CN101200764A - Method for rapid identification of liriomya huidobresis blanchard similar species by using PCR-RFLP technology - Google Patents
Method for rapid identification of liriomya huidobresis blanchard similar species by using PCR-RFLP technology Download PDFInfo
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Abstract
The invention discloses a method which can rapidly identify liriomyza approximate species by PCR-RFLP technology, which belongs to the insect species identification technical field. The method includes: (1) the DNA extraction of liriomyza trifolii, Americanliriomyza and South American liriomyza samples; (2) obtaining DNA sequence of 805bp by the PCR reaction of COII gene according to insect COII gene universal primers; (3) obtaining the COII sequence respectively through the clone, the transformation and the sequencing; (4) the selection of restriction endonuclease DraI; (5) the distinction of three approximate liriomyzas through a restriction map. The invention has the characteristics of accuracy, stability, fastness, cheapness, etc. And the invention can be applied in related departments of quarantine inspection, forecast, prevention, etc. to realize the rapid detection on the three approximate liriomyzas.
Description
Technical field
The present invention relates to caste authenticate technology field, relate in particular to the molecular method of a kind of PCR-RFLP of utilization technical evaluation liriomyza trifolii and allied species thereof.
Background technology
Liriomyza bryoniae belongs in (Liriomyza), and nearly kind more than 100 can cause harm or get food raise crop and ornamental crops, wherein has kind more than 20 to have important economic implications.From world wide, the maximum liriomyza bryoniae of harm has 3 kinds: Americal rice leaf miner (L.sativae Blanchard), south american leaf miner (L.huidobrensisBlanchard) and liriomyza trifolii (L.trifolii Burgess), these 3 kinds of liriomyza bryoniaes are all successfully invaded China at present, have brought enormous economic loss for the agriculture production of China; Wherein liriomyza trifolii has been defined as quarantine harmful organisms in China now, along with the frequent allocation and transportation of interzone agricultural-food, the trend that its distribution range and hazard rating more have expansion, increase the weight of.Because it is overlapping that liriomyza trifolii and Americal rice leaf miner and south american leaf miner have on ecological niche, several liriomyza bryoniaes often mix generation; Mainly the worm attitude with pupa and larva exists in the product allocation and transportation way of causing harm, and treats that larvae development is that adult carries out identification of morphology again and needs the above times in 2 weeks; Simultaneously, their adult build is small, and the formalness feature is closely similar, on identification of morphology, be difficult to accurate assurance, obscure easily, this gives quarantine, observe and predict and work such as control has brought difficulty, therefore presses for a kind of authentication method fast and accurately in quarantine.
After the eighties in 20th century, biochemical technology begins to be applied to the classification evaluation of liriomyza bryoniae, and Zehender (1993) has carried out the evaluation of trifolium liriomyza bryoniae, tomato liriomyza bryoniae and south american leaf miner with the starch gel technology; Collin (1996) utilizes the cellulose acetate electrophoresis to distinguish the south american leaf miner and the trifolium liriomyza bryoniae population of different areas.Along with development of science and technology, electron microscope also begins to be applied in the sort research of liriomyza bryoniae: Ma Ruiyan (1999) applying electronic microscope is observed the formalness feature of Americal rice leaf miner and south american leaf miner, but these technical evaluation cycles are longer, and complicated operation is unfavorable for the rapid detection of liriomyza bryoniae.In recent years, rise and widespread use based on the dna sequencing technology of PCR, therefore based on DNA, with molecular biology is laboratory facilities, the insect molecular systematics of the evaluation of research classification of insect, monoid genetic construction, phylogeny and molecular evolution has obtained very great development, and molecular marking technique is widely used in the evaluation of approximate species.
