CN102392075A - Method for identifying Frankliniella occidentalis species by utilizing PCR (Polymerase Chain Reaction)-RFLP (Restriction Fragment Length Polymorphism) technology - Google Patents
Method for identifying Frankliniella occidentalis species by utilizing PCR (Polymerase Chain Reaction)-RFLP (Restriction Fragment Length Polymorphism) technology Download PDFInfo
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Abstract
The invention relates to a method for identifying Frankliniella occidentalis species by utilizing PCR (Polymerase Chain Reaction)-RFLP (Restriction Fragment Length Polymorphism) technology, comprising the following steps: (1) extracting Frankliniella occidentalis genome DNA; (2) conducting PCR amplification to the mitochondrion COI gene of the Frankliniella occidentalis genome DNA by taking the Frankliniella occidentalis genome DNA as a template; (3) conducting enzyme cutting to the PCR product prepared in step (2) by adopting restriction enzyme EarI to obtain an enzyme cutting product; and (4) conducting agar gel electrophoresis analysis on the enzyme cutting production prepared in step (3). The restriction enzyme is the common restriction enzyme, therefore, the method provides a simple, convenient and stable enzyme cutting marker for screening two types of Frankliniella occidentalis, and simultaneously explores and establishes an identification technology for the two types of Frankliniella occidentalis, and lays a foundation for identification on the population dynamics of the two types of Frankliniella occidentalis and research on biology and invasion mechanisms.
Description
Technical field
The present invention relates to a kind of PCR-RFLP of utilization technology and differentiate the method for Frankliniella occidentalis strain, belong to agricultural biological technical field.
Background technology
Frankliniella occidentalis (Frankliniella occidentalis); It belongs to Thysanoptera (Thysanoptera) Thripidae (Thripidae) flower thrips and belongs to (Frankliniella); Originate from Western United States at first, begin to diffuse to rapidly each continent, the whole world eighties in 20th century, 69 countries and regions report is arranged at present; Be current in the world to one of the most serious worldwide insect of crop harm (Kirk, 2001; Kirk and Terry, 2003).This insect is not only got food host plant juice, shows the formation scar at fruit, reduces fruit quality, and can propagate tomato spotted wilf virus disease (TSWV) and garden balsam necrotic spot virus (INSV) (Kritzman A, 2002) with persistent mode.And the financial loss that virus caused of propagation is far longer than the loss that itself causes.China Ministry of Agriculture classified it as ground Plant Quarantine potentiality insect that enters the territory in 1996.
Brunner etc. (2010) find that based on the mtCOI gene order there are 2 hereditary offsprings in the Frankliniella occidentalis population all over the world; And advise these 2 hereditary offsprings are defined as 2 ecotypes (ecotypes), and Rugman-Jones etc. (2010) suggestion is defined as 2 kinds with it.In order to discuss conveniently, be referred to as G type and L type in this patent file respectively.Data shows that G type and L type Frankliniella occidentalis there are differences (deKogel et al., 1997 aspect the biology such as egg laying amount, host's flexibility, habitat flexibility, resistance; Brdsgaard, 1994; Brunner et al., 2010; Rugman-Jones et al., 2010).Therefore accurately distinguish two types, have important significance for theories and reference value for its invasion mechanism of further research and biology.At present, the differentiation that two types of Frankliniella occidentalis can't be clear and definite from the morphology only can be distinguished from the molecular genetics angle.Rugman-Jones etc. (2010) have designed one group of primer based on rDNA 28s, through pcr amplification, detect the polymorphum of PCR product and distinguish two types.This method has been distinguished the Frankliniella occidentalis in U.S. country of origin two types.
PCR-RFLP (restriction fragment length polymorphism polymerase chain reaction) technology is called CAPS technology (Cleaved Amplilfed Polymorphism Sequences) again, and it comes down to round pcr and a kind of method of RFLP technology bonded.Its ultimate principle is to utilize the dna sequence dna resource of known site (gene database, genome or cDNA clone and clone's RAPD band etc.) design one cover specific PCR primer (19～27bp) earlier; Use a certain dna fragmentation on this site of these primer amplifications then; Then with a kind of narrow spectrum restriction enzyme cutting gained amplified production; Gel electrophoresis separates endonuclease bamhi, dyes and carries out rflp analysis.What this technology disclosed is the information of the segmental restricted length variation of specific PCR.PCR-RFLP is one type of codominance molecule marker, and its advantage is to have avoided this step of film transfer printing in the rflp analysis, can keep the tolerance range of rflp analysis-can disclose the difference of single base again.In addition, because much restriction enzyme all can be cut with DNA cloning fragment enzyme, so detect polymorphum chance big (Zhao Shuqing, 2000).
