CN107541521B - A kind of identification method of radix coniti coreani DNA bar code and radix coniti coreani based on big data - Google Patents
A kind of identification method of radix coniti coreani DNA bar code and radix coniti coreani based on big data Download PDFInfo
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Abstract
The identification method of the invention discloses a kind of radix coniti coreani DNA bar code and radix coniti coreani based on big data, belongs to molecular identification technical field.Pass through the Aconitum distribution of medicinal plants area that takes a broad survey, and multiple place samplings are carried out in species distribution area as far as possible, build the bar code large database concept of the category medicinal plant ITS sequence, then by comparing the peculiar informative site of the distinctive ITS sequence of radix coniti coreani is found, its distinctive DNA bar code is further obtained：The DNA bar code is the sequence that the overall length expanded by primer sequence SEQ ID NO.1 and SEQ ID NO.2 is 629bp, and 89bp is A, and 443bp is A, and 447bp is that A, 491bp and 492bp are AT, and 499bp is T, and 577bp is C.The Rapid identification of medicinal plant radix coniti coreani will be greatly facilitated using the DNA bar code.
Description
Technical field
The present invention relates to molecular identification technical field more particularly to a kind of radix coniti coreani DNA bar code based on big data,
And the method using DNA bar code identification radix coniti coreani.
Background technology
Radix coniti coreani (Aconitum coreanum (H.L é veill é) Rapaics), also known as close monkshood, rhizoma typhonii, mountain loudspeaker
Flower and yellow black garland etc., are Ranunculaceae Aconitum perennial herb medicinal plant, are that Jilin Province especially Changbaishan area is genuine
Medicinal material.Its root tuber Korean aconite roof is used as medicine, and has the effect of wind-dispelling, eliminating dampness, resolving sputum analgesic.Aconitum plant has more than 200 to plant in China,
The platymiscium belongs to for poisonous plant simultaneously, homonym or generally existing the phenomenon that synonym in Aconitum medicinal material use.
Country's report Aconitum medicine poisoning is commonplace, and various aconitum plants are not easy to distinguish according to traditional form or Microscopic Identification method
Know.
DNA bar code technology (DNA Barcoding) is divided by the DNA sequence dna to a standard target gene
Analysis, to quickly and accurately carry out the technology of species identification.In recent years, DNA bar code technology had proven to a row
Effective bioassay means, can not only make strong supplement to conventional identification method, but also because it is more objective, accurate
Really, it breaks through and the transition of experience was relied in the past, can help to identify species, find novel species etc..
Currently, in the relevant molecule identification research of Aconitum medicinal plant, there are samples when species identification library is built to take
Sample is insufficient, fails to consider the problems of between different plant species or same species are interior there may be the variant sites similarities and differences between Different Individual, lead
Cause the poor reliability of identification, accuracy rate low.
Invention content
In view of this, the purpose of the present invention is to provide a kind of radix coniti coreani DNA bar code based on big data, and utilize
The method that the DNA bar code identifies radix coniti coreani, improves the reliability and accuracy rate of qualification result.
Technical scheme is as follows：
The present invention provides a kind of radix coniti coreani DNA bar code based on big data, and the DNA bar code is by primer sequence
The overall length that SEQ ID NO.1 and SEQ ID NO.2 are expanded is the sequence of 629bp, and 89bp is A, and 443bp is A, the
447bp is that A, 491bp and 492bp are AT, and 499bp is T, and 577bp is C.
Preferably, the sequence of the DNA bar code is as shown in SEQ ID NO.3.
The present invention also provides a kind of radix coniti coreani identification methods based on big data DNA bar code, include the following steps：
(1) extraction sample to be tested DNA；
(2) PCR amplification is carried out using DNA bar code primer, obtains the amplified production of 629bp, the DNA bar code primer
Sequence is SEQ ID NO.1 and SEQ ID NO.2；
(3) amplified production is sequenced, the sequence of radix coniti coreani species discriminating is carried out according to following specific position
Row aspect ratio pair：89bp is A, and 443bp is A, and 447bp is that A, 491bp and 492bp are AT, and 499bp is T, the
577bp is C；
If amplified production meets above-mentioned condition, which is radix coniti coreani.
Preferably, the amplification system of the PCR is 25 μ L, and component proportioning is as follows：10 μm of each 1.0 μ of the forward and reverse primers of ol/L
10 × PCRbuffer of L, 25mmol/L, 2.5 μ L, 2.5mM dNTP, 2.5 μ L, 5unit/ μ L Taq archaeal dna polymerases, 0.25 μ L,
50~100ng/L template DNAs, 1.0 16.75 μ L of μ L, ddH2O.
