CN107988232A - The DNA bar code standard sequence and its method for identifying molecules of a kind of open country Storehouse midge - Google Patents
The DNA bar code standard sequence and its method for identifying molecules of a kind of open country Storehouse midge Download PDFInfo
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Abstract
The present invention discloses the DNA bar code standard sequence and its method for identifying molecules of a kind of open country Storehouse midge, it is characterized in that I gene orders of CO of the Storehouse midge are obtained using the method for molecular biology DNA sequencing, compared again by gene order similitude, so as to carry out Identification of Species to the Storehouse midge according to comparison result.Storehouse midge method for identifying molecules in open country provided by the invention, is advantageously implemented open country Storehouse midge and fast and accurately identifies.
Description
Technical field
The present invention relates to insect species identify field, and in particular to the DNA bar code standard sequence of open country Storehouse midge and its point
Sub- identification method.
Background technology
Storehouse midge（CulicoidesLatreille, 1809）It is under the jurisdiction of Insecta（Insecta）Diptera（diptera）
Heleidae（Ceratopogonidae）, its build is small, species is various, widely distributed, the whole world it is 1368 kinds existing, China
Know 347 kinds, and worldwide also constantly find new species.Blood sucking midge refer to can by pierce inhale ruminant blood come
Propagate the midge of entomophila pathogen, including Bitting midgeCulicoides, LasioheleaLasiohelea, LeptoconopsLeptoconopsWith
Australia's midge categoryAustroconopsDeng.In Bitting midge blood sucking midge, the pathogen propagated by midge matchmaker have blue tongue virus (BTV),
Domestic animal epidemic hemorrhagic fever virus（EHDV）, apply Maron shellfish lattice virus（SBV）, African horse sickness virus（AHSV）Etc. many cause of diseases
Body.Wherein, open country Storehouse midge（Culicoides homotomus Kieffer, 1922）It has been confirmed to be potential blue tongue disease disease
Malicious communication media.Exactly because the fields such as blood sucking midge and health care and animal doctor are closely bound up, therefore once break out epizootic
Huge economic loss will be caused, such as abroad（Europe）Blue tongue disease just once occurred（BT）It is sick with Maron shellfish lattice are applied（SB）Big stream
OK, while save of domestic such as Yunnan, Xinjiang and the Inner Mongol detects blue tongue virus more in cattle and sheep（BTV）.Therefore, accurate mirror
It is that one step of key that disease is monitored, prevents is passed to midge to determine disease vector.
At present, many defects of generally existing in terms of the traditional form taxonomic history of Storehouse midge.First Storehouse midge ovum, larva and
The morphosis of the early development stages such as pupa and the polypide of mutilation are difficult to recognize, secondly the behaviour of traditional form identification
Make the practical experience that process is relatively complicated and need to possess stronger professional knowledge and enrich, be again limited by Storehouse midge gender, it is such as male
Property nonbloodsucking midge be easy to differentiate by morphological features such as external genital organs and cercoids, and female blood sucking midge is difficult to differentiate, last storehouse
The plasticity of midge phenotype and the changeability of heredity etc. are also easy to cause erroneous judgement.Therefore, there is an urgent need for seek a kind of fast and accurately side
Method, to make up the deficiency of traditional form sorting technique.
With the fast development of molecular biology and bioinformatics, using DNA sequence analysis as foundation Molecular Identification
As the common technology means of species identification, the defects of traditional taxonomy is identified greatly compensate for.DNA bar code technology refers to
A segment standard, have variation enough and certain conservative, the relatively short DNA fragmentation easily expanded in organism.Therefore, as
The standard target gene of DNA bar codes, it must is fulfilled for two conditions, first, must have certain conservative, easy to general
Design of primers and the large-scale PCR amplification of progress；Second, to have enough variability, and to distinguish different plant species, especially near edge
Kind.
