CN102392075B - 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 first Western United States, start to diffuse to rapidly each continent, the whole world eighties in 20th century, 69 countries and regions, report is arranged at present, one of the current world worldwide insect the most serious to crop harm (Kirk, 2001; Kirk and Terry, 2003).This insect not only takes food host plant juice, show to form scar at fruit, reduce fruit quality, and can propagate tomato spotted wilf virus disease (TSWV) and garden balsam necrotic spot virus (INSV) (Kritzman A, 2002) in persistent mode.And the financial loss that the virus of propagation causes is far longer than the loss itself caused.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 to 2 ecotypes (ecotypes), and Rugman-Jones etc. (2010) suggestion is defined as 2 kinds by it.In order to discuss conveniently, be referred to as respectively G type and L-type in this patent document.Data shows, G type and L-type Frankliniella occidentalis there are differences (deKogel et al., 1997 aspect the biology such as egg laying amount, host's adaptability, environmental adaptability, resistance; Brdsgaard, 1994; Brunner et al., 2010; Rugman-Jones et al., 2010).Therefore accurately distinguish two types, for its invasion mechanism of further research and biology, there is important theory significance and reference value.At present, the differentiation that two types of Frankliniella occidentalis can't be clear and definite from morphology, only can distinguish from the molecular genetics angle.Rugman-Jones etc. (2010) have designed one group of primer based on rDNA 28s, by pcr amplification, detect the polymorphism of PCR product and distinguish two types.This method has been distinguished the Frankliniella occidentalis in two types, U.S. country of origin.
PCR-RFLP (restriction fragment length polymorphism polymerase chain reaction) technology, be called again CAPS technology (Cleaved Amplilfed Polymorphism Sequences), and it is in fact a kind of method that round pcr is combined with the RFLP technology.Its ultimate principle is first to utilize the DNA sequence dna resource of known site (gene database, genome or cDNA clone and clone's RAPD band etc.) to design a set of specific PCR primer (19～27bp), then use a certain DNA fragmentation on this site of these primer amplifications, 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 restricted length variation of specific PCR fragment.PCR-RFLP is a class codominant marker, and its advantage is to have avoided this step of film transfer printing in rflp analysis, can keep again the tolerance range of rflp analysis-can disclose the difference of single base.In addition, because a lot of restriction enzymes all can be cut with DNA cloning fragment enzyme, so polymorphism chance large (Zhao Shuqing, 2000) detected.
The PCR-RFLP technology has been widely used in aspect (Wang Xiaoxuan, 2003 such as the species detection of plant, animal, microorganism and insect and evaluation, genetic variation and genetic differentiation evaluation; Zhang Ting, 2005; Horse rubine, 2006; Teng Qihui, 2006).Utilize this technology to distinguish and identified species (Moritz, 2000 such as Frankliniella occidentalis, U.S. sour jujube thrips, onion thrips and palm thrips; Brunner, 2002; Toda and Komazaki, 2002).The PCR-RFLP technology is equally applicable to the following first differentiation that is situated between of species.Zhou Jianfeng etc. (2003) have used the PCR-RFLP methods analyst 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 yet there are no report.
Summary of the invention
The present invention is directed to the deficiencies in the 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 at present Frankliniella occidentalis invasive species main body is called greenhouse system (greenhouse strain), is called G type Frankliniella occidentalis in this patent document.
The L-type Frankliniella occidentalis: because of this strain find the earliest with Zelanian lupine on, and to show with greenhouse be the biological characteristics of obvious difference, therefore be referred to as lupine system (lupin strain), is called the L-type Frankliniella occidentalis in this patent document.