Being widely used in classification evaluation of insect and the marker gene in the phylogeny research at present has: Mitochondrial DNA (Mitochondrial DNA, mtDNA), rDNA (Ribosomal DNA, rDNA) and nucleoprotein encoding gene (Nuclear protein coding genes) etc., wherein a plurality of marker gene are most widely used in the molecular system research of insect among mtDNA and the rDNA.Because evolutionary rate that mtDNA is little, fast relatively and maternal hereditary property make it be well suited for the research of evolving between the population history taxon close with sibship.MtDNA Terminal oxidase II (COII) gene is that the insect Molecular Phylogeny and Evolution is used one of more marker gene, wherein comprise the slower conservative fragments of evolutionary rate and the variable region faster of evolving, both can be used for phyletic evolution research, can be used for the comparative studies of kind differentiation or infraspecific category again, be suitable for planting the phylogenetic relationship research between interior and sibling species.Therefore, the COII gene is applied in the evaluation of insect more and more, for example evaluation of liriomyza bryoniae sibling species and phylogeny research.
The PCR-RFLP labeling technique is that (polymerase chain reaction is PCR) in conjunction with restriction fragment length polymorphism (Restricrion fragment length polymorphism, RFLP) technology for polymerase chain reaction.Because the PCR-RFLP technology is less demanding to sample purity, is applicable to ontoanalysis, and quick and easy, therefore be used widely at aspects such as the detection of species polymorphism, assortment, species evaluation, bacteriophage detections.Aspect the liriomyza bryoniae evaluation, [1] Antonio M, Andrea L, BarbaraM et al.Polymerase chain rection-restricrion fragment lengthpolymorphism assays to distinguish liriomyza huidobrensis (Diptera:Agromyzidae) from associated species on lettuce croppingsystems in Italy.Annals of the entomological society of America.2006,99 (4): 1268-1272. once utilized PvuII to follow the SnaBI restriction endonuclease to carry out south american leaf miner and identified, but these two kinds of restriction endonucleases are merely able to carry out the evaluation of south american leaf miner and can not be used for the differentiation of liriomyza trifolii with Americal rice leaf miner, and cost an arm and a leg a sample Xu Yao $5.[2] Scheffer SJ, Lewis ML.Two nucleargenes confirm mitochondrial evidence of cryptic species within liriomyzahuidobrensis.Annals of the entomological society of America.2001,94 (5): 1177-1182. and [3] L.F.F.Kox, H.E.van den Beld et al.Identification ofeconomically important liriomyza species by PCR-RFLP analysis.BulletinOEPP/EPPO 2005,35:79-85. also utilized correndonuclease to carry out the not evaluation of liriomyza bryoniae of the same race respectively, but restriction endonuclease or comparison costliness that they select are of little use, as SpeI, SspI etc.; Or can not carry out the accurate differentiation of multiple liriomyza bryoniae, as HinfI, EcoRV etc., and the sample overwhelming majority that they detected is from abroad, because insect has the phenomenon of geographical population difference, is still waiting further checking so whether their selected restriction endonuclease is suitable for the evaluation of the domestic allied species liriomyza bryoniae of China.
Summary of the invention
The present invention seeks to very easily to obscure at liriomyza trifolii and its formalness allied species---Americal rice leaf miner and south american leaf miner are distinguished when identifying, existing in the PCR-RFLP labeling technique that adopts, the restriction endonuclease of selecting for use or cost an arm and a leg or can not clearly distinguish the defective of these three kinds of allied species liriomyza bryoniaes; Proposing a kind of is appraisal basis with suitable aim sequence, by the difference that checks order between clear and definite domestic these three kinds common allied species liriomyza bryoniae corresponding sequence, and then select a kind of cheapness, stable restriction endonuclease to carry out Analysis and Identification, to reach quick, accurate, the low-cost method of distinguishing liriomyza trifolii and allied species thereof of identifying.
The object of the invention is achieved by the following technical programs.
COII (Mitochondrial DNA Terminal oxidase II) the specificity segment that the liriomyza bryoniae dna molecular is identified, its sequence is SEQ NO:1, SEQ NO:2 is shown in the SEQ NO:3.
