CN102687706B - Novel dasyhelea species and specific DNA (deoxyribonucleic acid) sequence thereof - Google Patents
Novel dasyhelea species and specific DNA (deoxyribonucleic acid) sequence thereof Download PDFInfo
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- CN102687706B CN102687706B CN2012101524151A CN201210152415A CN102687706B CN 102687706 B CN102687706 B CN 102687706B CN 2012101524151 A CN2012101524151 A CN 2012101524151A CN 201210152415 A CN201210152415 A CN 201210152415A CN 102687706 B CN102687706 B CN 102687706B
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- 108020004414 DNA Proteins 0.000 title abstract description 19
- 241000134323 Dasyhelea Species 0.000 title abstract description 10
- 102000053602 DNA Human genes 0.000 title abstract 4
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 13
- 238000003752 polymerase chain reaction Methods 0.000 claims description 28
- 101100329008 Artemia franciscana COI gene Proteins 0.000 claims description 13
- 230000003321 amplification Effects 0.000 claims description 9
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 9
- 238000001962 electrophoresis Methods 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 6
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 claims description 4
- 229960005542 ethidium bromide Drugs 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 4
- 238000003384 imaging method Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 238000012163 sequencing technique Methods 0.000 claims description 4
- 238000010186 staining Methods 0.000 claims description 4
- 229920000936 Agarose Polymers 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 101150062230 CO gene Proteins 0.000 claims 1
- 241000894007 species Species 0.000 abstract description 20
- 238000000034 method Methods 0.000 abstract description 15
- 238000006243 chemical reaction Methods 0.000 abstract description 9
- 230000000877 morphologic effect Effects 0.000 abstract description 5
- 102000000634 Cytochrome c oxidase subunit IV Human genes 0.000 abstract 2
- 108090000365 Cytochrome-c oxidases Proteins 0.000 abstract 2
- 238000012300 Sequence Analysis Methods 0.000 abstract 1
- 230000007613 environmental effect Effects 0.000 abstract 1
- 241000256135 Chironomus thummi Species 0.000 description 8
- 238000012408 PCR amplification Methods 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 241000238631 Hexapoda Species 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 238000000137 annealing Methods 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- HLXHCNWEVQNNKA-UHFFFAOYSA-N 5-methoxy-2,3-dihydro-1h-inden-2-amine Chemical compound COC1=CC=C2CC(N)CC2=C1 HLXHCNWEVQNNKA-UHFFFAOYSA-N 0.000 description 2
- 241000134426 Ceratopogonidae Species 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 241000255925 Diptera Species 0.000 description 2
- 241000255624 Psychoda Species 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
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- 230000001568 sexual effect Effects 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- 241000042458 Carpophilus obsoletus Species 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101150017040 I gene Proteins 0.000 description 1
- 241000124033 Salix Species 0.000 description 1
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- RSMUVYRMZCOLBH-UHFFFAOYSA-N metsulfuron methyl Chemical compound COC(=O)C1=CC=CC=C1S(=O)(=O)NC(=O)NC1=NC(C)=NC(OC)=N1 RSMUVYRMZCOLBH-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a novel dasyhelea species and a specific DNA (deoxyribonucleic acid) sequence thereof. The novel dasyhelea species belongs to dasyhelea and is named as dasyhelea silvatica Wang, Zhang and Yu, sp. nov. The novel species is researched by identifying methods such as morphological characteristics and sequence analysis, and results show that the species is novel in dasyhelea. In addition, a CO (cytochrome c oxidase) gene sequence of the novel dasyhelea species is obtained, and basis for molecular identification of the novel dasyhelea species is provided. The novel dasyhelea species can play a certain role in plant pollen spreading and environmental harmless reactions.
Description
Technical field
The present invention relates to a kind of moth fly, be specifically related to a kind of moth fly novel species and specific DNA sequence thereof.