The PCR-RFLP technology has been widely used in aspect (Wang Xiaoxuan, 2003 such as species detection and evaluation, the genetic variation and genetic differentiation evaluation of plant, animal, mikrobe and insect; Zhang Ting, 2005; Horse rubine, 2006; Teng Qihui, 2006).Species (Moritz, 2000 such as Frankliniella occidentalis, U.S. sour jujube thrips, onion thrips and palm thrips distinguished and identified to utilization should technology; Brunner, 2002; Toda and Komazaki, 2002).The PCR-RFLP technology is equally applicable to first differentiation that is situated between below the species.Zhou Jianfeng etc. (2003) utilization PCR-RFLP methods analyst the DNA5/6 section of 3 subspecies Mitochondrial DNAs, utilize 3 kinds of restriction endonucleases, distinguished 3 carp subspecies.But the method for utilizing the PCR-RFLP technique construction to distinguish two strains of Frankliniella occidentalis is not also appeared in the newspapers at present.
Summary of the invention
The present invention is directed to the deficiency of prior art, provide a kind of PCR-RFLP of utilization technology to differentiate the method for Frankliniella occidentalis strain.
Terminological interpretation:
G type Frankliniella occidentalis: the Frankliniella occidentalis strain that will form Frankliniella occidentalis invasive species main body at present is called greenhouse system (greenhouse strain), is called G type Frankliniella occidentalis in this patent file.
L type Frankliniella occidentalis: because of this strain find the earliest with Zelanian lupine on, and to show with the greenhouse be the biological characteristics of obvious difference, so be referred to as lupine system (lupin strain), is called L type Frankliniella occidentalis in this patent file.
A kind of method of utilizing the PCR-RFLP technology to differentiate the Frankliniella occidentalis strain, step is following:
(1) extracts the Frankliniella occidentalis genomic dna;
(2) be that template is carried out pcr amplification to its mitochondrial COI gene with the Frankliniella occidentalis genomic dna;
In the PCR system, primer sequence is following:
Sense primer: 5 ' GGATCACCTGATATAGCATTCCC3 '; SEQ ID NO.1
Antisense primer: 5 ' ACTGTAAATATATGATGAGCTCA3 '; SEQ ID NO.2
(3) the PCR product that step (2) is made is cut with restriction enzyme EarI enzyme, gets enzyme and cuts product;
(4) enzyme that step (3) is made is cut product and is carried out the agarose gel electrophoresis analysis, and when PCR product electrophoretogram showed that sample has the band of two different lengthss, then this sample to be detected was a L type Frankliniella occidentalis; When PCR product electrophoretogram shows that sample has a band, then be G type Frankliniella occidentalis.
Said pcr amplification system is:
Genomic dna 2 μ l, 20 μ M primers, 0.4 μ l, 5U/ μ l Taq enzyme 0.2 μ l, 10 * Taq Buffer, 5 μ l,
10mM dNTP 0.4 μ l, ddH
2O mends to 20 μ l;
Said pcr amplification condition is following: 94 ℃, and 5min; Carry out 94 ℃ of 50sec of 35 round-robin, 53 ℃ of 40sec, 72 ℃ of 50sec; 72 ℃ are extended 7min; 4 ℃, insulation.
EarI endonuclease reaction condition is following in the said step (3): 37 ℃, and 2hr.
Above-mentioned steps (1) can be with reference to (Frohlich; D.R.; I.Torres-Jerez; I.D.Bedford, P.G.Markham, and J.K.Brown.1999.A phylogeographical analysis of the Bemisia tabaci species complex based on mitochondrial DNA markers.Mol.Ecol.8:1683-1691.) in method operate.
Beneficial effect:
1, restriction enzyme of the present invention is restriction enzyme commonly used, for screening two types of Frankliniella occidentalis easy stable enzyme trimscript note is provided.
2, the PCR primer of the present invention Frankliniella occidentalis mitochondrial COI gene that is used to increase for identifying Frankliniella occidentalis, provides easy stable molecule marker, has solved the gordian technique of distinguishing two types of Frankliniella occidentalis.
3, the present invention has explored the difference of two types of Frankliniella occidentalis mitochondrial COI gene from molecular level; The authentication technique of two types of Frankliniella occidentalis has been set up in exploration, for the research of population dynamics evaluation, biology and the invasion mechanism of from now on two types of Frankliniella occidentalis is laid a good foundation.
Description of drawings
Fig. 1 is the electrophorogram of PCR product after the EarI enzyme is cut;
Wherein 1, the G type Frankliniella occidentalis PCR product cut of enzyme not; 2-5, EarI endonuclease digestion G type Frankliniella occidentalis PCR product; 6-8, the PCR product of EarI endonuclease digestion L type Frankliniella occidentalis, M, 100bp DNAMarker.