A kind of method of utilizing PCR-RFLP technology Rapid identification liriomyza bryoniae allied species, this method is carried out as follows:
(1) extracts liriomyza trifolii, Americal rice leaf miner and south american leaf miner sample total DNA respectively;
(2) with reference to the insect COII universal primer of Liu (1992) design, carry out the dna fragmentation (Fig. 1) that PCR (polymerase chain reaction) amplification length is 805bp, primer sequence is:
Upstream primer: 5 '-ATTTCTAATATGGCAGATTAGTGCA-3 '
Downstream primer: 5 '-ATATGGTTTAAGAGACCAGTACTT-3 ';
(3) obtain the COII sequence respectively by clone, conversion, three kinds of allied species liriomyza bryoniaes of order-checking, as SEQ NO:1, SEQ NO:2 is shown in the SEQ NO:3;
(4) by relevant molecular biology software three kinds of COII sequences of step (3) gained are spliced, are analyzed after, DraI is as restriction enzyme in selection;
(5) the PCR product after utilizing the DraI restriction endonuclease to purifying carries out that enzyme is cut, electrophoresis; Carry out the differentiation of three kinds of allied species liriomyza bryoniaes by restriction enzyme mapping;
(6) carry out confirmatory experiment by liriomyza bryoniae and the color fly of diving of allied species pea, to confirm the accurate, stable of this restriction endonuclease to different worm attitudes, different host, different geographical population;
Wherein, described PCR reaction conditions is as follows: system 50 μ l, reaction solution consist of 10 * Taq Buffer5 μ l, MgCl
2(25mmol/L) 4 μ l, dNTP (10mmol/L) 4 μ l, masterplate DNA (60ng/ μ l) 1 μ l, each 2.5 μ l of upstream and downstream primer (10 μ mol/L), Taq archaeal dna polymerase (TaKaRa) (5U/ μ l) 0.25 μ l, sterilization ddH
2O 30.75 μ l; Response procedures is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ are extended 60s; 35 circulations, last 72 ℃ are extended 10min, 4 ℃ of preservations; With ddH
2O replaces template to make blank.
The invention has the beneficial effects as follows:
1. utilize DraI to carry out liriomyza trifolii, Americal rice leaf miner and cut evaluation, can accurately distinguish domestic these three kinds of allied species liriomyza bryoniaes (Fig. 2) with the COII enzyme of south american leaf miner.
2. carried out the enzyme of three kinds of liriomyza bryoniae adults, pupa, larva and cut evaluation, the restriction enzyme digestion and electrophoresis band unanimity that obtains, the interference (Fig. 2) that therefore utilizes the DraI restriction endonuclease to analyze can not to be subjected to insect worm attitude.
3. utilize DraI to identify can not to be subjected to the influence comparatively similar with three kinds of liriomyza bryoniae forms, that the color fly of diving of pea of overlapping phenomenon is arranged with liriomyza bryoniae on the host, guarantee this restriction endonuclease accurately with stable (Fig. 3).
4. by to different areas, different host liriomyza trifolii, Americal rice leaf miner, south american leaf miner restriction analysis, show of the same race between Different Individual can keep very high stability (Fig. 4).
5. present method is identified fast only needs get final product 1 working days; Cost is lower simultaneously, and the detection of carrying out a sample only needs about 4 yuan.
In sum, the present invention is by carrying out the COII sequencing of domestic three kinds of allied species liriomyza bryoniaes, and then select stable, accurate, cheap DraI restriction endonuclease to identify, and verify with the liriomyza bryoniae of different worm attitudes by different geographical population, clear and definite the present invention has accurately, stablizes, fast, characteristics such as cheapness, can quarantine, observe and predict, relevant departments such as control use, to realize the rapid detection to these three kinds of allied species liriomyza bryoniaes.