Mitochondria In Developing Flight Muscle of Insects DNA is double-strand closed loop molecule, by ND1-6, ND4L, CO I-CO III, ATPase6, ATPase8,
13 protein genes such as Cytb, 22 tRNA genes and 2 rRNA genes（16S rRNA and 12S rRNA）Totally 37 genes
With comprising replicate promoter non-coding section composition, wherein CO I, CO II, Cytb, 12S rRNA, 16S rRNA, ND4,
ND5 is the highest molecular labeling of current applying frequency.There is substantial amounts of insertion and missing now because of it in 12S rRNA and 16S rRNA
As, and gene is highly conserved, evolves slower, therefore should not be used as bar coding gene.ND4 and ND5 evolves too fast, it is difficult to designs
Universal primer, limits their target genes as comprehensive DNA identification systems.It is big compared to CO II genes, CO I genes
Small and structure is more conservative, comprising containing much information, is easily expanded again by universal primer while can ensureing to make a variation enough, in thing
There was only the difference of 1%-2% in kind between Different Individual, and the difference between sibling species is big, and there is more systematic growth signal,
It is more suitable for parsing the close biological group of affiliation, is most popular molecular labeling.
So far, existing many researchs have shown that I genes of CO are suitable for the Molecular Identification of most biological groups, such as elder brother
The taxonomic identification of the monoids such as worm, birds, fish, Nemata.Therefore, the DNA bar codes techniques pair based on I genes of CO can be used
Blood sucking midge carries out Molecular Identification.This method is proved each other with traditional form sorting technique, can not only realize blood sucking midge it is quick,
Precise Identification, and can help to find the cryptic species or novel species in Bitting midge using DNA bar code technology.At present, the party is passed through
Method has obtained I gene orders of CO of a variety of blood sucking midges and has been uploaded in GenBank, but there is no I genes of CO of open country Storehouse midge so far
Sequence.Open country Storehouse midge DNA bar code standard gene sequence provided by the invention is advantageously implemented quick, the accurate mirror of open country Storehouse midge
It is fixed, shorten the species identification time.
The content of the invention
DNA bar code standard gene sequence and its Molecular Identification side it is an object of the invention to provide a kind of open country Storehouse midge
Method.Open country Storehouse midge DNA bar code standard gene-I gene orders of CO are obtained by the method for molecular biology DNA sequencing, are passed through
The comparison of gene order similitude, realizes quick, the precise Identification of open country Storehouse midge.
To achieve the above object, the present invention is achieved through the following technical solutions：
Prepared by DNA profiling and PCR reacts, by I genes of CO of the primer amplification open country Storehouse midge synthesized, PCR is produced after amplification
Thing can detect amplification with agarose gel electrophoresis, and specific amplification is gone out the band sequence that size is about 650bp and is sent specially
Industry biotech firm is sequenced.Sequencing result carries out Blast similarity searchings by manual check and correction, sequence assembly in NCBI, really
It is target gene sequence to protect gained sequence, while the Identification of Species of open country Storehouse midge is realized by Morphological Identification, and through authority
Expert checks, so as to ensure that the reliability of result.Open country Storehouse midge DNA bar code standard gene of the present invention is I bases of CO
Because of partial sequence, I gene orders of CO of the open country Storehouse midge are as shown in SEQ ID No .1：
tttatttttg gagcttgagc aggaatagta ggtacttctc taagtatttt aattcgtgct 60
gaattaggac acccaggagc tttaattgga aatgatcaaa tttataatgt cattgtaaca 120
gcccatgctt ttattataat tttttttata gttataccta ttataattgg aggatttggt 180
aactgattag ttcctttaat attaggagcc ccagacatag ctttccctcg tataaataat 240
ataagatttt gaatacttcc cccttcacta tctttattac ttattagaag tttagtagaa 300
aatggggcag gaactggatg aactgtttac cctcctttgt cagctaatgt ttcgcatgca 360
ggagcttctg tagatttagc aattttttct ttacatttag ctggtatttc ttcaatctta 420
ggggctatta acttcattac tacaattatt aatatacgat ctaacggtat ttctttcgac 480
cgaatacctt tatttgtttg gtctgttttt attacagcca ttttattatt attatcatta 540
cctgtattag caggagcaat cacaatactt ttaacagatc gtaatattaa tacatctttt 600
tttgaccctg ctggaggagg agaccctatt ctttaccaac atttattttg 650。
The PCR primer of the DNA bar code standard gene sequence of the open country Storehouse midge, it is characterised in that PCR primer sequence
Respectively：
Forward primer：5’TAAACTTCAGGGTGACCAAAAAATCA 3’.