A kind of method of utilizing the PCR-RFLP technology to differentiate the Frankliniella occidentalis strain, step is as follows:
(1) extract the Frankliniella occidentalis genomic dna;
(2) take the Frankliniella occidentalis genomic dna carries out pcr amplification to its mitochondrial COI gene as template;
In the PCR system, primer sequence is as follows:
Sense primer: 5 ' GGATCACCTGATATAGCATTCCC3 '; SEQ ID NO.1
Antisense primer: 5 ' ACTGTAAATATATGATGAGCTCA3 '; SEQ ID NO.2
(3) PCR product step (2) made is cut with restriction enzyme EarI enzyme, obtains enzyme and cuts product;
(4) enzyme step (3) made is cut product and is carried out the agarose gel electrophoresis analysis, when PCR product electrophoretogram shows that sample has the band of two different lengthss, should detected sample be the L-type Frankliniella occidentalis; When PCR product electrophoretogram shows that sample has a band, it is G type Frankliniella occidentalis.
Described pcr amplification system is:
10mM dNTP 0.4 μ l, ddH
2O mends to 20 μ l;
Described pcr amplification condition is as follows: 94 ℃, and 5min; Carry out 94 ℃ of 50sec of 35 circulations, 53 ℃ of 40sec, 72 ℃ of 50sec; 72 ℃ are extended 7min; 4 ℃, insulation.
In described step (3), EarI endonuclease reaction condition is as follows: 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.) method in is operated.
Beneficial effect:
1, restriction enzyme of the present invention is restriction enzyme commonly used, for screening two types of Frankliniella occidentalis, provides easy stable enzyme trimscript note.
2, PCR primer of the present invention, for increasing the Frankliniella occidentalis mitochondrial COI gene, 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 two types of Frankliniella occidentalis is from now on laid a good foundation.
The accompanying drawing explanation
Fig. 1 is the electrophorogram of PCR product after the EarI enzyme is cut;
Wherein 1, the G type Frankliniella occidentalis PCR product that enzyme is not cut; 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 example and accompanying drawing, content of the present invention is described further, but institute of the present invention protection domain is not limited to this.
The type of G described in embodiment Frankliniella occidentalis was collected in Shandong Province in 2011, the L-type Frankliniella occidentalis was collected in respectively Qingdao of Shandong province, urban district, Weihai and Rongcheng City in 2011; The EarI restriction endonuclease is purchased from New England Biolabs (NEB) company.
The analysis of Frankliniella occidentalis specimen material
(1) extraction of Frankliniella occidentalis genomic dna
Single head thrips individuality is placed in to the centrifuge tube containing the 0.2ml of 60 μ l alkaline lysis liquid, and alkaline lysis liquid is: 50mmolL
-1Tris-HCl (pH8.0), 20mmolL
-1NaCl, 1mmolL
-1EDTA, 1%SDS, after fully grinding homogenate with sealing rifle head, be placed in 65 ℃ of water-bath 15min of water-bath, then, after 95 ℃ of water-bath 10min, makes Frankliniella occidentalis genomic dna solution.
(2) pcr amplification of Frankliniella occidentalis COI gene
The Frankliniella occidentalis genomic dna of take carries out pcr amplification as template to its COI gene, obtains the PCR product;
Described 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 as follows:
Sense primer: 5 ' GGATCACCTGATATAGCATTCCC3 '; SEQ ID NO.1
Antisense primer: 5 ' ACTGTAAATATATGATGAGCTCA3 '; SEQ ID NO.2
The pcr amplification condition is as follows: 94 ℃, and 5min; Carry out 94 ℃ of 50sec of 35 circulations, 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 in the 620bp left and right if detect, and the PCR product is carried out to two-way order-checking, obtains sequence as shown in SEQ ID NO.3 or SEQ ID NO.4.
(4) (this analysis software can log in 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 two types, fragment 179bp place Frankliniella occidentalis, 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 SEQ ID NO.3, and the G type Frankliniella occidentalis that is collected in Shandong Province has nucleotide sequence shown in SEQ ID NO.4.
A kind of method of utilizing the PCR-RFLP technology to differentiate the Frankliniella occidentalis strain, step is as follows:
(1) extraction of Frankliniella occidentalis genomic dna
Single head thrips individuality is placed in to the centrifuge tube containing the 0.2ml of 60 μ l alkaline lysis liquid, and alkaline lysis liquid is: 50mmolL
-1Tris-HCl (pH8.0), 20mmolL
-1NaCl, 1mmolL
-1EDTA, 1%SDS, after fully grinding homogenate with sealing rifle head, be placed in 65 ℃ of water-bath 15min of water-bath, then, after 95 ℃ of water-bath 10min, makes Frankliniella occidentalis genomic dna solution.