Embodiment
Below in conjunction with instance and accompanying drawing content of the present invention is done further explanation, but institute of the present invention protection domain is not limited thereto.
The type of G described in embodiment Frankliniella occidentalis was collected in the Shandong Province in 2011, L type Frankliniella occidentalis was collected in Qingdao of Shandong province, urban district, Weihai and Rongcheng City respectively in 2011; The EarI restriction endonuclease is available from New England Biolabs (NEB) company.
Embodiment 1
The analysis of Frankliniella occidentalis specimen material
(1) extraction of Frankliniella occidentalis genomic dna
Single head thrips individuality is placed the centrifuge tube of the 0.2ml that contains 60 μ l alkaline lysis liquid, and alkaline lysis liquid is: 50mmolL
-1Tris-HCl (pH8.0), 20mmolL
-1NaCl, 1mmolL
-1EDTA, 1%SDS with sealing after the rifle head fully grinds homogenate, place 65 ℃ of water-bath 15min of water-bath, behind 95 ℃ of water-bath 10min, make Frankliniella occidentalis genomic dna solution then.
(2) pcr amplification of Frankliniella occidentalis COI gene
With the Frankliniella occidentalis genomic dna is that template is carried out pcr amplification to its COI gene, gets the PCR product;
Said pcr amplification system is:
Genomic dna: 3 μ l; 20 μ M primers: 0.5 μ l; 5U/ μ l Taq enzyme: 0.5 μ l; 10 * Taq Buffer:5 μ l;
10mM dNTP:1 μ l; DdH
2O mends to 50 μ l;
The amplimer sequence is following:
Sense primer: 5 ' GGATCACCTGATATAGCATTCCC3 '; SEQ ID NO.1
Antisense primer: 5 ' ACTGTAAATATATGATGAGCTCA3 '; SEQ ID NO.2
The pcr amplification condition is following: 94 ℃, and 5min; Carry out 94 ℃ of 50sec of 35 round-robin, 53 ℃ of 40sec, 72 ℃ of 50sec; 72 ℃ are extended 7min; 4 ℃ of insulations.
(3) the PCR product detects with 2% agarose gel electrophoresis, band and length is arranged all about 620bp if detect, and the PCR product is carried out two-way order-checking, obtains sequence shown in SEQ ID NO.3 or SEQ ID NO.4.
(4) (this analysis software can land following network address and use to utilize restriction enzyme digestion sites analysis software WatCut
Http:// watcut.uwaterloo.ca/watcut/watcut/template.php? Act=restriction_new), analysis can have base difference at the two types of Frankliniella occidentalis in fragment 179bp place, and finds restriction enzyme EarI (CTCTTC).Through compare of analysis, the L type Frankliniella occidentalis that is collected in Qingdao of Shandong province, urban district, Weihai and Rongcheng City has nucleotide sequence shown in the SEQ ID NO.3, and the G type Frankliniella occidentalis that is collected in the Shandong Province has nucleotide sequence shown in the SEQ ID NO.4.
Embodiment 2
A kind of method of utilizing the PCR-RFLP technology to differentiate the Frankliniella occidentalis strain, step is following:
(1) extraction of Frankliniella occidentalis genomic dna
Single head thrips individuality is placed the centrifuge tube of the 0.2ml that contains 60 μ l alkaline lysis liquid, and alkaline lysis liquid is: 50mmolL
-1Tris-HCl (pH8.0), 20mmolL
-1NaCl, 1mmolL
-1EDTA, 1%SDS with sealing after the rifle head fully grinds homogenate, place 65 ℃ of water-bath 15min of water-bath, behind 95 ℃ of water-bath 10min, make Frankliniella occidentalis genomic dna solution then.
(2) pcr amplification of Frankliniella occidentalis COI gene
With the Frankliniella occidentalis genomic dna is that template is carried out pcr amplification to its COI gene, gets the PCR product;
Said pcr amplification system is:
Genomic dna 2 μ l, 20 μ M primers, 0.4 μ l, 5U/ μ lTaq enzyme 0.2 μ l, 10 * Taq Buffer, 2 μ l,
10mM dNTP 0.4 μ l, ddH
2O mends to 20 μ l;
The amplimer sequence is following:
Sense primer: 5 ' GGATCACCTGATATAGCATTCCC3 '; SEQ ID NO.1
Antisense primer: 5 ' ACTGTAAATATATGATGAGCTCA3 '; SEQ ID NO.2
The pcr amplification condition is following: 94 ℃, and 5min; Carry out 94 ℃ of 50sec of 35 round-robin, 53 ℃ of 40sec, 72 ℃ of 50sec; 72 ℃ are extended 7min; 4 ℃ of insulations.