Background technology
Moth fly is miniature insect common in urban green belts and gardens, belongs to Diptera (Diptera) Heleidae (Ceratopogonidae) moth fly subfamily.So far as is known, the moth fly subfamily only comprise Psychoda (
DasyheleaKieffer, 1911) 1 genus., because moth fly is non-blood sucking midge class,, to the person poultry harmless, often get the brush-off.But the effect of moth fly in propagating plant pollen and environmentally friendlyization reaction paid close attention to by people gradually.
For a long time, evaluation to midge classes such as moth flys mainly relies on morphological feature to realize, but in recent years, progress along with technology such as DNA sequencings, people adopt molecular biology method to study the characteristic DNA of midge class successively,, to setting up new sorting technique, with the traditional classification method, mutually testify.
Plastosome CO I gene is the molecule marker that is most widely used in midge class identification research, be mainly used at present the Molecular Identification research of blood sucking midge, successfully identify the species in a plurality of kinds of groups such as greyish black storehouse midge and C.obsoletus, can be applied to equally in the Molecular Identification of moth fly.
Summary of the invention
The object of the present invention is to provide a kind of moth fly novel species and characteristic DNA sequence thereof.The diversity of world's species has been enriched in the discovery of this novel species, the acquisition of its characteristic DNA sequence, and the Rapid identification that can be this novel species provides scientific basis.
For achieving the above object, the present invention is achieved through the following technical solutions:
The gained sample is made slide, observe under biomicroscope, draw morphological specificity figure, and consult domestic and international related documents,, according to the morphological specificity of sample, carry out species and identify.This midge kind belongs to Psychoda, called after woods moth fly (
Dasyhelea silvaticaWang, Zhang and Yu, sp. nov.).Type specimen is stored in Military Medical Science Institute's medical insect sample shop.
Described moth fly novel species has following identification of morphology feature:
A, long 1.15 mm of ♂ wing, wide 0.35 mm;
B, body brown, medium-sized;
Distance between c, two compound eyes is about 1/3 of facet diameter, and between facet, pubescence is carefully small and dense;
It is heart-shaped that d, volume sheet are;
E, feeler flagellum rag are obvious, and the wheel staple length is and close, and the 15th joint distal process is obvious, each joint long 21:14:15:15:15:16:16:16:16:36:31:26:36 that is that compares; AR 1.13;
F, antenna respectively save the long 9:10:17:11:14 that is that compares;
G, chaperon peltate, nearly upper limb has 2, hair on the neck, each 3 of every sides;
H, cercoids 9-tergum trailing edge pleurapophysis are flourishing, embrace the acra joint and be the microbrachia shape, aed-aedeagus without in prominent, the top of a pair of pleurapophysis is the bird indictment; Paramere middle period willow leaf shape.
Adopt small insects trace DNA templet preparation method, by improved PCR reaction conditions, by the CO I gene of synthetic this moth fly novel species of primer amplification, PCR product sequence is sent by professional biotech firm and is measured.Sequencing result by craft proofread, sequence assembly, and carry out the Blast similarity searching in NCBI, guarantee that institute's calling sequence is target sequence.The CO of described moth fly novel species
Gene order (does not contain primer) as shown in SEQ ID No.1:
cttaatgcta ggagcccctg atatagcttt tcctcgaata aataatataa gattctggat 60
actcccacct tctttaaccc ttctactggt aagttcacta gttgaaaatg gggctgggac 120
aggatgaaca gtttaccctc cattggccag caatatcgcc cacggaggat cttcagttga 180
cttagcaatt ttttccttac atttagcagg tatttcatct attttaggag ccgtaaattt 240
cattacaact attattaata tgcgatcaaa tgggattaca tttgatcgaa tacctttatt 300
tgtttgatct gttttaatta ctgctatttt actcctttta tctcttcctg tcttagcagg 360
agctattact atactattaa ctgatcgaaa tttaaataca tcattttttg accctgctgg 420
gggaggagat cctattttat accaacattt attctggttt tttggccacc ca 472。
The PCR primer of the DNA standard detection gene of described moth fly novel species is characterized in that the PCR primer sequence is:
Forward primer 5 ' GGA GGA TTT GGA AAT TGA TTA GTT CC3 ';
Reverse primer 5 ' CCC GGT AAA ATT AAA ATA TAA ACT TC3 '.