It is further preferred that the amplification condition of the PCR is：94 DEG C of pre-degeneration 2min, 1 cycle；94 DEG C denaturation 30s, 52
DEG C annealing 1min, 72 DEG C extension 2min, 35 cycle；72 DEG C of extension 7min.
Preferably, it is of the present invention sequencing use bidirectional sequencing, the primer sequence such as SEQ ID NO.1 of the sequencing and
Shown in SEQ ID NO.2.
It is further preferred that the reaction system of the sequencing is 5 μ L：Wherein ddH23 μ L of O, 10 μm of forward and reverse primers of ol/L
Each 0.25 μ L, 0.5 μ L, PCR purified product of sequencing reaction mixture, 1 μ L.
It is furthermore preferred that the reaction condition of the sequencing is：96 DEG C of pre-degenerations 10s, 50 DEG C of annealing 5s, 60 DEG C of extension 4min,
33 cycles.
Compared with prior art, the present invention has the following advantages：
The present invention is carried out multiple places in species distribution area and is adopted by the Aconitum distribution of medicinal plants area that takes a broad survey
Sample builds the bar code large database concept of the category medicinal plant ITS sequence, then by comparing the ITS sequence for finding radix coniti coreani
Peculiar informative site further obtains its distinctive DNA bar code.
Compared to previous morphology or method for identifying molecules, the present invention is to be carried out extensively to Aconitum medicinal plant for the first time
On the basis of general sampling, radix coniti coreani is identified using ITS sequence as DNA bar code, by carrying out PCR amplification to ITS sequence
And sequencing, qualification result is directly obtained, detection process is quick, and as a result accuracy is high.This method will greatly facilitate medicinal plant
The Rapid identification of radix coniti coreani.
Description of the drawings
Fig. 1 is the pcr amplification product collection of illustrative plates for building the sample segment used in Aconitum medicinal plant, wherein is located at the right
" M " of position is GeneRuler 100bp Plus DNA Ladder；
Fig. 2 is the sample segment aligned sequences result used in Aconitum medicinal plant.
Specific implementation mode
The present invention is by the Aconitum distribution of medicinal plants area that takes a broad survey, and progress is more as far as possible in species distribution area
A place sampling, builds the bar code large database concept of the category medicinal plant ITS sequence, then special by comparing discovery radix coniti coreani
Some peculiar informative sites of ITS sequence, further obtain its distinctive DNA bar code.
The radix coniti coreani DNA bar code of the present invention is to expand to obtain by primer sequence SEQ ID NO.1 and SEQ ID NO.2
Overall length be 629bp sequence, in the sequence, specific recognition site is：89bp is A, and 443bp is A, 447bp
It is AT for A, 491bp and 492bp, 499bp is T, and 577bp is C.The amplimer of the present invention is using ITS sequences
In the sequence of the 629bp expanded, chrysanthemum can be identified according to the base type of above-mentioned specific position for the universal primer of row
The rhizome of Chinese monkshood.
Since the radix coniti coreani DNA bar code of the present invention is obtained based on the Aconitum medicinal herbs obtained as far as possible more
It arrives, it is contemplated that be one there may be between Different Individual the problem of the variant sites similarities and differences between different plant species or in same species
Radix coniti coreani DNA bar code of the kind based on big data.Above-mentioned radix coniti coreani DNA bar code using the present invention can be quick and precisely
Radix coniti coreani is identified, overcome in the prior art identify poor reliability defect.
The present invention is extracted by the DNA to sample to be tested, by the way that amplified production is sequenced, by amplified production
Sequence is compared with the specific recognition sites in radix coniti coreani DNA bar code of the present invention, directly obtains qualification result.It detected
Journey is quick, and as a result accuracy is high, can be used for the Rapid identification of medicinal plant radix coniti coreani.
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
In specific embodiment, the total DNA of sample to be tested is extracted using 4 × CTAB methods of improvement.The present invention is to the sources DNA in sample to be tested
It is not particularly limited, can be leaf, root or the stem of sample to be tested.
PCR amplification and when amplimer is sequenced sequence used are the universal sequence of ITS in the present invention, specifically
For：
ITS4(SEQ ID NO.1)：5'-TCCTCCGCTTATTGATATGC-3',
ITS5(SEQ ID NO.2)：5'-GGAAGTAAAAGTCGTAACAAGG-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, component proportioning is：10 μm of each 1.0 μ L of the forward and reverse primers of ol/L,
10 × PCR of 25mmol/L buffer, 2.5 μ L, 2.5mM dNTP, 2.5 μ L, 5unit/ μ L Taq DNA polymerase
0.25 μ L, 50~100ng/L template DNA 1.0 μ L, ddH2O 16.75μL.The present invention does not have the reagent source in amplification system
There is a particular determination, Commercial reagents well-known to those skilled in the art can be used in agents useful for same in amplification system.