Reverse primer：5’GGTCAACAAATCATAAAGATATTGG 3’.
The method for identifying molecules of the open country Storehouse midge of the present invention, step include：
1）The separation and Extraction DNA from midge tissue to be measured；
2）Using the DNA as template, the primer that uses for：Forward primer： 5’TAAACTTCAGGGTGACCAAAAAATCA 3’；Instead
To primer：5’GGTCAACAAATCATAAAGATATTGG 3’.Pass through PCR（PCR）Amplify Storehouse midge CO I
Gene Partial sequence；
3）Then appropriate step 2 is taken）PCR amplification go out Storehouse midge CO I gene agarose gel electrophoresis point
From, purpose band is determined whether according to electrophoretic band, will if energy specific amplification goes out the band that size is about 650bp
PCR product cuts glue purification, send biotech firm to be sequenced；
4）Analyzed by the comparison of sequencing result, if the similitude of corresponding I gene orders of CO and SEQ ID No .1 98% with
On, you can judge the test serum as open country Storehouse midge.
It is using the beneficial effect of above-mentioned technical proposal：
1）The present invention has combined traditional Morphological Identification and method for identifying molecules, the Storehouse midge is identified, it can be ensured that thing
The accuracy and reliability of kind identification.
2）The open country Storehouse midge method for identifying molecules of the invention compared with traditional Morphological Identification method, established can be significantly
Shorten the qualification time of open country Storehouse midge.
3）The present invention has filled up blank of Storehouse midge CO I gene orders in open country in GenBank databases.
Brief description of the drawings
Fig. 1 is the techniqueflow chart of the present invention.
Fig. 2 is the electrophoresis pattern of I gene PCR amplifications of open country Storehouse midge CO.Numbering is described as follows：Swimming lane 1 is negative right
According to 2-4 is I genes of CO of open country Storehouse midge female adult, detects the band that size is about 650bp.M is 250bp DNA Ladder
Marker。
Fig. 3 is the electrophoresis pattern of unknown I gene PCR amplifications of Storehouse midge female adult CO.Numbering is described as follows：Swimming lane 1 is the moon
Property control, swimming lane 2 be unknown Storehouse midge female adult I genes of CO, detection size be about 650bp band.M is 250bp DNA
Ladder Marker。
Embodiment
The acquisition of 1 open country Storehouse midge CO of embodiment, I gene orders
1st, the collection and preservation of open country Storehouse midge sample
The field habitat that open country Storehouse midge sample sweeps method by net and lamp lures method to be collected in the provinces such as Guizhou, Sichuan, and be stored in
In 95% alcohol.Dissected with dissecting needle under stereoscope, Permanent slide is made in remaining in addition to chest, reflects through authoritative expert
It is fixed, it is ensured that the accuracy of qualification result.Molecule experiments will be carried out after the independent label of open country Storehouse midge chest.
2nd, extracting genome DNA
After the open country Storehouse midge chest grinding of preservation, carried out according to QIAGEN DNeasy Blood＆ Tissue Kit specifications
Single Storehouse midge extracting genome DNA, and extremely -20 DEG C of preservation is spare.
3rd, primer synthesizes
The present embodiment the primer is as follows：
Forward primer：5’TAAACTTCAGGGTGACCAAAAAATCA 3’.
