CN106119394A - The nucleotide sequence authentication method of a kind of generation latitude lettuce tongue and the application of nucleotide sequence - Google Patents
The nucleotide sequence authentication method of a kind of generation latitude lettuce tongue and the application of nucleotide sequence Download PDFInfo
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- CN106119394A CN106119394A CN201610725888.4A CN201610725888A CN106119394A CN 106119394 A CN106119394 A CN 106119394A CN 201610725888 A CN201610725888 A CN 201610725888A CN 106119394 A CN106119394 A CN 106119394A
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention discloses nucleotide sequence authentication method and the application of nucleotide sequence of a kind of generation latitude lettuce tongue, comprise the following steps: S1: obtain plant tissue sample and extract sample DNA；S2: use ITS primer and/or sample DNA is carried out PCR amplification, after electrophoresis detection pcr amplification product, pcr amplification product being checked order.S3: the sequence that 3 order-checkings obtain is compared by DNAMAN software, cuts off the insincere sequence that two ends are inconsistent, obtain ITS sequence and the trnL F sequence of plant tissue sample, be used for identifying whether this plant tissue sample is generation latitude lettuce tongue.This authentication method analyzes the hereditary material of generation latitude lettuce tongue from molecular level analysis, so can plant identification generation latitude lettuce tongue fast and accurately, protection and medical value research for generation latitude lettuce tongue lay the foundation.
Description
Technical field
The invention belongs to Medicinal Plant Germplasm Resources identification technology field, be specifically related to the nucleotides sequence of a kind of generation latitude lettuce tongue
Row authentication method and the application of nucleotide sequence.
Background technology
Generation latitude lettuce tongue Tengia scopulorum Chun, has another name called Guizhou Province lettuce tongue, and the peculiar Precious, Rare, Endangered of Guizhou In China is medicinal plants
Thing.Mr. Deng Shiwei catches an illness, in succession to gather this batch of plant field with assistant Yang Changhan, Xu Fangcai, yellow diligent civilian four people then
Die at one's post, paid the cost of life.Nowadays the proterotype place of production (Guiding flat cuts down) population is doubtful has withered away.With regard to bio-diversity
Speech, Endangered species is priority protection object.And generation latitude lettuce tongue has for the research of Gesneriaceae system classification and evo-devo
There are very important scientific value and meaning, are irreplaceable valuable materials.Limited by stock number, the medicine of generation latitude lettuce tongue simultaneously
Still belong to blank with research.Protection and further scientific research, the matter of utmost importance faced is all the qualification of former plant.And it is traditional
Phytomorph is identified have to wait until the florescence, has bigger time restriction, and the distinguishing ability of operator is had higher wanting
Ask.Simultaneously because sample size and correlational study are less, existing document is also undisclosed the DNA Identification bar for generation latitude lettuce tongue
Shape code sequence.
Summary of the invention
There is the restriction of discriminating time period and differentiate that the technology that difficulty is bigger is asked in the species identification for existing generation latitude lettuce tongue
Topic, the invention discloses nucleotide sequence authentication method and the application of nucleotide sequence, this authentication method of a kind of generation latitude lettuce tongue
Analyze the hereditary material of generation latitude lettuce tongue from molecular level analysis, so can plant identification generation latitude lettuce tongue fast and accurately, for generation
The protection of latitude lettuce tongue and medical value research lay the foundation.
The nucleotide sequence authentication method of a kind of generation latitude lettuce tongue, it is characterised in that comprise the following steps:
S1: obtain plant tissue sample and extract sample DNA；
S2: using ITS primer that sample DNA carries out PCR amplification, described ITS primer includes on the ITS shown in SEQ ID NO.1
ITS downstream primer shown in trip primer and SEQ ID NO.2, after electrophoresis detection pcr amplification product, is carried out pcr amplification product
Order-checking；
And/or using trnL-F primer that sample DNA carries out PCR amplification, described trnL-F primer includes shown in SEQ ID NO.3
TrnL-F forward primer and SEQ ID NO.4 shown in trnL-F downstream primer, electrophoresis detection isolates pcr amplification product
After, pcr amplification product is checked order；
S3: the sequence obtained repeatedly checking order is compared by DNAMAN software, cuts off the insincere sequence that two ends are inconsistent,
Obtain ITS sequence and the trnL-F sequence of plant tissue sample, ITS sequence is compared with the sequence shown in SEQ ID NO.13
Right, trnL-F sequence is compared with the sequence shown in SEQ ID NO.14, to identify that whether this plant tissue sample is as generation
Latitude lettuce tongue.