The molecular assay method of moth fly novel species of the present invention comprises:
1) separation and Extraction DNA from moth fly novel species tissue to be measured;
2) take this DNA as the template pair of primers, forward primer 5 ' GGA GGA TTT GGA AAT TGA TTA GTT CC3 ', reverse primer 5 ' CCC GGT AAA ATT AAA ATA TAA ACT TC3 ', go out this moth fly novel species CO I gene by polymerase chain reaction (PCR) amplification;
3) then get appropriate step 2) the CO I gene that goes out of polymerase chain reaction (PCR) amplification separate with agarose electrophoresis, detect in gel imaging system after ethidium bromide staining,, according to the big or small result of determination of amplified production,, if the energy specific amplification goes out the band of 524bp, send biotech firm's order-checking;
4) according to sequencing result, if with the homology of described gene order SEQ ID NO.1 more than 98%, can judge that described tissue to be measured is the woods moth fly.
The present invention can detect amplification with the agarose gel electrophoresis detection method.
The present invention adopts traditional Morphological Identification to combine with the molecular classification method, and described moth fly novel species is identified, can guarantee accuracy and reliability that species are identified.
Description of drawings
Fig. 1 is woods moth fly feeler figure of the present invention.
Fig. 2 is woods moth fly antenna figure of the present invention.
Fig. 3 is woods moth fly volume sheet figure of the present invention.
Fig. 4 is woods moth fly chaperon figure of the present invention.
Fig. 5 is woods moth fly cercoids of the present invention (removing aed-aedeagus) figure.
Fig. 6 is woods moth fly aed-aedeagus figure of the present invention.
Fig. 7 is woods moth fly CO I gene PCR amplified production electrophoresis evaluation figure of the present invention.Numbering is described as follows: swimming lane 1-2 is the CO I gene of woods moth fly male worm, detects size and is the band of 524bp.M is the DNA marker of DL-2000.
The unknown moth fly CO of Fig. 8 I gene PCR amplified production electrophoresis is identified figure.Numbering is described as follows: swimming lane 1 is the CO I gene of unknown moth fly male worm, detects size and is the band of 524bp.M is the DNA marker of DL-2000.
Embodiment
1, the collection of sample and preservation
Employing is waved the net method and is gathered, and adopts the sample that obtains and directly is stored in 75% alcohol after freezing execution.
2, slide sample is made
Taking-up is stored in the sample in 75% alcohol, with dissecting needle, head, sexual organ part and wing is downcut, and is convenient to film-making.Preparation of specimen carries out with reference to the described method in " Chinese Ceratopogonidae " (Yu Yixin chief editor, 2005).
The sample remainder moves in the PCR pipe, and 95% alcohol is preserved, one of every pipe, and carry out exclusive number, be used for DNA extraction., if need the long period to preserve, the PCR pipe that the residue tissue site is housed should be placed in-20 ℃ of refrigerators frozen.
3, DNA profiling preparation
Adopt small insects trace DNA templet preparation method to extract woods moth fly DNA(Wen Lizhang chief editor. entomology research method and technology introduction [M]. Beijing: Science Press, 2010. 278-286), the DNA sample of extraction saves backup in-20 ℃.
4, primer is synthetic
The present embodiment the primer is as follows:
Forward primer 5 ' GGA GGA TTT GGA AAT TGA TTA GTT CC3 '
Reverse primer 5 ' CCC GGT AAA ATT AAA ATA TAA ACT TC3 '
5, pcr amplification
The PCR reaction system of the present embodiment is as shown in table 1.