Description of drawings
Three kinds of liriomyza bryoniaes of Fig. 1 carry out the 805 bpDNA sequence chart that the reaction of COII gene PCR obtains respectively;
Wherein: the Ls Americal rice leaf miner; The Lt liriomyza trifolii; The Lh south american leaf miner
Fig. 2 three kinds of liriomyza bryoniae adults, pupa and larvas are through DraI endonuclease digestion analytical results figure;
Wherein: the Ls Americal rice leaf miner; The Lt liriomyza trifolii; The Lh south american leaf miner
The liriomyza trifolii of Fig. 3 different areas, Americal rice leaf miner and south american leaf miner are through DraI endonuclease digestion analytical results figure;
The color fly of diving of the pea of Fig. 4 different areas is through DraI endonuclease digestion analytical results figure.
Annotate: UM is not digested COII PCR product, and long 805bp, M are 100bp DNA ladder (TaKaRa)
Specific embodiments
The present invention is described in further detail by the following examples.
Explanation to related worm source, reagent etc.:
The experimental population that 3 kinds of used liriomyza bryoniae adults keep for this laboratory in the test, the host plant that live box is used are Kidney bean (kind: heavenly steed ground beans).Wherein liriomyza trifolii picks up from the Zhejiang Cixi in April, 2007, identifies affirmation through the Chen Naizhong researcher of CIQ academy of sciences; Americal rice leaf miner and south american leaf miner are so kind as to give by Liu Wanxue assistant researcher of Institute of Plant Protection of the Chinese Academy of Agricultural Sciences and professor Pang Baoping of Agricultural University of the Inner Mongol respectively.
Tris：AMRESCO
SDS：AMRESCO
Proteinase K: AMRESCO
The saturated phenol of Tris: Beijing ancient cooking vessel state
Embodiment 1:(utilizes DraI that the COII enzyme of liriomyza trifolii, Americal rice leaf miner and south american leaf miner is cut evaluation)
Carry out according to the following steps:
One, the acquisition of DNA extraction and correlated series:
Get each 1 of liriomyza trifolii, Americal rice leaf miner and south american leaf miner adult sample, and extract DNA respectively according to the following steps: get 1 of sample and place the 1.5ml centrifuge tube of containing sterilized water to soak 1h; Take out sample, (SDS:1% grinds in PH=8.0), adds 2.5 μ l Proteinase Ks (20mg/ml) in 56 ℃ of water-baths digestion 2h for Tris:10mM, EDTA:1mM in 100 μ l extracting solutions; Add isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1) mixed solution extracting, get supernatant liquor and add 2 times of volume dehydrated alcohols, deposit D NA; Use the sterilized water dissolving DNA at last;-20 ℃ of preservations are as the template of PCR reaction.
The PCR reaction conditions is as follows: system 50 μ l, reaction solution consist of 10 * Taq Buffer, 5 μ l, MgCl
2(25mmol/L) 4 μ l, dNTP (10mmol/L) 4 μ l, masterplate DNA (60ng/ μ l) 1 μ l, with reference to Liu H., Beckenbach AT.Evolution of the mitochondrial cytochrome oxidase IIgene among 10 orders of insects.Molecular Phylogenetics and Evolution.1992,1:41-52. design, on the synthetic insect COII of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, each 2.5 μ l of downstream primer (10 μ mol/L), Taq archaeal dna polymerase (5U/ μ l) 0.25 μ l (TaKaRa), sterilization ddH
2O 30.75 μ l; Response procedures is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ are extended 60s; 35 circulations; Last 72 ℃ are extended 10min, 4 ℃ of preservations.With ddH
2O replaces template to make blank.The PCR product is at 1.0% agarose electrophoresis 1h, and bromination second pyridine (EB) is dyeed, and gel imaging system is taken pictures, and 1kbp DNA ladder makes molecular weight marker (Fig. 1).