The amplification condition of PCR is：94 DEG C of pre-degeneration 2min, 1 cycle；94 DEG C of denaturation 30s, 52 DEG C of annealing 1min, 72 DEG C are prolonged
Stretch 2min, 35 cycles；72 DEG C of extension 7min.The sample DNA sequence of all standby screenings under above-mentioned amplification system and amplification condition
It can Successful amplification.Those skilled in the art can based on the above technical solution carry out amplification system and amplification condition
Appropriate rationally adjustment, such as change the volume, the concentration of component, the temperature and time for adjusting amplification of amplification system, belong to this
The protection domain of invention.The present invention preferably purifies amplified production.Using purifying side well-known to those skilled in the art
Method carries out.If amplification obtains the amplified production of 629bp, sample to be tested is possible to as radix coniti coreani, passes through to be sequenced and compares
Whether specific recognition site is that radix coniti coreani is further identified to sample to be tested.
The present invention is not particularly limited the mode of sequencing, using sequencing approach well-known to those skilled in the art, such as
Unidirectional sequencing or bidirectional sequencing.In the specific embodiment of the invention, it is preferred to use bidirectional sequencing.
The sequencing reaction system of 5 μ L is preferably used in the present invention：ddH2O 3 μ L, 10 μm of each 0.25 μ of the forward and reverse primers of ol/L
L, 0.5 μ L, PCR purified product of sequencing reaction mixture, 1 μ L.Wherein sequencing agents useful for same can be used well known in the art
Commercial reagents.The reaction condition of sequencing is preferably：96 DEG C of pre-degeneration 10s, 50 DEG C of annealing 5s, 60 DEG C of extension 4min, 33 recycle.
It is appropriate reasonable that those skilled in the art can based on the above technical solution carry out sequencing reaction system and sequencing condition
Adjustment, such as change the temperature and time that the volume, the concentration of component, adjustment of reaction system are sequenced, belong to the guarantor of the present invention
Protect range.After the completion of reaction, product is settled, is purified, after thermal denaturation, upper machine is sequenced.The present invention to sedimentation, it is pure
Change, thermal denaturation and sequencing procedure are not particularly limited, using the method known to skilled person.
Sequencing result is compared with the radix coniti coreani DNA bar code of the present invention, test sample is waited for if meeting following condition
This is radix coniti coreani：89bp is A, and 443bp is A, and 447bp is that A, 491bp and 492bp are AT, and 499bp is T,
577bp is C.
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, detection process is quick, result accuracy is high.
To make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiment to the present invention into
Row detailed description, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1：
1, the acquisition and preservation of Aconitum medicinal plant sample
The holding items of consulting literatures and domestic major specimen museum are formulated field according to the sampling request of DNA bar code and are adopted
Sample strategy, each species acquire multiple individuals (1-13) of separate sources in its distributed area, the leaf of all individuals as far as possible
Piece is preserved with silica dehydrator.The kind of Aconitum more than 70 (containing mutation) 206 parts of blade material, the sequence of rest part sample are obtained altogether
Information downloads (totally 23) from NCBI gene pools.Wherein, the sample of radix coniti coreani is from Heilongjiang Province, Jilin Province and Liaoning Province
5 places, cover the main distributed area of the species, and each sampling point respectively acquires 3 parts of leaf samples.The bill of materials and sample source are shown in
Table 1.
1 Aconitum medicinal plant sample of table and source
2, Genome DNA extraction
The total DNA that above-mentioned aconitum plant blade is extracted using 4 × CTAB methods of improvement, is as follows：
(1) mortar is cleaned and is dried for standby in baking oven, use alcohol calcination mortar before extracting total DNA, pestle, spoon is to go out
Bacterium.
(2) it chooses clean blade to be placed in mortar, with liquid nitrogen coolant after moisture content completely loss, firmly grinds material
Material is allowed to be in powdered, is then transferred to ground material in the centrifuge tube of the 2ml of precooling.
(3) 4 × 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 set to be thoroughly dispersed in extracting solution, during which warm bath about 1.5 hours in 65 DEG C of water-baths shake up 3~5 times.
