CN110438215A - A method of identifying cottonweed kind using ITS2 sequence - Google Patents
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Abstract
Step 1 a kind of method identifying cottonweed kind using ITS2 sequence disclosed by the invention extracts the DNA of plant sample blade to be identified；Step 2, the DNA for treating plant identification leaf samples carries out the PCR amplification of ITS2 nucleotide sequence；Step 3, bidirectional sequencing is carried out to PCR product, obtains plant sample blade ITS2 spacer sequence to be identified；Step 4, the rDNA ITS2 sequence of homologous sequence and outer rim kind sequence is obtained；Step 5, K2P genetic distance is calculated, phylogenetic tree construction identifies cottonweed kind.A kind of method using ITS2 Sequence Identification cottonweed kind of the present invention, Molecular Identification foundation is provided for the identification of affine cudweed medicinal material, affine cudweed and its easily mixed product can be quickly and accurately detected from several kinds of Chinese medicinal materials, are improved the accuracy of detection, are suitable for wide popularization and application.
Description
Technical field
The invention belongs to bioassay method and technology fields, and in particular to a kind of to identify Gnaphalium plant using ITS2 sequence
The method of article kind.
Background technique
Gnaphalium (Gnaphalium L.) belongs to Angiospermae, Dicotyledoneae, composite family, mostly annual grass
This, minority is perennial herb, and stem is herbaceous stem or base portion slightly with wooden, close cotton wool or villus by white；Leaf is alternate leaf,
Full edge, nothing or tool short handle；Inflorescence is capitulum, and in the coniform of sympodium or development, involucre is in oval or bell, phyllary
Mostly golden yellow, faint yellow or yellowish-brown, minority are bronzing, have 2-4 or 5 layer, the back side is by cotton wool, corolla yellow or yellowish
Color；Achene.
The plant whole world for recording Gnaphalium in " Chinese Plants will " has nearly 200 kinds, and China has 19 kinds, cottonweed
It is many kinds of, it is much like on mode of appearance, it is difficult to distinguish, affine cudweed, which is taken as the Chinese bulbul with potentilla chinensis in some areas, to be made
With reason has: one is related with the color of affine cudweed plant；Two in some places there are homonym phenomenon, affine cudweed is called
The Chinese bulbul；Third is that the identification of Ji Yuan, character etc. needs the identification personnel of profession, and it is affected by the subjective consciousness of people, so
Simpler, more accurate method is needed to carry out the identification of type.
DNA bar code (DNA barcoding) technology be by comparing one section of general DNA fragmentation, to species carry out quickly,
Accurate identification and identification have the characteristics that efficient, quick, accuracy rate is high, are the main sides of animals and plants Med Mat Appreciation in recent years
One of method.ITS2 is the noncoding region between rRNA encoding gene 5.8S and 28S, be moderate conserved sequence and length not
It is long, the conservative with higher in planting, while evolutionary rate is very fast, there is different degrees of variation again in different inter-species.This hair
It is bright to carry out Molecular Identification by the ITS2 sequence of Gnaphalium medicinal plant, and calculate in its kind, the affiliation of inter-species, it is
The research of Gnaphalium medicinal plant and further development and utilization lay the foundation.
Summary of the invention
The object of the present invention is to provide a kind of methods for identifying cottonweed kind using ITS2 sequence, solve existing
The problem for having affine cudweed discrimination method accuracy rate low.