Concrete steps are as follows: pcr amplification product utilizes gel to reclaim test kit (U-GENE
) the recovery purifying; This experiment Promega pGEM
-T easy Vector test kit is finished ligation, prepares competent cell with bacterial classification DH5 α, according to blue hickie screening positive clone, uses the positive colony bacterium liquid extracting plasmid that filters out, and cuts with the EcoRI enzyme and determines whether to contain the purpose fragment.Containing the segmental clone of purpose bacterium liquid the most at last serves extra large English fine horse (invitrogen) Bioisystech Co., Ltd and carries out sequencing.Accuracy in order to ensure sequence, this research is carried out cloning and sequencing to sample, and every DNA gets the recombinant plasmid order-checking of three correspondences, and each recombinant plasmid carries out positive and negative two-way order-checking, final sequence is comprehensively decided by the sequence of three plasmids, to guarantee the accuracy of sequence.Through above step, obtain SEQ NO:1 (liriomyza trifolii COII sequence) respectively; SEQ NO:2 (Americal rice leaf miner COII sequence); Three kinds of sequences of SEQ NO:3 (south american leaf miner COII sequence).
Two, restriction endonuclease is selected and restriction analysis:
The editor of sequence, analysis of statistical data etc. are finished by the LaserGene software of DNASTAR company; The splicing of sequence is finished by SeqMan software; The search work of homologous sequence utilizes blast program to finish in the GenBank database; Searching by DNAman software of restriction enzyme finished.
The amplified production COII of 3 kinds of liriomyza bryoniaes cuts under the system at 20 μ l enzymes behind the purifying, and reaction solution consists of: sterilization deionized water 10 μ l, 10 * buffer, 2 μ l, PCR product 7 μ l behind the purifying, DraI (10U/ μ l) 1 μ l; 37 ℃ are carried out DraI (TaKaRa) enzyme and cut 2h, and 10 * loading buffer termination reaction is got enzyme and cut product 5 μ L, carry out electrophoresis with 2.0% sepharose, finish bromination second pyridine (EB) dyeing of electrophoresis, gel imaging system is taken pictures, and 100bp DNA ladder makes molecular weight marker (Fig. 1).Three kinds of liriomyza bryoniaes can obviously be distinguished in the electrophoretic band position from electrophorogram: Americal rice leaf miner only is a 805bp electrophoretic band; The COII gene of liriomyza trifolii can digestedly be 92bp, 270bp, 443bp three bands; South american leaf miner COII gene can digestedly be 194bp, 249bp, 362bp three bands.
Three kinds of COII sequence chart that the liriomyza bryoniae adult obtains respectively by clone, conversion, order-checking, as SEQ NO:1, SEQ NO:2, shown in the SEQ NO:3, attached.
Embodiment 2:(utilizes the checking of DraI restriction endonuclease to the different worm attitude of three kinds of liriomyza bryoniaes homology)
The way of the DNA extraction of liriomyza trifolii, Americal rice leaf miner and south american leaf miner pupa and larva and the acquisition of correlated series and embodiment 1 adult is identical; Enzyme is cut evaluation, and the restriction enzyme digestion and electrophoresis band that obtains is consistent with embodiment 1 adult, therefore, utilizes the DraI restriction endonuclease to distinguish the interference (Fig. 2) that analysis can not be subjected to insect worm attitude.
Embodiment 3:(utilizes the DraI restriction endonuclease to pea color dive fly and three kinds of checkings that liriomyza bryoniae is not of the same race)
Owing on the host, with liriomyza bryoniae overlapping phenomenon is arranged with three kinds of comparatively similar color latent flies of pea of liriomyza bryoniae form again, therefore the color fly of diving of pea need be planted as checking and be verified; The color fly (identical with embodiment 1) after the DraI enzyme is cut of diving of pea, its electrophoretic band obviously is different from three kinds of liriomyza bryoniae restriction enzyme digestion and electrophoresis bands, illustrates that utilizing the DraI restriction endonuclease to identify can not be subjected to the color fly influence (Fig. 3) of diving of miscellaneous pea.