(4) taking-up of the material of warm bath is placed in be cooled at room temperature after room temperature and isometric chloroform-isoamyl alcohol is added
(volume ratio 24:1) solution is shaken up 5~10 minutes, is then centrifuged 5 minutes with 10000~12000 revs/min.
(5) supernatant (about 700~800 μ l) is transferred in a new centrifuge tube to (attention should not be miscellaneous in drawing
Matter sucks up), add isometric chloroform-isoamyl alcohol (volume ratio 24:1), shake up 5~10 minutes, then with 10000~
12000 revs/min centrifuge 5~10 minutes.
(6) supernatant (about 450~600 μ l) is transferred in a new centrifuge tube, the isopropanol of 70% volume is added, sunk
DNA is dropped, is gently overturned 2~3 times, it is seen that 30 minutes or more are stood in white flock precipitate, room temperature or 4 DEG C of refrigerators, then 10000
~12000 leave the heart 5~10 minutes, abandon supernatant.
(7) it is respectively washed 2 times with 70% ethyl alcohol of 200 μ l and absolute ethyl alcohol, supernatant is abandoned after 20~30s of brief centrifugation, then
Centrifuge tube is placed in 37 DEG C of baking ovens (or at room temperature) ethyl alcohol is made to volatilize.Add 30~50 μ l TE solution and 1 after total DNA drying
~2 μ l RNase A are digested 2~3 hours in 37 DEG C of baking ovens with ribalgilase (RNaseA), then in -20 DEG C (or 4 DEG C)
It is saved backup in refrigerator.
3, pcr amplification reaction
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 ITS sequence：
ITS4(SEQ ID NO.1)：5'-TCCTCCGCTTATTGATATGC-3',
ITS5(SEQ ID NO.2)：5'-GGAAGTAAAAGTCGTAACAAGG-3'.
PCR reaction systems are 25 μ L, and component proportioning is as follows：Forward and reverse primer (10 μm of ol/L) each 1.0 μ L, 10 ×
PCRbuffer(Mg2+Plus, 25mmol/L) 2.5 2.5 μ L, Taq DNA polymerase (5unit/ μ of μ L, dNTP (2.5mM)
L) 0.25 μ L, 1.0 μ L of template DNA (50~100ng/ μ L), ddH2O16.75μL。
The primer of all standby screenings can Successful amplification under following amplification condition：94 DEG C of pre-degeneration 2min, 1 cycle；94
DEG C denaturation 30s, 52 DEG C annealing 1min, 72 DEG C extension 2min, 35 cycle；After 72 DEG C extension 7min, 4 DEG C preservation.
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 Aconitum 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
ReactionKit, bidirectional sequencing.
Sequencing reaction system is 5 μ L：Wherein ddH23 μ L of O, sequencing each 0.25 μ L of upstream and downstream primer (10 μm of ol/L), sequencing
1 μ L of reaction mixture (mix, Big Dye v3.1) 0.5 μ L, PCR purified product.
Amplimer sequence in the sequence synchronization rapid 3 of sequencing primer, i.e. SEQ ID NO.1 and SEQ ID NO.2.
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 after purification through 70% alcohol
20μL ddH2Loading after O is denaturalized 2 minutes under the conditions of 95 DEG C, is sequenced on 3730 automatic sequencers of ABI.
5, data analysis
Sequencing result is spliced and compared respectively with software SeqMan (DNAstar) and BioEdit (ver 7.0.0)
Right, the insertion and deletion in matrix is replaced with "-".The ITS sequence matrix of Aconitum medicinal plant is built, ITS sequence data are obtained
Library.
6, the sequence signature of sequence alignment and radix coniti coreani
The result of sequence alignment is：Entire matrix overall length 640bp, by 229 Sequence compositions.Wherein, 23 are downloaded from NCBI
Item, the present invention obtain 206, and sequence adheres to 72 kinds of Aconitum (containing mutation) common plant of being used as medicine separately.In a matrix, radix coniti coreani institute
There is the ITS sequence recognition site of sample to be：It is A in 92bp bases, the positions 206bp and 207bp are missing, 452bp and 456bp
It is set to A, remaining species is G；500-501bp has AT insertions, remaining species sample site deletion；The positions 508bp are T, remaining
Species sample is G；The positions 587bp are C, remaining is T.
If only seeing the sequence of single species, the radix coniti coreani extension increasing sequence of all separate sources is consistent, with primer I TS4 and
The amplified production sequence that ITS5 is expanded is as shown in SEQ ID NO.3, and the overall length of the sequence is 629bp, and 89bp is base
A, 443bp A, 447bp A, 491bp and 492bp are AT, 499bp T, 577bp C.