Further, in step sl, described plant tissue sample is the leaf tissue sample with chloroplast, and uses
Described plant tissue sample is dried by silica gel.
Further, step S1 includes: the silica dehydrator blade taking 0.5-2cm3 is placed in the EP pipe of 2mL, puts into little magnetic
Pearl, mills 30s with grater after liquid nitrogen freezing, adds CTAB solution preheated for 800 μ L65 DEG C, add the β of 0.5% concentration-
Mercaptoethanol 10 μ L, 65 DEG C of water-bath 2h；Adding the CI solution of 800 μ L, described CI solution is to include the chloroform that volume ratio is 24:1
And isoamyl alcohol, shake up 10min；Then 10 DEG C, 12000rpm, centrifugal 10min；Take supernatant in the EP pipe of 2mL, add CI solution,
Shake up, centrifugal；Take supernatant in the EP pipe of 1.5mL, add CI solution, shake up, 4 DEG C, 12000rpm, centrifugal 7min；Take supernatant, turn
EP to 1.5mL manages；Add the isopropanol of equivalent-20 DEG C, stand 1-2h at-20 DEG C, make DNA be precipitated out；After standing, 4 DEG C,
12000rpm, centrifugal 10min, abandon supernatant；Add 300 μ L 75% ethanol, wash twice, each 4 DEG C, 12000rpm, centrifugal
10min；Natural air drying about 30min, adds the DDH of 60 μ L2O dissolving DNA about 30min, obtains sample DNA；Use gel electrophoresis
Detection STb gene.
Further, in step S2, the reaction system of PCR amplification includes: 10 × Buffer 2 μ L, dNTP 1.6 μ L,
DDH2O 11.3 μ L, Taq enzyme 0.1 μ L, sample DNA 1 μ L, ITS forward primer or trnL-F forward primer 2 μ L, ITS
Downstream primer or trnL-F downstream primer 2 μ L；
The concentration of described dNTP is 2.5 mmol/L, described ITS forward primer, trnL-F forward primer, ITS downstream primer,
The concentration of trnL-F downstream primer is 2 mmol/L.
Further, in step S2, the course of reaction of PCR amplification includes: enters after 94 DEG C of denaturations 4min and circulates, 94 DEG C
Degeneration 45s, 54 DEG C of annealing 45s, 72 DEG C extend 45s, circulate 32 times, and 72 DEG C extend 10min.
Further, step S3 also includes: by the ITS sequence obtained and trnL-F sequence inputting plant genetic sequence number
Compare according to storehouse, determine nearly edge species.
Application in SEQ ID NO.13 sequence alive latitude lettuce tongue qualification as above.
Application in SEQ ID NO.14 sequence alive latitude lettuce tongue qualification as above.
According to the nucleotide sequence authentication method of the generation latitude lettuce tongue that the present invention provides, it is that the present inventor passes through different primers
Find after PCR amplification test, use ITS sequence and trnL-F sequence that ITS primer and trnL-F primer obtain after carrying out PCR amplification
Row have the genetic sequence of the distinguishing characteristics bigger with other plants, i.e. ITS sequence and trnL-F sequence and other species
There is bigger discrimination, be applied to the species identification of generation latitude lettuce tongue, compared with traditional identification of morphology, this DNA sequence
Authentication method by vegetation period limited little, plant tissue materials needed for qualification is few, is particularly well suited for use in rare or endangered species
Qualification；On the other hand this method can reduce the unstable factor identifying qualification process, simultaneously as the quantization characteristic of sequence
And decrease artificial subjective intervention so that the qualification process of generation latitude lettuce tongue more quick and precisely, for generation latitude lettuce tongue protection with
Research provides reliable basis.
Detailed description of the invention
Technical scheme in the present invention will be clearly and completely described below, it is clear that described is only this
Bright a part of embodiment rather than whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art are not having
There is on the premise of making creative work the every other embodiment obtained, broadly fall into the scope of protection of the invention.