Three kinds of liriomyza bryoniaes of embodiment 4:(are the checking of different areas, host insect source homology separately)
Adopt the method for embodiment 1 simultaneously liriomyza trifolii, Americal rice leaf miner and the south american leaf miner of different areas, different host to be carried out confirmatory experiment (table 1), draw of the same race between Different Individual can keep very high stability, also show with this and adopt the DraI restriction endonuclease that this evaluations worm kind is carried out the accuracy and stable (Fig. 4) that enzyme is cut evaluation.
By embodiment 1,2,3 and 4 explanations, utilize DraI to carry out liriomyza trifolii, Americal rice leaf miner and cut evaluation with the COII enzyme of south american leaf miner, can distinguish three kinds of domestic allied species liriomyza bryoniaes accurately and rapidly.
Three kinds of liriomyza bryoniaes of table 1 different areas, different host and the checking result of the color fly of diving
| Numbering | Kind | Gather ground | The host | Worm attitude/male and female |
| 1 | The color fly of diving of pea | Hangzhou | Little rape | Adult ♂ |
| 2 | The color fly of diving of pea | Hangzhou | Little rape | Adult ♀ |
| 3 | The color fly of diving of pea | Hangzhou | Pea | Adult ♂ |
| 4 | The color fly of diving of pea | Hangzhou | Pea | Adult ♀ |
| 5 | The color fly of diving of pea | Hangzhou | Pea | Pupa |
| 6 | The color fly of diving of pea | The Quzhou | Pea | Adult ♂ |
| 7 | The color fly of diving of pea | Jinhua | Pea | Adult ♂ |
| 8 | Liriomyza trifolii | Hangzhou | Pea | Adult ♂ |
| 9 | Liriomyza trifolii | Hangzhou | Pea | Adult ♀ |
| 10 | Liriomyza trifolii | Cixi | Celery | Adult ♂ |
| 11 | Liriomyza trifolii | Ningbo | Celery | Adult ♂ |
| 12 | Liriomyza trifolii | Zhuhai | The cylinder beans | Adult ♂ |
| 13 | Liriomyza trifolii | Zhuhai | Green vegetables | Adult ♂ |
| 14 | Americal rice leaf miner | Hangzhou | Sponge gourd | Adult ♂ |
| 15 | Americal rice leaf miner | Hangzhou | French beans | Adult ♂ |
| 16 | Americal rice leaf miner | Guangdong | The cylinder beans | Adult ♂ |
| 17 | Americal rice leaf miner | Beijing | Kidney bean | Adult ♂ |
| 18 | Americal rice leaf miner | Beijing | Kidney bean | Adult ♀ |
| 19 | Americal rice leaf miner | Guangxi | The cylinder beans | Adult ♂ |
| 20 | South american leaf miner | The Inner Mongol | Cucumber | Adult ♂ |
| 21 | South american leaf miner | The Inner Mongol | Kidney bean | Adult ♂ |
| 22 | South american leaf miner | The Inner Mongol | Kidney bean | Adult ♀ |
| 23 | South american leaf miner | The Inner Mongol | Pumpkin | Adult ♂ |
| 24 | South american leaf miner | The Inner Mongol | Pumpkin | Pupa |
Sequence table
＜110〉China Measures Institute
＜120〉a kind of method of utilizing PCR-RFLP technology Rapid identification liriomyza bryoniae allied species
<160>3
<210>1
<211>805
<212>DNA
＜213〉derive from liriomyza trifolii (Liriomyza trifolii (Burgess))
<400>1
atttctaata tggcagatta gtgcaatgga tttaagctcc atatataaag tgtttaactt 60
ttattagaat aaatgtcaac atgagctaat ttaaatttac aaaatagagc ctctccgtta 120