Table 1 is woods moth fly CO I gene PCR reaction system (50uL system)
| Composition | Volume (uL) |
| Template | 10 |
| |
1 |
| Primer 2 | 1 |
| 2×Mastermix | 25 |
| ddH 2O | 13 |
The response procedures of the present embodiment is as shown in table 2.
Table 2 is woods moth fly CO I gene PCR response procedures
| Step | Temperature of reaction (℃) | Time | Cycle number |
| Denaturation | 94 | 3 min | — |
| Sex change | 94 | 30 s | 5 |
| Annealing | 45 | 90 s | 5 |
| Extend | 68 | 60 s | 5 |
| Sex change | 94 | 30 s | 35 |
| Annealing | 51 | 90 s | 35 |
| Extend | 68 | 60 s | 35 |
| Extend | 68 | 10 min | — |
The PCR product saves backup in 4 ℃.
6, pcr amplification product detects
Get 5 μ L pcr amplification products, with 1% agarose gel electrophoresis (120V voltage, electrophoresis 30 min).Ethidium bromide staining.Gel imaging system detects, and electrophoretogram is referring to accompanying drawing 7.
7, gene sequencing
Choose 2-3 effect preferably pcr amplification product send by order-checking (order-checking the primer identical with the PCR the primer) after biotech firm's purifying, be the CO of this moth fly novel species after the calibrated splicing of gained gene order
Gene order-SEQ ID No.1.
Embodiment 2
1, the collection of sample and preservation
Employing is waved the net method and is gathered, and adopts the sample that obtains and directly is stored in 75% alcohol after freezing execution.
2, sample chooses and and pre-treatment
From the midge class sample that collects, random choose goes out a moth fly, with dissecting needle, head, sexual organ part and wing is downcut, and is convenient to film-making and carries out identification of morphology.Remainder moves in the PCR pipe, and 95% alcohol is preserved, one of every pipe, and carry out exclusive number, be used for DNA extraction.
3, DNA profiling preparation
Adopt small insects trace DNA templet preparation method to extract unknown moth fly DNA(Wen Lizhang chief editor. entomology research method and technology introduction [M]. Beijing: Science Press, 2010. 278-286), the DNA sample of extraction saves backup in-20 ℃.
4, primer is synthetic
The present embodiment the primer is as follows:
Forward primer 5 ' GGA GGA TTT GGA AAT TGA TTA GTT CC3 '
Reverse primer 5 ' CCC GGT AAA ATT AAA ATA TAA ACT TC3 '
5, pcr amplification
The PCR reaction system of the present embodiment is as shown in table 3.
Table 3 is unknown moth fly CO I gene PCR reaction system (50uL system)
| Composition | Volume (uL) |
| Template | 10 |
| |
1 |
| Primer 2 | 1 |
| 2×Mastermix | 25 |
| ddH 2O | 13 |
The response procedures of the present embodiment is as shown in table 4.
Table 4 is unknown moth fly CO I gene PCR response procedures
| Step | Temperature of reaction (℃) | Time | Cycle number |
| Denaturation | 94 | 3 min | — |
| Sex change | 94 | 30 s | 5 |
| Annealing | 45 | 90 s | 5 |
| Extend | 68 | 60 s | 5 |
| Sex change | 94 | 30 s | 35 |
| Annealing | 51 | 90 s | 35 |
| Extend | 68 | 60 s | 35 |
| Extend | 68 | 10 min | — |
The PCR product saves backup in 4 ℃.
6, pcr amplification product detects
Get 5 μ L pcr amplification products, with 1% agarose gel electrophoresis (120V voltage, electrophoresis 30 min).Ethidium bromide staining.Gel imaging system detects, and electrophoretogram is referring to accompanying drawing 8.The unknown moth fly of finding out embodiment 2 by electrophorogram 8 also can specific amplification goes out the product of 524bp, can tentatively judge describedly should the unknown moth fly be
The woods moth fly.The woods moth flys in order more clearly to prove, willThe product of 524bp
Send biotech firm's order-checking, test result shows that with the homology of gene order SEQ ID NO. 1 be 99.9%, thereby can confirm that this unknown moth fly is the woods moth fly.