Embodiment 1
The material that the present embodiment uses is the generation latitude lettuce that Guiyang College of Traditional Chinese Medicine He Shunzhi professor finds in Guiyang and identifies
The stochastical sampling sample of tongue population, voucher specimen deposits in Wang Yin political affairs seminar of Plant Inst., specimen numbering LMT-
2012-016.Choose each 1 of the fresh blade of the population diverse location multiple plant of point, seal up for safekeeping with silica dehydrator immediately.
Take the blade of generation latitude lettuce tongue silica dehydrator, use the CTAB method after improvement to extract STb gene.
Extracting STb gene operation is: the silica dehydrator blade taking 0.5-2cm3 is placed in the EP pipe of 2mL, puts into little magnetic bead, liquid
Milling 30s with grater after chilled nitrogen, (cetyl trimethylammonium bromide is molten to add CTAB solution preheated for 800 μ L65 DEG C
Liquid), add the beta-mercaptoethanol 10 μ L of 0.5% concentration, 65 DEG C of water-bath 2h；Adding the CI solution of 800 μ L, described CI solution is
Including the chloroform that volume ratio is 24:1 and isoamyl alcohol, shake up 10min；Then 10 DEG C, 12000rpm, centrifugal 10min；Take supernatant extremely
In the EP pipe of 2mL, add CI solution, shake up, centrifugal；Take supernatant in the EP pipe of 1.5mL, add CI solution, shake up, 4 DEG C,
12000rpm, centrifugal 7min；Take supernatant, go to the EP pipe of 1.5mL.Add the isopropanol of equivalent-20 DEG C, at-20 DEG C, stand 1-
2h, makes DNA be precipitated out；After standing, 4 DEG C, 12000rpm, centrifugal 10min, abandon supernatant；Add 300 μ L 75% ethanol, washing
Twice, each 4 DEG C, 12000rpm, centrifugal 10min；Natural air drying about 30min, adds the DDH of 60 μ L2O(distilled water) dissolve
About DNA30min, obtains sample DNA；With detected through gel electrophoresis STb gene (taking 2 μ L mother solutions, with 6 × Loading buffer).
Using ITS primer that sample DNA carries out PCR amplification, described ITS primer includes on the ITS shown in SEQ ID NO.1
ITS downstream primer shown in trip primer and SEQ ID NO.2.
The reaction system of PCR amplification includes: 10 × Buffer 2 μ L, dNTP(2.5 mM) 1.6 μ L, DDH2O 11.3
μ L, Taq enzyme 0.1 μ L, sample DNA 1 μ L, ITS forward primer (2 mM) 2 μ L, ITS downstream primer (2 mM) 2 μ L.
The course of reaction of PCR amplification includes: enter circulation, 94 DEG C of degeneration 45s, 54 DEG C of annealing after 94 DEG C of denaturations 4min
45s, 72 DEG C extend 45s, circulate 32 times, and 72 DEG C extend 10min, obtain pcr amplification product.
After the detected through gel electrophoresis pcr amplification product of 1% agarose, pcr amplification product send Hua Da gene do two-way survey
Sequence.
Record sequence with DNAMAN by 3 times to compare, cut off the insincere sequence that two ends are inconsistent, obtain SEQ ID
NO.13 sequence, long 617 bases.
The data base of the SEQ ID NO.13 sequence inputting " Chinese crude drug DNA bar code identification systems " obtained will carry out thing
Plant and identify, show that immediate species are shown as Tengia scopulorum [Petrocodon scopulorus], sequence phase
Like property 99.2%.
The data base of the BLAST webpage of the SEQ ID NO.14 sequence inputting NCBI obtained will carry out species identification,
Close species are shown as Tengia scopulorum, sequence similarity 99%.
Embodiment 2
The present embodiment include such as embodiment 1 in most of technical characteristic, its difference is:
Using trnL-F primer that sample DNA carries out PCR amplification in the present embodiment, described trnL-F primer includes SEQ ID
TrnL-F forward primer shown in NO.3 and the trnL-F downstream primer shown in SEQ ID NO.4.