The technical scheme adopted by the invention is that a method of identify cottonweed kind using ITS2 sequence,
The following steps are included:
Step 1, it takes plant sample blade to be identified several, and extracts the DNA of plant sample blade to be identified；
Step 2, to the DNA of plant sample blade to be identified in step 1, the PCR amplification of ITS2 nucleotide sequence is carried out, is obtained
To PCR product；
Step 3, bidirectional sequencing is carried out to the PCR product that step 2 obtains by sequenator, obtains sequencing result, will be sequenced
As a result splice through Seqman software and proofread, then annotated by Hidden Markov Model and remove both ends 5.8S and 28S section, obtain
Plant sample blade ITS2 spacer sequence to be identified saves；
Wherein the gene of plant sample blade ITS2 spacer sequence to be identified is encoded as shown in sequence 1；
Step 4, the plant sample blade ITS2 spacer sequence to be identified obtained based on step 3, in ncbi database
Blast sequence alignment is carried out, the homologous sequence and outer rim kind sequence of cottonweed are obtained, obtains homologous sequence and outer rim kind
The rDNA ITS2 sequence of sequence；
Step 5, the ITS2 sequence obtained for step 3 and step 4, calculating K2P genetic distance, phylogenetic tree construction,
Identify cottonweed kind.
It is of the invention to be further characterized in that,
Step 1 specifically:
Step 1.1, it takes plant sample blade to be identified several, is respectively put into the centrifuge tube of volume 1.5mL, is placed in liquid
It is quick-frozen in nitrogen, then take out plant sample blade to be identified, be put into the plastic foam container containing liquid nitrogen and be milled into it is powdered,
Until restoring to room temperature；
Step 1.2, the CTAB solution water-bath that concentration is 2% (m/V) is preheated to 65 DEG C, 700ul is taken to be added to through step 1
In treated plant sample blade to be identified, then in 65 DEG C of heating water bath 30min, takes out, be placed in room temperature environment；
Step 1.3, the solution to handle through step 1.2 is restored to room temperature, 450ul phenol chloroform is added, in 14000rpm item
Twice, each 10min is stood for whirlpool concussion under part, draws the centrifuge tube that 420ul supernatant liquid merging volume is 1.5mL, then
Isometric chloroform is added, vortex centrifugal vibrates 10min under the conditions of 14000rpm, stands, and layering takes supernatant；
Step 1.4, the supernatant 300ul in step 1.3 is taken, isometric isopropanol for being cooled to -20 DEG C in advance is added, in temperature
It is stood under the conditions of -20 DEG C of degree and is no less than 10min, 10min is then centrifuged under the conditions of 14000rpm, stood, abandoned supernatant, obtain pipe
Bottom white precipitate；
Step 1.5,500ul is added in the white precipitate of step 1.4 and has been cooled to -20 DEG C of volume fraction in advance for 70%
Ethyl alcohol, suspend precipitating, shakes 2min under the conditions of 14000rpm, is stored at room temperature 10min, abandons supernatant, 50ul is then added
ddH2O is centrifuged 1min under the conditions of 14000rpm, saves at 4 DEG C of temperature, that is, extracts plant sample leaf DNA to be identified.
The reaction system of the PCR amplification of step 2 are as follows:
2 × Taq PCR Mix 12.5ul, universal primer ITS2F and ITS3R select each 1ul of concentration 10umol/L, step
Rapid 1 obtained DNA 1ul, ddH2O 9.5ul.
The reaction condition of PCR amplification in step 2 are as follows:
94 DEG C of temperature, it is denaturalized 5min；94 DEG C of denaturation 30s of temperature, 56 DEG C of annealing 30s of temperature, 72 DEG C of temperature extension 45s, 40
A circulation；72 DEG C of extension 2min of temperature.
PCR reacts amplimer in step 2 are as follows:
ITS2F:5 '-ATCGATGCGATACTTGGTGTGAAT-3 ',
ITS3R:5 '-ATCGGACGCTTCTCCAGACTACAAT-3 '.
Sequenator is specially ABI3730xl DNA Analyzer sequenator in step 3.
The beneficial effects of the present invention are: a kind of method for identifying cottonweed kind using ITS2 sequence of the present invention,
Provide Molecular Identification foundation for the identification of affine cudweed medicinal material, can quickly and accurately be detected from several kinds of Chinese medicinal materials affine cudweed and
Its easily mixed product, improves the accuracy of detection, is suitable for wide popularization and application.