Claims (3)
1. the DNA standard detection gene of a moth fly novel species, is characterized in that, the DNA standard detection gene of described moth fly novel species is CO
Gene is gene order SEQ ID No.1 as described below:
cttaatgcta ggagcccctg atatagcttt tcctcgaata aataatataa gattctggat
actcccacct tctttaaccc ttctactggt aagttcacta gttgaaaatg gggctgggac
aggatgaaca gtttaccctc cattggccag caatatcgcc cacggaggat cttcagttga
cttagcaatt ttttccttac atttagcagg tatttcatct attttaggag ccgtaaattt
cattacaact attattaata tgcgatcaaa tgggattaca tttgatcgaa tacctttatt
tgtttgatct gttttaatta ctgctatttt actcctttta tctcttcctg tcttagcagg
agctattact atactattaa ctgatcgaaa tttaaataca tcattttttg accctgctgg
gggaggagat cctattttat accaacattt attctggttt tttggccacc ca 。
2. the PCR primer of the DNA standard detection gene of moth fly novel species claimed in claim 1 is characterized in that the PCR primer sequence is:
Forward primer 5 ' GGA GGA TTT GGA AAT TGA TTA GTT CC3 ';
Reverse primer 5 ' CCC GGT AAA ATT AAA ATA TAA ACT TC3 '.
3. the molecular assay method of a moth fly novel species comprises:
1) separation and Extraction DNA from moth fly novel species tissue to be measured;
2) take this DNA as the template pair of primers, forward primer 5 ' GGA GGA TTT GGA AAT TGA TTA GTT CC3 ', reverse primer 5 ' CCC GGT AAA ATT AAA ATA TAA ACT TC3 ', go out this moth fly novel species CO I gene by polymerase chain reaction (PCR) amplification;
3) then get appropriate step 2) the CO I gene that goes out of polymerase chain reaction (PCR) amplification separate with agarose electrophoresis, detect in gel imaging system after ethidium bromide staining,, according to the big or small result of determination of amplified production,, if the energy specific amplification goes out the band of 524bp, send biotech firm's order-checking;
4) according to sequencing result, if with the homology of the DNA standard detection gene order SEQ ID NO.1 of moth fly novel species more than 98%, can judge that moth fly novel species tissue to be measured is the woods moth fly.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105420399A (en) * | 2016-01-08 | 2016-03-23 | 福建国际旅行卫生保健中心 | Specific gene of brown atrichopogon and molecular identification method of brown atrichopogon |
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| CN108486121B (en) * | 2018-01-25 | 2020-02-11 | 遵义医学院 | Specific DNA sequence of Cuicoides spinuloides and molecular identification method thereof |
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| DNA条形码识别III媒介蚊类DNA条形码芯片的初步研究;赵明 等;《中国媒介生物学及控制杂志》;20080430(第2期);第99-103页 * |
| Linto,Y.M.等.Accession Number AF069242.《Genbank》.2005,1页. * |
| Molecular differentiation and diversity of Forcipomyia taiwana (Diptera:Ceratopogonidae) based on the mitochondrial cytochrome oxidase II sequence.;Yeh WB等;《Journal of Medical Entomology》;20090331(第2期);249-256页 * |
| Yeh WB等.Molecular differentiation and diversity of Forcipomyia taiwana (Diptera:Ceratopogonidae) based on the mitochondrial cytochrome oxidase II sequence..《Journal of Medical Entomology》.2009,(第2期),249-256页. |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105420399A (en) * | 2016-01-08 | 2016-03-23 | 福建国际旅行卫生保健中心 | Specific gene of brown atrichopogon and molecular identification method of brown atrichopogon |
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