The reaction system of PCR amplification includes: 10 × Buffer 2 μ L, dNTP(2.5 mM) 1.6 μ L, DDH2O 11.3
μ L, Taq enzyme 0.1 μ L, sample DNA 1 μ L, trnL-F forward primer (2 mM) 2 μ L, trnL-F downstream primer (2 mM) 2
μL。
Finally give SEQ ID NO.14 sequence, long 838 bases.
The data base of the BLAST webpage of the SEQ ID NO.14 sequence inputting NCBI obtained will carry out species identification,
With " ident " as priority ordering under the conditions of, immediate species are shown as Tengia scopulorum, sequence similarity 99%.
Comparative example 1
This comparative example include such as embodiment 1 in most of technical characteristic, its difference is:
Using rbcL primer that sample DNA carries out PCR amplification in the present embodiment, described rbcL primer includes rbcL forward primer
(as shown in SEQ ID NO.5) and rbcL downstream primer (as shown in SEQ ID NO.6).
The reaction system of PCR amplification includes: 10 × Buffer 2 μ L, dNTP(2.5 mM) 1.6 μ L, DDH2O 11.3
μ L, Taq enzyme 0.1 μ L, sample DNA 1 μ L, rbcL forward primer (2 mM) 2 μ L, rbcL downstream primer (2 mM) 2 μ L..
Obtain SEQ ID NO.15 sequence, long 636 bases.
By the data base of the BLAST webpage of SEQ ID NO.15 sequence inputting NCBI carries out species identification, immediate
Species are shown as Primulina liboensis, sequence similarity 99%.
Comparative example 2
This comparative example include such as embodiment 1 in most of technical characteristic, its difference is:
Using matK primer that sample DNA carries out PCR amplification in the present embodiment, described matK primer includes rbcL forward primer
(as shown in SEQ ID NO.7) and matK downstream primer (as shown in SEQ ID NO.8).
The reaction system of PCR amplification includes: 10 × Buffer 2 μ L, dNTP(2.5 mM) 1.6 μ L, DDH2O 11.3
μ L, Taq enzyme 0.1 μ L, sample DNA 1 μ L, matK forward primer (2 mM) 2 μ L, matK downstream primer (2 mM) 2 μ L.
Obtaining SEQ ID NO.16 sequence, (after comparison, end has done the length after nearly 200 bases are wiped out to long 847 bases
Degree).
By the data base of the BLAST webpage of SEQ ID NO.16 sequence inputting NCBI carries out species identification, immediate
Species are shown as Petrocodon coriaceifolius, sequence similarity 99%.
Comparative example 3
This comparative example include such as embodiment 1 in most of technical characteristic, its difference is:
Using atpI-H primer that sample DNA carries out PCR amplification in the present embodiment, described matK primer includes that atpI-H upstream is drawn
Thing (as shown in SEQ ID NO.9) and atpI-H downstream primer (as shown in SEQ ID NO.10).
The reaction system of PCR amplification includes: 10 × Buffer 2 μ L, dNTP(2.5 mM) 1.6 μ L, DDH2O 11.3
μ L, Taq enzyme 0.1 μ L, sample DNA 1 μ L, atpI-H forward primer (2 mM) 2 μ L, atpI-H downstream primer (2 mM) 2 μ
L。
Obtaining SEQ ID NO.17 sequence, (after comparison, the length after about 70 bases are wiped out has been done in front end to long 867 bases
Degree).
By the data base of the BLAST webpage of SEQ ID NO.17 sequence inputting NCBI carries out species identification, immediate
Species are shown as Petrocosmea iodioides, sequence similarity 98%.
Comparative example 4
This comparative example include such as embodiment 1 in most of technical characteristic, its difference is:
Using rps16 primer that sample DNA carries out PCR amplification in the present embodiment, described rps16 primer includes that atpI-H upstream is drawn
Thing (as shown in SEQ ID NO.11) and rps16 downstream primer (as shown in SEQ ID NO.12).
The reaction system of PCR amplification includes: 10 × Buffer 2 μ L, dNTP(2.5 mM) 1.6 μ L, DDH2O 11.3
μ L, Taq enzyme 0.1 μ L, sample DNA 1 μ L, rps16 forward primer (2 mM) 2 μ L, rps16 downstream primer (2 mM) 2 μ L.