Reverse primer：5’GGTCAACAAATCATAAAGATATTGG 3’.
4th, PCR amplification
The PCR reaction systems of the present embodiment are as follows：
DNA profiling 1.5uL
Sense primer（10uM） 1uL
Anti-sense primer（10uM） 1uL
2×PCR MasterMix 12.5uL
ddH2O 9uL
Total system 25uL
PCR reaction conditions are 94 DEG C of 5 min of pre-degeneration；94 DEG C of denaturation 1min, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, are circulated
35 times；Last 72 DEG C of extensions 5min.
5th, PCR product identification and recycling
PCR product determines whether purpose band, electrophoresis pattern is referring to attached drawing into row agarose gel electrophoresis according to electrophoretic band
2.PCR product is carried out according to Ago-Gel DNA QIAquick Gel Extraction Kits specification and cuts glue purification, and product after purification is sent to biological public
Department carries out bidirectional sequencing.
6th, CO I genes sequencing
By manually proofread and bioinformatics software open country Storehouse midge CO I gene sequences are compared, edited and spliced after i.e.
For SEQ ID No .1.
The identification of 2 unknown Storehouse midge of embodiment
1st, the collection and preservation of midge sample
Midge sample sweeps method by net and lamp lures method to be collected in field habitat, and is stored in 95% alcohol.With dissecting needle stereoscopic
Dissected under mirror, remaining is made Permanent slide and is used for Morphological Identification in addition to chest.It will be divided after the independent label of midge chest
Son experiment.
2nd, extracting genome DNA
After the midge chest of preservation is ground, single is carried out not according to QIAGEN DNeasy Blood＆ Tissue Kit specifications
Know midge extracting genome DNA, and extremely -20 DEG C of preservation is spare.
3rd, primer synthesizes
The present embodiment the primer is as follows：
Forward primer：5’TAAACTTCAGGGTGACCAAAAAATCA 3’.
Reverse primer：5’GGTCAACAAATCATAAAGATATTGG 3’.
4th, PCR amplification
The PCR reaction systems of the present embodiment are as follows：
DNA profiling 1.5uL
Sense primer（10uM） 1uL
Anti-sense primer（10uM） 1uL
2×PCR MasterMix 12.5uL
ddH2O 9uL
Total system 25uL
PCR reaction conditions are 94 DEG C of 5 min of pre-degeneration；94 DEG C of denaturation 1min, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, circulate 35
It is secondary；Last 72 DEG C of extensions 5min.
5th, PCR product identification and recycling
PCR product determines whether purpose band, electrophoresis pattern is referring to attached drawing into row agarose gel electrophoresis according to electrophoretic band
3.Attached drawing 3 shows that the unknown Storehouse midge of embodiment 2 can also go out the product that size is about 650bp by PCR specific amplifications.According to fine jade
Sepharose DNA QIAquick Gel Extraction Kits specification carries out PCR product and cuts glue purification, and product after purification is sent to biotech firm's progress pair
To sequencing.
6th, CO I genes sequencing
By manually proofread and bioinformatics software unknown Storehouse midge CO I gene sequences are compared, edited and spliced after with
SEQ ID No .1 carry out similitude comparison, its similitude with gene order SEQ ID NO 1 of the results show is more than 99%,
Thus it can determine that the unknown Storehouse midge is open country Storehouse midge.