(2) pcr amplification of Frankliniella occidentalis COI gene
The Frankliniella occidentalis genomic dna of take carries out pcr amplification as template to its COI gene, obtains the PCR product;
Described pcr amplification system is:
10mM dNTP 0.4 μ l, ddH
2O mends to 20 μ l;
The amplimer sequence is as follows:
Sense primer: 5 ' GGATCACCTGATATAGCATTCCC3 '; SEQ ID NO.1
Antisense primer: 5 ' ACTGTAAATATATGATGAGCTCA3 '; SEQ ID NO.2
The pcr amplification condition is as follows: 94 ℃, and 5min; Carry out 94 ℃ of 50sec of 35 circulations, 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 as follows that enzyme is cut system: 10 * NEB damping fluid, 2 μ l; PCR product 5 μ l; EarI restriction endonuclease 0.5 μ l; Sterilizing distilled water to 15 μ l.
Reaction conditions is as follows: 37 ℃ of incubations 2 hours.
After restriction enzyme EarI enzyme is cut, obtain enzyme and cut product;
(4) enzyme is cut to product and separated with 2% agarose gel electrophoresis, imaging on ultraviolet gel imaging instrument after EB dyeing, observe its polymorphism.Found 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.Result as shown in Figure 1.
Claims (4)
1. a method of utilizing the PCR-RFLP technology to differentiate the Frankliniella occidentalis strain, is characterized in that, step is as follows:
(1) extract the Frankliniella occidentalis genomic dna;
(2) take the Frankliniella occidentalis genomic dna carries out pcr amplification to its mitochondrial COI gene as template;
In the PCR system, primer sequence is as follows:
Sense primer: 5 ' GGATCACCTGATATAGCATTCCC3 '; SEQ ID NO.1
Antisense primer: 5 ' ACTGTAAATATATGATGAGCTCA3 '; SEQ ID NO.2
(3) PCR product step (2) made is cut with restriction enzyme EarI enzyme, obtains enzyme and cuts product;
(4) enzyme step (3) made is cut product and is carried out the agarose gel electrophoresis analysis, when PCR product electrophoretogram shows that sample has the band of two different lengthss, should detected sample be the L-type Frankliniella occidentalis; When PCR product electrophoretogram shows that sample has a band, it is G type Frankliniella occidentalis.
2. the method for claim 1, is characterized in that, described pcr amplification system is:
Genomic dna 2 μ l, 20 μ M primer 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, described pcr amplification condition is as follows: 94 ℃, and 5min; Carry out 94 ℃ of 50sec of 35 circulations, 53 ℃ of 40sec, 72 ℃ of 50sec; 72 ℃ are extended 7min; 4 ℃, insulation.
4. the method for claim 1, is characterized in that, in described step (3), EarI endonuclease reaction condition is as follows: 37 ℃, and 2hr.
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| 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 |
| CN103290131B (en) * | 2013-06-18 | 2014-10-08 | 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 |
| 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 |
| CN114317768B (en) * | 2021-12-21 | 2023-04-07 | 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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| CN101805782A (en) * | 2009-08-05 | 2010-08-18 | 云南出入境检验检疫局检验检疫技术中心 | Method for fast identifying frankliniella occidentalis |
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Non-Patent Citations (3)
| Title |
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| A molecular identification key for economically important thrips species (Thysanoptera: Thripidae) using direct sequencing and a PCR‐RFLP‐based approach;PC Brunner et al;《Agricultural and Forest Entomology》;20021231;第4卷(第2期);127-136 * |
| 西花蓟马(Frankliniella occidentalis) rDNA ITS2和COI基因5 末端序列的克隆与比较分析;武晓云等;《浙江大学学报(农业与生命科学版)》;20090430;第35卷(第4期);355-364 * |
| 鹅MC4R基因RFLP及其与胴体和;陈宏权等;《畜牧兽医学报》;20081231;第39卷(第7期);全文 * |
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