Obtain SEQ ID NO.18 sequence, long 760 bases
The data base of the BLAST webpage of SEQ ID NO.18 sequence inputting NCBI will carry out species identification, immediate species
It is shown as Primulina dryas, sequence similarity 98%.
Interpretation of result
ITS primer employed in embodiment 1-2, comparative example 1-4, trnL-F primer, rbcL primer, matK primer, atpI-H
Primer and rps16 primer are existing primer, according to identification result analysis, determine the DNA Identification bar code of generation latitude lettuce tongue
ITS sequence (SEQ ID NO.13 sequence) and the trnL-F sequence (SEQ ID NO.14 sequence) of chloroplast DNA for core DNA.
It is direct and effective that DNA fragmentation is applied to plant identification, and its key link is prestoring of data.Just as form
The flora of appraisal basis, pre-stored and the identification result of the DNA of plants data base needed for DNA qualification are evaluated most important.Right
Ratio sequence, such as generation latitude lettuce tongue rbcL, matK, atpI-H, rps16, lacks the nearlyest edge DNA data stored in advance, so
Cannot be applied to identify.On the other hand, some fragment is too low to the discrimination of concrete species, also cannot be applied to identify；Such as generation
The matK sequence of latitude lettuce tongue, has reached more than 20 the most with the species sequence of its sequence similarity 99%, it is difficult to distinguish；rbcL、
AtpI-H, rps16 sequence also finds, DNA of plants data base, other species that sequence similarity is higher, and discrimination is inadequate.
DNA identifies that the advantage of identification of morphology of ratio mainly has: by vegetation period limited material that is minimum, that take needed for qualification
Material is few, simultaneously as the quantization characteristic of sequence and decrease artificial subjective intervention.Wherein minimum to trophophase and material
Require feature, be particularly well suited for use in the qualification of rare or endangered species.
Unique sequence fragment is limited to the discrimination of extensive species identification, needs more reliable multisequencing combination to identify bar shaped
Code, reaches the purpose of precise Identification.Therefore ITS and the trnL-F sequence bar code originally determined be combined with each other and can reach treasure
The quick and precisely qualification of dilute Endangered Medicinal Herb generation latitude lettuce tongue, protection and research for generation latitude lettuce tongue provide reliable basis.
Above content is to combine concrete preferred implementation further description made for the present invention, it is impossible to assert
Being embodied as of the present invention is confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of present inventive concept, it is also possible to make some simple deduction or replace, all should be considered as belonging to the present invention's
Protection domain.
<110>Liu, bright great waves
<120>the nucleotide sequence authentication method of a kind of generation latitude lettuce tongue and the application of nucleotide sequence
<130> 2016
<160> 18
<170> PatentIn version 3.5
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<210> 6
<211> 20
<212> DNA
<213>artificial sequence
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<210> 7
<211> 23
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ttggaacaag ggtatcgaac 20
<210> 9
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ccaayccagc agcaataac 19
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gaaggacacg atccgytgtg gat 23
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<212> DNA
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cgatagacgg ctcattggga ta 22
<210> 13
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<212> DNA
<213>generation latitude lettuce tongue (Tengia scopulorum Chun)
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<213>generation latitude lettuce tongue (Tengia scopulorum Chun)
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<213>generation latitude lettuce tongue (Tengia scopulorum Chun)
