactagagcggtcgaagttagggtcggaatcatttaccttgtcctattcaattat540
ctctcagcacactgatgattccaaagagtagcccagatatacgaagcaaaatcatcaagt600
tgcataaaaaagctaacataatttataatattatgctgtacaaataagttcgacaccccc660
atggtcagtatgaaggctgacaatcccatggtcagtatagctggcacccctatggccggt720
ggtagccatggt732
<210>2
<211>18
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<213> synthetic
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cccataaatctgccttct18
<210>3
<211>18
<212>DNA
<213> synthetic
<400>3
gggtgccagctatactga18
Claims (3)
1. differentiate a nucleotide sequence for purple striae pocket orchid, it is characterized in that this nucleotides sequence is classified as:
ACCATGGCTACCACCGGCATGTTAAGTTCCTCTATAAATCCACCCTATTCTTTGGAAGATAGAGGGCCTAGTGAATTCATAGTCCCCATCAACTCACCTGGATGCCCCATAAATCTGCCTTCTTCCCTAAGAAGCTTTAGTGAATGATCAAGTCAAAATTAGTCGGCACCCCCAATGTCGGCCTGGGTAACATCCCCGTATCCCTGTAATTATTATTTTTGGCATGACTTCTATTCAAGTATGACGTACTACTTGCTTTCGCAATAATTTTATTTGTTTTTGCACTTTATGCATGGGGCACTATTCCAGTCCTAGTTTAATAATATAATTTGCAGCTATTTAAATCTTTGCAGAGACTCTTTGAAATTACTTAGTGTCATGAGAATAATTCCGAACAACTCAATGTATGGGAAACCCACTTAACCTACTCAATTGACACTCTTATCATCAGTCTTGCCCACGCACAACTGTGATAGCTGAGTTCAAACTAGAGCGGTCGAAGTTAGGGTCGGAATCATTTACCTTGTCCTATTCAATTATCTCTCAGCACACTGATGATTCCAAAGAGTAGCCCAGATATACGAAGCAAAATCATCAAGTTGCATAAAAAAGCTAACATAATTTATAATATTATGCTGTACAAATAAGTTCGACACCCCCATGGTCAGTATGAAGGCTGACAATCCCATGGTCAGTATAGCTGGCACCCCTATGGCCGGTGGTAGCCATGGT。
2., based on a kind of Auele Specific Primer ZWF/ZWR differentiating the nucleotide sequence design of purple striae pocket orchid as claimed in claim 1, it is characterized in that, this specific primer sequence is as follows:
Upstream primer ZWF:5 '-GAAATGAAACCGAAAGAAAG-3 ';
Downstream primer ZWR:5 '-AATGGCTACCACCCTCCT-3 '.
3. differentiate a method for purple striae pocket orchid, it is characterized in that, adopt Auele Specific Primer ZWF/ZWR according to claim 2 as PCR primer, purple striae pocket is blue to utilize PCR method to identify, concrete steps routinely PCR method are carried out.
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