Detailed description of the invention
Fig. 1, which is that the present invention is a kind of, identifies affine cudweed universal primer in the method for cottonweed kind using ITS2 sequence
ITS2F/3R expands electrophoretogram；
Fig. 2, which is that the present invention is a kind of, identifies K2P genetic distance figure in the method for cottonweed kind using ITS2 sequence；
Fig. 3 is a kind of Neighor for the method for identifying cottonweed kind using ITS2 sequence of the present invention
Joining Tree figure.
Specific embodiment
The following describes the present invention in detail with reference to the accompanying drawings and specific embodiments.
A kind of method for identifying cottonweed kind using ITS2 sequence of the present invention, comprising the following steps:
Step 1, it takes plant sample blade to be identified several, and extracts the DNA of plant sample blade to be identified, such as 1 institute of table
Show, wherein sample source can be 8 kinds of different geographical affine cudweed (serial number 1-8 in table 1), a kind of spire affine cudweed (serial number in table 1
For 35), a kind of edelweiss (serial number 38 in table 1), for the plant identification sample of selection；
1 plant sample source to be identified table of table
Step 1.1, take respectively it is above-mentioned take plant sample blade to be identified several, be respectively put into the centrifuge tube of volume 1.5mL
In, it is placed in quick-frozen in liquid nitrogen, then takes out plant sample blade to be identified, be put into the plastic foam container containing liquid nitrogen and grind
It clays into power shape, until restoring to room temperature；
Step 1.2, it is that 2% (m/V) CTAB solution water-bath is preheated to 65 DEG C by concentration, 700ul is taken to be added to through step 1 place
In plant sample blade to be identified after reason, then in 65 DEG C of heating water bath 30min, takes out, be placed in room temperature environment；
Step 1.3, the solution to handle through step 1.2 is restored to room temperature, 450ul phenol chloroform is added, in 14000rpm item
Twice, each 10min is stood for whirlpool concussion under part, draws the centrifuge tube that 420ul supernatant liquid merging volume is 1.5mL, then
Isometric chloroform is added, vortex centrifugal vibrates 10min under the conditions of 14000rpm, stands, and layering takes supernatant；
Step 1.4, the supernatant 300ul in step 1.3 is taken, isometric isopropanol for being cooled to -20 DEG C in advance is added, in temperature
It is stood under the conditions of -20 DEG C of degree and is no less than 10min, 10min is then centrifuged under the conditions of 14000rpm, stood, abandoned supernatant, obtain pipe
Bottom white precipitate；
Step 1.5,500ul is added in the white precipitate of step 1.4 and has been cooled to -20 DEG C of volume fraction in advance for 70%
Ethyl alcohol, suspend precipitating, shakes 2min under the conditions of 14000rpm, is stored at room temperature 10min, abandons supernatant, 50ul is then added
ddH2O is centrifuged 1min under the conditions of 14000rpm, saves at 4 DEG C of temperature, that is, extracts plant sample leaf DNA to be identified.
It is wherein from left to right followed successively by marker band, Mt. Huang in Anhui affine cudweed AH, Hanzhong flowers and fruits as shown in Figure 1:
Mountain affine cudweed H, Mount Taibai edelweiss TX；DNA marker band be from top to bottom followed successively by 2000bp, 1000bp, 750bp,
500bp,250bp,100bp；As shown in Figure 1, DNA cloning success, can be sequenced.
Step 2, to the DNA of plant sample blade to be identified in step 1, the PCR amplification of ITS2 nucleotide sequence is carried out, is obtained
To PCR product；
The reaction system of PCR amplification are as follows: 2 × Taq PCR Mix 12.5ul, universal primer ITS2F and ITS3R are selected
Each 1ul of concentration 10umol/L, DNA 1ul, the ddH2O 9.5ul that step 1 obtains；
The reaction condition of PCR amplification are as follows: 94 DEG C of temperature, be denaturalized 5min；94 DEG C of denaturation 30s of temperature, 56 DEG C of temperature annealing
30s, 72 DEG C of extension 45s of temperature, 40 circulations；72 DEG C of extension 2min of temperature.