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<213>generation latitude lettuce tongue (Tengia scopulorum Chun)
<400> 16
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tcaatatttc cctttttaga ggacaatttt tcacatttca cttttgtgtt agatatattc 300
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tctcatttac gatcaacatc ttgtgaagtt cttcttgaac gaatctattt ctatgtaaaa 600
atagaacgtc ttgtgaacgt ctttgttaag gttaactatt ttcagacgaa cttatggttg 660
gtgaaggaat cttgcatgca ttatgttagg tatcaaagaa aatccatttt ggcttcaaaa 720
gggacgtctc attttatgaa taaatggaaa tgttaccttg taactttttg gcaatggcat 780
ttttcgctgt ggtttcatcc aagaaggatt tatataaacc aattatccaa tcattccctt 840
gaatttt 847
<210> 17
<211> 867
<212> DNA
<213>generation latitude lettuce tongue (Tengia scopulorum Chun)
<400> 17
ctaagattcc ccttttcgtc cacttaatat catatcgtat ttttattcaa ggaatattta 60
ctatatatta tattggtccg ccaatcttct taattcatca aatcataatt tcaataagat 120
atatgtttac gacgtgtgag taaataaaaa accggagaac ctattctact ttacagttca 180
ttttattcaa caattgacca aaagaaaata ttttattaat aaataaaata ataaattgca 240
tgaacttaga tcaatcaaat aatgatattt ctatccaata atgggcctta atcaatttca 300
atttaatttg cccatttaga ttagattgta ttcggttgga atggtaataa cgaaaacgga 360
aggaagaaaa aaaatttaca aaacaaaaat gggattccta aaaaaataga tagattctcg 420
aatctgattg aatttaatgg tttacgttat ggaagaaaca cgtgtatatg tgatattaga 480
tattgactag ttatatatga actaaagata tatttaattt ctcctactac tgtagggggg 540
ttctaacaga agtcctttcg tctcgtttcg actgtgactt gcctgaataa agagatgaaa 600
tcaagaaaag ctgaaagaat tgtgaaataa cagtttggaa ccaagaaatg taaaaacgag 660
aattctatgg attcgcgaag actctgtgga tagaaacgaa aaaggatata tcgaagtagt 720
tctgataatt caataatatt attactgaaa ttcgaagttc ttagttactt cgactagatt 780
aatcctatcg atcgaataat taagtcataa ttcattggtt gattgtatca ttaaccattt 840
ctttttgttg gtacgaggaa cttatca 867
<210> 18
<211> 760
<212> DNA
<213>generation latitude lettuce tongue (Tengia scopulorum Chun)
<400> 18
tgaagatgct cttgtctcga catcgtttgt tctgttacac ctgaatcctt tgtttttttg 60
ttgggttgta aatagtctac gatggagctc gagtcgaaaa gtcgaaagga ttaattcatt 120
tctggggggc aaggatctag ggttaatgcc aatcaatcaa ttggaacaac ttcgtaaacg 180
tatcttctat atataataat atagaaatca aaaggatcca atcaaatttc aacaagtttt 240
ctttttaaat tggaaacttg ttataattga tcaaactttt tcgattcaaa gtgtattacg 300
cgggaatcaa ctgtccatcg gattctttga tagaaagaaa tcaaatttca aatatttcca 360
attcaaatga aagggtatgt tgctgccatt ttgaaagtat taagaagcac cgaagtaatg 420
tctaaaccca atgatttaaa ataaagatta aaggatccaa gaacaagtaa atccatttta 480
attgtctcga tagtctcaat aactggatca tccaaatcta attttaaacg agacaaaaaa 540
agcgggtaaa gacaactcaa taaatgaaat tgactaaaga gaagaatttc cttttgagct 600
atttgagagt tattcaactt gagttatgag tacgaatggt ttcttttaaa ttttcaggaa 660
ggacaaaaaa cgaatgaaat taaagtctaa ttgattttct aggttcattc gaatcaaatc 720
attttttctc gagccgtacg aggagaaaac ttcctatacg 760
Claims (8)
1. the nucleotide sequence authentication method of a generation latitude lettuce tongue, it is characterised in that comprise the following steps:
S1: obtain plant tissue sample and extract sample DNA；
S2: using ITS primer that sample DNA carries out PCR amplification, described ITS primer includes on the ITS shown in SEQ ID NO.1
ITS downstream primer shown in trip primer and SEQ ID NO.2, after electrophoresis detection pcr amplification product, is carried out pcr amplification product
Order-checking；
And/or using trnL-F primer that sample DNA carries out PCR amplification, described trnL-F primer includes shown in SEQ ID NO.3
TrnL-F forward primer and SEQ ID NO.4 shown in trnL-F downstream primer, electrophoresis detection isolates pcr amplification product
After, pcr amplification product is checked order；
S3: the sequence obtained repeatedly checking order is compared by DNAMAN software, cuts off the insincere sequence that two ends are inconsistent,
Obtain ITS sequence and the trnL-F sequence of plant tissue sample, ITS sequence is compared with the sequence shown in SEQ ID NO.13
Right, trnL-F sequence is compared with the sequence shown in SEQ ID NO.14, to identify that whether this plant tissue sample is as generation
Latitude lettuce tongue.