atagaacaac taattttttt tcacgatcac gcattaataa ttttaataat aattactacc 180
ttagtgggat atttaataat tactttatta tttaataaat ttacaaatcg ttatcttcta 240
catggacaaa taattgaaat aatttgaaca atcttaccag caattatttt actatttatt 300
gcctttcctt ctcttcgact tttatatctt cttgatgaaa ttaatgagcc ttcaatcact 360
ttaaaagcta ttggtcatca atgatactga agctatgaat attcggattt caacaaccaa 420
atagaatttg attcttatat aatcccaact aatgaattaa aaagaaatga atttcgactt 480
ctagatgttg ataatcgagt tgttctacct ataaatactc aaatccgaat tttagtaact 540
gcagcagatg ttttacattc ttgaactgtg ccagctttag gggttaaaat tgatggaaca 600
ccaggccgat taaatcaaac taattttttt attaatcgac ctggtctatt ttatggacaa 660
tgttctgaaa tctgtggtac aaatcatagt tttataccaa ttgtaattga aagagttccc 720
tcaaatattt ttatcaaatg aatcataaat aactcaaact aatcattagg tggctgaaag 780
caagtactgg tctcttaaac catat 805
<210>2
<211>805
<212>DNA
＜213〉derive from Americal rice leaf miner (Liriomyza sativae (Blanchard))
<400>2
atttctaata tggcagatta gtgcaatgga tttaagctcc atatataaag tattttactt 60
ttattagaat aaatgtcaac atgagctaac ttaaatttac aaaatagtgc ctctccttta 120
atagaacaat taattttctt tcatgatcac gcattaataa ttttagtaat aattactact 180
ttagtgggat atttaataat tactttgtta tttaataaat ttacaaatcg ttatctttta 240
cacggacaaa taattgaaat aatttgaaca attttaccag caattatttt attatttatt 300
gcttttccat ctcttcgact tttatattta cttgatgaaa ttaatgaacc atcaatcacc 360
ttaaaggcta ttggtcatca atgatattga agctatgaat actccgattt caacaataat 420
atagaatttg attcttatat aatcccaaca aacgaattaa aggaagatga attccgactt 480
ttagatgttg ataatcgggt tgttcttcct ataaacactc aaattcgaat tttagtaact 540
gcagcagatg ttcttcattc ttgaactgtg ccagccctag gggttaaaat tgatggaaca 600
ccgggtcgat taaatcaaac caattttttt atcaatcgac ctggtttatt ttatggacaa 660
tgttctgaaa tttgtggtgc aaatcatagt tttatgccaa ttgtgattga aagagttcct 720
tcaaatattt ttatcaaatg aatcataaat aactcaaact aatcattagg tggctgaaag 780
caagtactgg tctcttaaac catat 805
<210>3
<211>805
<212>DNA
＜213〉derive from south american leaf miner (Liriomyza huidobrensis (Blanchard))
<400>3
atttctaata tggcagatta gtgcaatgga tttaagctcc atatataaag tatttaactt 60
ttattagaat aaatgtcaac atgagctaat ctaaatcttc aaaatagagc ttccccttta 120
atagaacaat taattttttt tcatgatcat gctttaataa ttttagttat aattacagta 180
ttagtcggtt acgtaataat tactttattt tttaataaat ttacaaaccg actattatta 240
cacggacaaa taattgaaat aatctgaact attttacctg ctattattct tttatttatt 300
gcttttccct ctcttcgatt gctttattta ttagatgaga ttaatgaacc ttcaattact 360
ttaaaggcta ttggtcacca atgatactga agttatgaat attctgattt taataataat 420
atagaatttg attcttatat aattccaact aatgaactaa aaaatgatga atttcgatta 480
ttagatgtag ataatcgaac tgtattacca ataaatactc aaattcgaat tttagttaca 540
gctgcagatg tattacactc ttgaacagtt cctgctctag gggtaaaggt tgatgctacg 600
ccaggccgtt taaatcaaac taatttcttt attaatcgac caggattatt ttatggacag 660
tgttcagaaa tttgtggggc taatcatagt tttataccaa ttgtaattga aagagttcct 720
tctgatattt ttattaagca catcaaaaat aattctaact aatcattaga tgactgaaag 780
caagtactgg tctcttaaac catat 805
Claims (2)
1. the COII specificity segment identified of liriomyza trifolii, Americal rice leaf miner and south american leaf miner dna molecular, its sequence is SEQ NO:1, SEQ NO:2 is shown in the SEQ NO:3.