It can be used for as the DNA bar code of radix coniti coreani according to the specific information site of the obtained radix coniti coreani of the present invention
The identification of radix coniti coreani.The identification method of the present invention is merely with general versatility primer, by PCR amplification and sequencing approach,
By above-mentioned 7 specific recognition sites Rapid identification can be carried out to radix coniti coreani.
Embodiment 2
The radix coniti coreani having no result without flower and other Aconitum plant leafs acquired using medicinal material growing area and field is material
Material carries out DNA extractions using the method for embodiment 1, and PCR amplification and sequencing, qualification result are shown, are planted in known radix coniti coreani
The 89bp of the obtained ITS sequence of object be base A, 443bp A, 447bp A, 491bp and 492bp be AT, 499bp T,
577bp is C, and other unknown aconitum plants have no above-mentioned specific position, it is possible thereby to identify radix coniti coreani 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.
Sequence table
<110>Kunming Inst. of Botany, Chinese Academy of Sciences
<120>A kind of identification method of radix coniti coreani DNA bar code and radix coniti coreani based on big data
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
tcctccgctt attgatatgc 20
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ggaagtaaaa gtcgtaacaa gg 22
<210> 3
<211> 629
<212> DNA
<213> Aconitum coreanum
<400> 3
cgaaacctgc ccagcagagc gacccgcgaa caagtgaaaa caaaaccgga cggaccgaag 60
aggggcgcat gcccccgatc gcccgcccat cggaccgcga cctcttctgc gaccgcactg 120
atttgtgggt ggaggggtgg gttgttgagt ccgcacaaaa ccaaaaaccg gcgcgacagg 180
cgccaaggaa atcttagcgg aaagagggct tccccgttcg cggaggcagt cttcagaatc 240
cgatactcaa acgactctcg gcaacggata tctcggctct tgcatcgatg aagaacgtag 300
cgaaatgcga tacttggtgt gaattgcaga atcccgtgaa ccatcgagtc tttgaacgca 360
agttgcgccc gaggccatta ggtcgagggc acgtctgcct gggcgtcaca cacagcgtcg 420
caccccgtca accacgttgt cgaggaacgg agattggccc cccgggcccc tgcgggcacg 480
gtcggcacaa atatgttttt ccccggcggc gagcgtcgcg gtcagcggtg gttgtatttc 540
tcatcctcca aagacatcaa gacgcgtcgt cctcgtcgca tgttgggaca catcgacccc 600
acgaagtcgc tttgcgcgac attcaccct 629
Claims (6)
1. a kind of radix coniti coreani identification method for identifying site based on big data DNA bar code, which is characterized in that including following step
Suddenly：
（1）Extract 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 SEQ
ID NO.1 and SEQ ID NO.2；
（3）The amplified production is sequenced, sequencing result is compared with SEQ ID NO.3, and specific position is：89bp is
A, 443bp are A, and 447bp is that A, 491bp and 492bp are AT, and 499bp is T, and 577bp is C；
If amplified production meets above-mentioned specific position sequence information, which is radix coniti coreani.
2. according to the method described in claim 1, it is characterized in that, the amplification system of the PCR is 25 μ L, component is with such as
Under：Each 1.0 μ, 10 × PCR of L, 25mmol/L buffer solutions, 2.5 μ L, 2.5mM dNTP of 10 μm of forward and reverse primers of ol/L 2.5 μ L, 5
0.25 μ L, 50 ~ 100ng/L template DNA of unit/ μ L Taq archaeal dna polymerases 1.0 μ L, ddH2O 16.75µL。
3. according to the method described in claim 2, it is characterized in that, the amplification condition of the PCR is：94 DEG C of 2 min of pre-degeneration,
1 cycle；94 DEG C of denaturation 30s, 52 DEG C of annealing 1min, 72 DEG C of extension 2min, 35 recycle；72 DEG C of extension 7min.
4. according to the method described in claim 1, it is characterized in that, the sequencing is bidirectional sequencing, the primer sequence of the sequencing
Row are as shown in SEQ ID NO.1 and SEQ ID NO.2.
5. according to the method described in claim 4, it is characterized in that, the reaction system of the sequencing is 5 μ L：Wherein ddH2O 3µ
L, 10 μm of each 0.25 μ L of the forward and reverse primers of ol/L, 0.5 μ L, PCR purified product of sequencing reaction mixture, 1 μ L.
6. according to the method described in claim 5, it is characterized in that, the reaction condition of the sequencing is：96 DEG C of pre-degeneration 10s,
50 DEG C of annealing 5s, 60 DEG C of extension 4min, 33 recycle.
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