PCR reacts amplimer are as follows:
ITS2F:5 '-ATCGATGCGATACTTGGTGTGAAT-3 ',
ITS3R:5 '-ATCGGACGCTTCTCCAGACTACAAT-3 '.
Step 3, two-way survey is carried out to the PCR product that step 2 obtains by ABI3730xl DNA Analyzer sequenator
Sequence obtains sequencing result, and sequencing result is spliced through Seqman software and is proofreaded, and is then annotated and is removed by Hidden Markov Model
Both ends 5.8S and 28S section obtains plant sample blade ITS2 spacer sequence to be identified, saves；
Wherein the gene of the plant sample blade ITS2 spacer sequence to be identified is encoded as shown in sequence 1；
Step 4, the plant sample blade ITS2 spacer sequence to be identified obtained based on step 3, in ncbi database
Blast sequence alignment is carried out, the homologous sequence and outer rim kind sequence of cottonweed are obtained, obtains homologous sequence and outer rim kind
The rDNA ITS2 sequence of sequence is as shown in table 1 the source of homologous sequence and outer rim kind sequence；
Step 5, the ITS2 sequence obtained for step 3 and step 4 carries out data analysis using MEGA7.0 software, calculates
K2P genetic distance, phylogenetic tree construction identify cottonweed kind.
As shown in Fig. 2, calculating kind of an interior, Genetic distance using 2 parameter model of Kimura；Genetic distance range
It is 0.0226~0.1468, to be 0~0.0100, inter-species minimum genetic distance is greater than maximum in its kind inbred genetic distance range
Genetic distance genetic distance.
As shown in figure 3, being evident that affine cudweed for the phylogenetic tree of building by figure and its belonging to easily mixed kind and all divide
Not in different branch, it can obviously identify and separate.
A kind of method using ITS2 Sequence Identification cottonweed kind of the present invention, mentions for the identification of affine cudweed medicinal material
Molecular Identification foundation has been supplied, affine cudweed and its easily mixed product can be quickly and accurately detected from several kinds of Chinese medicinal materials, improve detection
Accuracy, be suitable for wide popularization and application.
<110>Xi'an Medical University
<120>a kind of method for identifying cottonweed kind using ITS2 sequence
<130> 2019
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 219
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 1
cgcatcgtgt cgccccctac aactcctcaa atggatgttt ggtgtggggg cggatattgg 60
tctcccgttt ctccgatatg gttggccaaa atacgagtcc cattcgatgg atgcacgact 120
agtggtggtt gctaaaacct tcgtcttcgg ttgtgcatct tcattcgtaa gggatgctta 180
aagaccccaa tgtgttgtct tctgatgacg cttcgaccg 219
Claims (6)
1. a kind of method for identifying cottonweed kind using ITS2 sequence, which comprises the following steps:
Step 1, it takes plant sample blade to be identified several, and extracts the DNA of plant sample blade to be identified；
Step 2, to the DNA of plant sample blade to be identified in step 1, the PCR amplification of ITS2 nucleotide sequence is carried out, is obtained
PCR product；
Step 3, bidirectional sequencing is carried out to the PCR product that the step 2 obtains by sequenator, obtains sequencing result, will be sequenced
As a result splice through Seqman software and proofread, then annotated by Hidden Markov Model and remove both ends 5.8S and 28S section, obtain
Plant sample blade ITS2 spacer sequence to be identified saves；
Wherein the gene of the plant sample blade ITS2 spacer sequence to be identified is encoded as shown in sequence 1；
Step 4, the plant sample blade ITS2 spacer sequence to be identified obtained based on step 3, is carried out in ncbi database
Blast sequence alignment obtains the homologous sequence and outer rim kind sequence of cottonweed, obtains homologous sequence and outer rim kind sequence
RDNA ITS2 sequence；
Step 5, the ITS2 sequence obtained for step 3 and step 4 calculates K2P genetic distance, phylogenetic tree construction, identification
Cottonweed kind.