The nucleotide sequence authentication method of a kind of generation latitude lettuce tongue the most according to claim 1, it is characterised in that in step S1
In, described plant tissue sample is the leaf tissue sample with chloroplast, and uses silica gel to enter described plant tissue sample
Row is dried.
The nucleotide sequence authentication method of a kind of generation latitude lettuce tongue the most according to claim 1 and 2, it is characterised in that step
S1 includes: the silica dehydrator blade taking 0.5-2cm3 is placed in the EP pipe of 2mL, puts into little magnetic bead, uses grater after liquid nitrogen freezing
Mill 30s, adds CTAB solution preheated for 800 μ L65 DEG C, adds the beta-mercaptoethanol 10 μ L of 0.5% concentration, 65 DEG C of water-baths
2h；Adding the CI solution of 800 μ L, described CI solution is to include chloroform and the isoamyl alcohol that volume ratio is 24:1, shakes up 10min；So
Latter 10 DEG C, 12000rpm, centrifugal 10min；Take supernatant in the EP pipe of 2mL, add CI solution, shake up, centrifugal；Take supernatant extremely
In the EP pipe of 1.5mL, add CI solution, shake up, 4 DEG C, 12000rpm, centrifugal 7min；Take supernatant, go to the EP pipe of 1.5mL；Add
The isopropanol of equivalent-20 DEG C, stands 1-2h, makes DNA be precipitated out at-20 DEG C；After standing, 4 DEG C, 12000rpm, centrifugal
10min, abandons supernatant；Add 300 μ L 75% ethanol, wash twice, each 4 DEG C, 12000rpm, centrifugal 10min；Natural air drying
About 30min, adds the DDH of 60 μ L2O dissolving DNA about 30min, obtains sample DNA；Use detected through gel electrophoresis STb gene.
The nucleotide sequence authentication method of a kind of generation latitude lettuce tongue the most according to claim 1, it is characterised in that step S2
In, the reaction system of PCR amplification includes: 10 × Buffer 2 μ L, dNTP 1.6 μ L, DDH2O 11.3 μ L, Taq enzyme 0.1
μ L, sample DNA 1 μ L, ITS forward primer or trnL-F forward primer 2 μ L, ITS downstream primer or trnL-F downstream primer
2μL；
The concentration of described dNTP is 2.5 mmol/L, described ITS forward primer, trnL-F forward primer, ITS downstream primer,
The concentration of trnL-F downstream primer is 2 mmol/L.
The nucleotide sequence authentication method of a kind of generation latitude lettuce tongue the most according to claim 4, it is characterised in that step S2
In, the course of reaction of PCR amplification includes: enters after 94 DEG C of denaturations 4min and circulates, 94 DEG C of degeneration 45s, 54 DEG C of annealing 45s, 72
DEG C extend 45s, circulate 32 times, 72 DEG C of extension 10min.
The nucleotide sequence authentication method of a kind of generation latitude lettuce tongue the most according to claim 1, it is characterised in that in step S3
Also include: the ITS sequence obtained and trnL-F sequence inputting plant genetic sequence library are compared, determines nearly edge thing
Kind.
7. the application in identifying according to the SEQ ID NO.13 sequence alive latitude lettuce tongue described in any one in claim 1-6.
8. the application in identifying according to the SEQ ID NO.14 sequence alive latitude lettuce tongue described in any one in claim 1-6.
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| CN109486984A (en) * | 2018-08-03 | 2019-03-19 | 广东天保参茸有限公司 | A kind of combination bar code sequence and its accurate identification method identified for ginseng and American Ginseng |
| WO2020212190A1 (en) | 2019-04-16 | 2020-10-22 | Indena S.P.A. | Method and kit for the identification of vaccinium myrtillus |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109486984A (en) * | 2018-08-03 | 2019-03-19 | 广东天保参茸有限公司 | A kind of combination bar code sequence and its accurate identification method identified for ginseng and American Ginseng |
| WO2020212190A1 (en) | 2019-04-16 | 2020-10-22 | Indena S.P.A. | Method and kit for the identification of vaccinium myrtillus |
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