Sequence table
<110>Zunyi Medical College
<120>The DNA bar code standard sequence and its method for identifying molecules of a kind of open country Storehouse midge
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 650
<212> DNA
<213>I genes of open country Storehouse midge CO
<400> 1
tttatttttg gagcttgagc aggaatagta ggtacttctc taagtatttt aattcgtgct 60
gaattaggac acccaggagc tttaattgga aatgatcaaa tttataatgt cattgtaaca 120
gcccatgctt ttattataat tttttttata gttataccta ttataattgg aggatttggt 180
aactgattag ttcctttaat attaggagcc ccagacatag ctttccctcg tataaataat 240
ataagatttt gaatacttcc cccttcacta tctttattac ttattagaag tttagtagaa 300
aatggggcag gaactggatg aactgtttac cctcctttgt cagctaatgt ttcgcatgca 360
ggagcttctg tagatttagc aattttttct ttacatttag ctggtatttc ttcaatctta 420
ggggctatta acttcattac tacaattatt aatatacgat ctaacggtat ttctttcgac 480
cgaatacctt tatttgtttg gtctgttttt attacagcca ttttattatt attatcatta 540
cctgtattag caggagcaat cacaatactt ttaacagatc gtaatattaa tacatctttt 600
tttgaccctg ctggaggagg agaccctatt ctttaccaac atttattttg 650
<210> 2
<211> 26
<212> DNA
<213>Forward primer
<400> 2
taaacttcag ggtgaccaaa aaatca 26
<210> 3
<211> 25
<212> DNA
<213>Reverse primer)
<400> 3
ggtcaacaaa tcataaagat attgg 25
Claims (3)
1. a kind of DNA bar code standard sequence of open country Storehouse midge, it is characterised in that the DNA bar code standard gene is I bases of CO
Cause, is gene order SEQ ID No .1 as described below：
tttatttttg gagcttgagc aggaatagta ggtacttctc taagtatttt aattcgtgct 60
gaattaggac acccaggagc tttaattgga aatgatcaaa tttataatgt cattgtaaca 120
gcccatgctt ttattataat tttttttata gttataccta ttataattgg aggatttggt 180
aactgattag ttcctttaat attaggagcc ccagacatag ctttccctcg tataaataat 240
ataagatttt gaatacttcc cccttcacta tctttattac ttattagaag tttagtagaa 300
aatggggcag gaactggatg aactgtttac cctcctttgt cagctaatgt ttcgcatgca 360
ggagcttctg tagatttagc aattttttct ttacatttag ctggtatttc ttcaatctta 420
ggggctatta acttcattac tacaattatt aatatacgat ctaacggtat ttctttcgac 480
cgaatacctt tatttgtttg gtctgttttt attacagcca ttttattatt attatcatta 540
cctgtattag caggagcaat cacaatactt ttaacagatc gtaatattaa tacatctttt 600
tttgaccctg ctggaggagg agaccctatt ctttaccaac atttattttg 650。
2. the PCR primer of the DNA bar code standard gene sequence of the open country Storehouse midge described in claim 1, it is characterised in that PCR draws
Thing sequence is respectively：
Forward primer：5’TAAACTTCAGGGTGACCAAAAAATCA 3’；
Reverse primer：5’GGTCAACAAATCATAAAGATATTGG 3’.
3. a kind of method for identifying molecules of open country Storehouse midge, step include：
1）The separation and Extraction DNA from midge tissue to be measured；
2）Using the DNA as template, the primer that uses for：Forward primer：5’TAAACTTCAGGGTGACCAAAAAATCA 3’；Instead
To primer：5’GGTCAACAAATCATAAAGATATTGG 3’；Pass through PCR（PCR）Amplify Storehouse midge CO I
Gene；
3）Then appropriate step 2 is taken）PCR amplification go out Storehouse midge CO I gene agarose gel electrophoresis point
From, purpose band is determined whether according to electrophoretic band, will if energy specific amplification goes out the band that size is about 650bp
PCR product cuts glue purification, send biotech firm to be sequenced；
4）Analyzed by the comparison of sequencing result, if the similitude of corresponding I gene orders of CO and SEQ ID No .1 98% with
On, you can judge the test serum as open country Storehouse midge.
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| CN111378764A (en) * | 2020-04-14 | 2020-07-07 | 遵义医科大学 | Identification method of seven species of coe (coe of species) genes based on coe (cytochrome oxidase I) vector culicoides |
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