2. a kind of method for being identified cottonweed kind using ITS2 sequence according to claim 1, feature are existed
In the step 1 specifically:
Step 1.1, it takes plant sample blade to be identified several, is respectively put into the centrifuge tube of volume 1.5mL, is placed in liquid nitrogen
It is quick-frozen, then take out plant sample blade to be identified, be put into the plastic foam container containing liquid nitrogen and be milled into it is powdered, until
Restore to room temperature；
Step 1.2, the CTAB solution water-bath that concentration is 2% mass/volume is preheated to 65 DEG C, 700ul is taken to be added to through step 1
In treated plant sample blade to be identified, then in 65 DEG C of heating water bath 30min, takes out, be placed in room temperature environment；Wherein
The concentration of CTAB solution is that CTAB content is 0.02 in every liter of solution
Step 1.3, the solution to handle through step 1.2 is restored to room temperature, 450ul phenol chloroform is added, under the conditions of 14000rpm
Whirlpool shakes twice, and each 10min is stood, and draws the centrifuge tube that 420ul supernatant liquid merging volume is 1.5mL, is then added
Isometric chloroform, vortex centrifugal vibrates 10min under the conditions of 14000rpm, stands, and layering takes supernatant；
Step 1.4, the supernatant 300ul in step 1.3 is taken, isometric isopropanol for being cooled to -20 DEG C in advance is added, in temperature -20
It is stood under the conditions of DEG C and is no less than 10min, 10min is then centrifuged under the conditions of 14000rpm, stood, abandoned supernatant, it is white to obtain tube bottom
Color precipitating；
Step 1.5,500ul is added in the white precipitate of step 1.4 and has been cooled to -20 DEG C of volume fraction in advance for 70% second
Alcohol, suspend precipitating, shakes 2min under the conditions of 14000rpm, is stored at room temperature 10min, abandons supernatant, 50ul ddH is then added2O,
It is centrifuged 1min under the conditions of 14000rpm, is saved at 4 DEG C of temperature, that is, extracts plant sample leaf DNA to be identified.
3. a kind of method for being identified cottonweed kind using ITS2 sequence according to claim 1, feature are existed
In the reaction system of the PCR amplification of the step 2 are as follows:
2 × Taq PCR Mix 12.5ul, universal primer ITS2F and ITS3R select each 1ul of concentration 10umol/L, and step 1 obtains
DNA 1ul, the ddH2O 9.5ul arrived.
4. a kind of method for being identified cottonweed kind using ITS2 sequence according to claim 3, feature are existed
In the reaction condition of PCR amplification in the step 2 are as follows:
94 DEG C of temperature, it is denaturalized 5min；94 DEG C of denaturation 30s of temperature, 56 DEG C of annealing 30s of temperature, 72 DEG C of extension 45s of temperature, 40 are followed
Ring；72 DEG C of extension 2min of temperature.
5. a kind of method for being identified cottonweed kind using ITS2 sequence according to claim 3, feature are existed
In PCR reacts amplimer in the step 2 are as follows:
ITS2F:5 '-ATCGATGCGATACTTGGTGTGAAT-3 ',
ITS3R:5 '-ATCGGACGCTTCTCCAGACTACAAT-3 '.
6. a kind of method for being identified cottonweed kind using ITS2 sequence according to claim 1, feature are existed
In sequenator is specially ABI3730xl DNA Analyzer sequenator in the step 3.
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| CN110331194B (en) * | 2019-06-28 | 2023-03-31 | 西安医学院 | Method for identifying affine cudweed variety by utilizing psbA-trnH sequence |
| CN113160893A (en) * | 2021-06-09 | 2021-07-23 | 中国科学院昆明植物研究所 | Mining plant ITSs sequence from second generation sequencing data and using the same for identifying variety families |
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