2. method of utilizing PCR-RFLP technology Rapid identification liriomyza bryoniae allied species is characterized in that this method carries out as follows:
(1) extracts liriomyza trifolii, Americal rice leaf miner and south american leaf miner sample total DNA respectively;
(2) insect COII gene universal primer carries out the dna fragmentation that pcr amplification length is 805bp, and primer sequence is:
Upstream primer: 5 '-ATTTCTAATATGGCAGATTAGTGCA-3 '
Downstream primer: 5 '-ATATGGTTTAAGAGACCAGTACTT-3 ';
(3) obtain the COII sequence respectively by clone, conversion, order-checking, as SEQ NO:1, SEQ NO:2 is shown in the SEQ NO:3;
(4) by relevant molecular biology software three kinds of COII sequences of step (3) gained are spliced, are analyzed after, DraI is as restriction enzyme in selection;
(5) the PCR product after utilizing the DraI restriction endonuclease to purifying carries out that enzyme is cut, electrophoresis, carries out the differentiation of three kinds of allied species liriomyza bryoniaes by restriction enzyme mapping;
(6) carry out confirmatory experiment by liriomyza bryoniae and the color fly of diving of allied species pea, to confirm the accurate, stable of this restriction endonuclease to different worm attitudes, different host, different geographical population;
Wherein, described PCR reaction conditions is as follows: system 50 μ l, reaction solution consist of 10 * Taq Buffer, 5 μ l, the MgCl of 25mmol/L
24 μ l, the dNTP 4 μ l of 10mmol/L, the masterplate DNA 1 μ l of 60ng/ μ l, each 2.5 μ l of the upstream and downstream primer of 10 μ mol/L, the Taq archaeal dna polymerase 0.25 μ l of 5U/ μ l, sterilization ddH
2O30.75 μ l; Response procedures is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ are extended 60s; 35 circulations, last 72 ℃ are extended 10min, 4 ℃ of preservations; With ddH
2O replaces template to make blank.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102605085A (en) * | 2012-03-29 | 2012-07-25 | 广东出入境检验检疫局检验检疫技术中心 | Identification method for L. sativae Blanchard, L. huidobrensis and L. trifolii |
| CN103290131A (en) * | 2013-06-18 | 2013-09-11 | 徐鹏 | Primer pair and kit for distinguishing Channa argus and Channa maculata, and PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) detection method |
| CN103882116A (en) * | 2014-01-22 | 2014-06-25 | 西北农林科技大学 | Molecular identification primer for four fruit tree core-eating insects and using method of molecular identification primer |
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2007
- 2007-11-28 CN CNA2007101570676A patent/CN101200764A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102605085A (en) * | 2012-03-29 | 2012-07-25 | 广东出入境检验检疫局检验检疫技术中心 | Identification method for L. sativae Blanchard, L. huidobrensis and L. trifolii |
| CN102605085B (en) * | 2012-03-29 | 2016-03-09 | 广东出入境检验检疫局检验检疫技术中心 | The authentication method of Americal rice leaf miner, south american leaf miner, Liriomyza trifolii |
| CN103290131A (en) * | 2013-06-18 | 2013-09-11 | 徐鹏 | Primer pair and kit for distinguishing Channa argus and Channa maculata, and PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) detection method |
| CN103882116A (en) * | 2014-01-22 | 2014-06-25 | 西北农林科技大学 | Molecular identification primer for four fruit tree core-eating insects and using method of molecular identification primer |
| CN103882116B (en) * | 2014-01-22 | 2015-12-02 | 西北农林科技大学 | Four planting fruit-trees heart-eating worm Molecular Identification primer and using method |
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