(3) the PCR product carries out the EarI enzyme and cuts
It is following that enzyme is cut system: 10 * NEB damping fluid, 2 μ l; PCR product 5 μ l; EarI restriction endonuclease 0.5 μ l; Sterilization distilled water to 15 μ l.
Reaction conditions is following: 37 ℃ of incubations 2 hours.
After restriction enzyme EarI enzyme is cut, get enzyme and cut product;
(4) enzyme is cut product and separate with 2% agarose gel electrophoresis, forming images on ultraviolet gel imaging appearance in EB dyeing back, observes its polymorphum.The result finds that the COI fragment of L type Frankliniella occidentalis is cut open, and the COI fragment of G type Frankliniella occidentalis is not cut open.The result is as shown in Figure 1.
Claims (4)
1. one kind is utilized the technological method of differentiating the Frankliniella occidentalis strain of PCR-RFLP, it is characterized in that step is following:
(1) extracts the Frankliniella occidentalis genomic dna;
(2) be that template is carried out pcr amplification to its mitochondrial COI gene with the Frankliniella occidentalis genomic dna;
In the PCR system, primer sequence is following:
Sense primer: 5 ' GGATCACCTGATATAGCATTCCC3 '; SEQ ID NO.1
Antisense primer: 5 ' ACTGTAAATATATGATGAGCTCA3 '; SEQ ID NO.2
(3) the PCR product that step (2) is made is cut with restriction enzyme EarI enzyme, gets enzyme and cuts product;
(4) enzyme that step (3) is made is cut product and is carried out the agarose gel electrophoresis analysis, and when PCR product electrophoretogram showed that sample has the band of two different lengthss, then this sample to be detected was a L type Frankliniella occidentalis; When PCR product electrophoretogram shows that sample has a band, then be G type Frankliniella occidentalis.
2. the method for claim 1 is characterized in that, said pcr amplification system is:
Genomic dna 2 μ l, 20 μ M primers, 0.4 μ l, 5U/ μ l Taq enzyme 0.2 μ l, 10 * Taq Buffer, 5 μ l,
10mM dNTP 0.4 μ l, ddH
2O mends to 20 μ l.
3. the method for claim 1 is characterized in that, said pcr amplification condition is following: 94 ℃, and 5min; Carry out 94 ℃ of 50sec of 35 round-robin, 53 ℃ of 40sec, 72 ℃ of 50sec; 72 ℃ are extended 7min; 4 ℃, insulation.
4. the method for claim 1 is characterized in that, EarI endonuclease reaction condition is following in the said step (3): 37 ℃, and 2hr.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103255223A (en) * | 2013-05-22 | 2013-08-21 | 青岛农业大学 | Primer and method for authenticating frankliniella occidentalis by using expressed sequence tag (EST) microsatellite markers |
| 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 |
| CN109439761A (en) * | 2018-06-26 | 2019-03-08 | 中国计量大学 | Application of the COI sequence in Rapid identification river Puffer and its fish product |
| CN112301136A (en) * | 2020-12-07 | 2021-02-02 | 中国水产科学研究院珠江水产研究所 | Molecular marker for identifying flower and clupanodon based on mitochondrial COI gene and application thereof |
| CN114317768A (en) * | 2021-12-21 | 2022-04-12 | 广西壮族自治区农业科学院 | Dual PCR detection primer and method for identifying frankliniella occidentalis and frankliniella occidentalis |
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Cited By (7)
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| CN103255223A (en) * | 2013-05-22 | 2013-08-21 | 青岛农业大学 | Primer and method for authenticating frankliniella occidentalis by using expressed sequence tag (EST) microsatellite markers |
| CN103255223B (en) * | 2013-05-22 | 2014-08-06 | 青岛农业大学 | Primer and method for authenticating frankliniella occidentalis by using expressed sequence tag (EST) microsatellite markers |
| 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 |
| CN109439761A (en) * | 2018-06-26 | 2019-03-08 | 中国计量大学 | Application of the COI sequence in Rapid identification river Puffer and its fish product |
| CN112301136A (en) * | 2020-12-07 | 2021-02-02 | 中国水产科学研究院珠江水产研究所 | Molecular marker for identifying flower and clupanodon based on mitochondrial COI gene and application thereof |
| CN112301136B (en) * | 2020-12-07 | 2021-07-13 | 中国水产科学研究院珠江水产研究所 | Molecular marker for identifying flower and clupanodon based on mitochondrial COI gene and application thereof |
| CN114317768A (en) * | 2021-12-21 | 2022-04-12 | 广西壮族自治区农业科学院 | Dual PCR detection primer and method for identifying frankliniella occidentalis and frankliniella occidentalis |
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