CN105255883B - A kind of PCR primer differentiating carpocapsa pononella clone and discrimination method thereof - Google Patents
A kind of PCR primer differentiating carpocapsa pononella clone and discrimination method thereof Download PDFInfo
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- CN105255883B CN105255883B CN201510814414.2A CN201510814414A CN105255883B CN 105255883 B CN105255883 B CN 105255883B CN 201510814414 A CN201510814414 A CN 201510814414A CN 105255883 B CN105255883 B CN 105255883B
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Abstract
The present invention provides a kind of PCR primer differentiating carpocapsa pononella clone and discrimination method thereof.The present invention expands carpocapsa pononella and the PCR primer pair of other several insect cell line COI Genes, and its forward primer sequence is SEQ ID NO:1, and reverse primer sequences is SEQ ID NO:2.Forward primer sequence after optimization is SEQ ID NO:3, and reverse primer sequences is SEQ ID NO:4.The present invention also provides the discrimination method of a kind of carpocapsa pononella clone.It is different from other several insect cell line COI Gene sequences that the present invention explores carpocapsa pononella clone from molecular level, establishes the discrimination method of the clone such as carpocapsa pononella and Holotrichia parallela, beet armyworm and cabbage looper.
Description
Technical field
The invention belongs to agricultural biological technical field, be specifically related to a kind of PCR primer differentiating carpocapsa pononella clone and
Its discrimination method.
Background technology
Carpocapsa pononella (Cydia pomonella) belongs to Lepidoptera, Olethreutidae, originates in the Europe middle and south.20th century
The fifties invades Xinjiang of China, has invaded the areas such as Xinjiang, Gansu, Ningxia, the Inner Mongol, Heilungkiang at present, has directly threatened China
East apple producing region, constitutes a serious threat to China's Apple Industry, it has also become a kind of important quarantine harmful organisms of China.Grind
Study carefully the biology of carpocapsa pononella and physiology characteristic has great importance for the comprehensive regulation of this worm, but Pest-or disease-free area at home
Raise carpocapsa pononella and there is certain difficulty and potential risk, set up carpocapsa pononella clone and do not result in carpocapsa pononella to protection
The invasion in district, can by cultivate carpocapsa pononella cell carry out above-mentioned correlative study, clone be established as correlative study
Provide an approach convenient, quick.
The foundation of clone and incubation may be changed into the thin of other castes by other cell contaminations
Born of the same parents system or the clone that coexists of multiple variety classes insect cell, this will to the Entomological Physiology of later stage research carpocapsa pononella and
Biological characteristics causes very big impact.And the method for current identification of cell system, such as cell line dna amplified production finger-print
(DAF), randomly amplified polymorphic DNA (RAPD) etc., its qualification result is easily affected by many factors, also exist poor repeatability and
The problems such as sensitivity is low.
Content of the invention
The present invention provides a kind of PCR primer differentiating carpocapsa pononella clone and discrimination method thereof, thus makes up existing skill
The deficiency of art.
Present invention firstly provides a kind of PCR primer pair for detecting the moth-eaten clone of apple, its primer sequence information is as follows:
Forward primer LCO1490:5 '-GGTCAACAAATCATAAAGATATTGG-3 ' (SEQ ID NO:1),
Reverse primer HCO2198:5 '-TAAACTTCAGGGTGACCAAAAAATCA-3 ' (SEQ ID NO:2).
Being optimized above-mentioned primer, the sensitivity making the primer amplification after optimization is higher more preferable with repeatability, excellent
The sequence information of the primer after change is as follows:
Forward primer MU1:5 '-GGTCAACAAATCATAAAGATACTGG-3 ' (SEQ ID No:3),
Reverse primer MD2:5 '-TAAACTTCAGGGTGACCAAATAATA-3 ' (SEQ ID No:4).
The present invention also provides the discrimination method of a kind of carpocapsa pononella clone, and its step is as follows:
1) extract the genomic DNA of sample to be identified, obtain DNA extract；
2) utilize above-mentioned primer to step 1) in DNA extract enter performing PCR amplification；
3) with restriction enzyme Hinf I to step 2) pcr amplification product be digested；
4) by step 3) digestion products enters row agarose gel electrophoresis analysis, when PCR primer electrophoresis pattern shows detected sample
Product are when 320bp and 380bp has band, then sample is defined as carpocapsa pononella clone；
The PCR amplification system of described step (2) is as follows:
The reverse primer solution 1 μ L of the forward primer solution 1 μ L of Genomic DNA solution 1 μ L, 10 μm of ol/L, 10 μm of ol/L,
5U/ μ L Taq enzyme 0.5 μ L, 10 × Taq buffer solution 5 μ L, 2.5mmol/L dNTP 4 μ L, sterilize pure water 37.5 μ L.
Described step 2) PCR amplification condition be:
94 DEG C of denaturations 5 minutes；94 DEG C of denaturation 1 minute, anneal 1 minute for 54 DEG C, and 72 DEG C extend 2 minutes, carry out 35 and follow
Ring；72 DEG C extend 5 minutes.
Described step 3) in restriction enzyme Hinf I endonuclease reaction condition be: 37 DEG C are digested 1 hour.
Restriction enzyme of the present invention is conventional restriction enzyme, for differentiating that carpocapsa pononella clone provides
Easy, stable is digested discrimination method.
The present invention explores carpocapsa pononella clone with other several insects (such as Holotrichia parallela, beet from molecular level
Noctuid and cabbage looper) difference of clone COI Gene sequence, establish carpocapsa pononella and Holotrichia parallela,
The discrimination method of the clone such as beet armyworm and cabbage looper.
Brief description
Fig. 1 is the agarose gel electrophoresis after carpocapsa pononella expands with other several insect cell line COI genetic fragments PCR
Figure；Wherein M, DL5000 DNA Marker；1-5 is carpocapsa pononella ovum, carpocapsa pononella clone, Holotrichia parallela clone, sweet
Dish frugiperda cell system and Trichopllusia ni cell line.
Fig. 2 is the agarose gel electrophoresis figure after a few strain carpocapsa pononella clone COI genetic fragment PCR expands；Wherein M,
DL5000 DNA Marker；1-7, carpocapsa pononella clone Cp-E-4, Cp-E-6, Cp-E-9, Cp-E-10, Cp-E-11, Cp-
E-12 and Cp-E-13.
Fig. 3 is the agarose gel electrophoresis figure after a few strain carpocapsa pononella clone pcr amplification product is digested through Hinf I；Its
Middle M, DL5000 DNA Marker；1-7, carpocapsa pononella clone Cp-E-4, Cp-E-6, Cp-E-9, Cp-E-10, Cp-E-11,
Cp-E-12 and Cp-E-13.
Fig. 4 is carpocapsa pononella with Holotrichia parallela, beet armyworm and Trichopllusia ni cell line pcr amplification product through Hinf I
Agarose gel electrophoresis figure after being digested；Wherein M, DL5000 DNA Marker；1-4, carpocapsa pononella clone, black dull gill gold
Tortoise clone, beet armyworm clone and Trichopllusia ni cell line.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in detail.
Embodiment 1: the amplification of carpocapsa pononella clone and other several insect cell line genome C OI genetic fragments and sequence
Row are analyzed
Applicant analyzes carpocapsa pononella clone COI Gene Partial sequence (SEQ ID No:5) and the apple that amplification obtains
The COI Gene Partial sequence (SEQ ID No:6) of the moth-eaten moth ovum genome amplification of fruit；Carry out Blast comparison in ncbi database,
Utilize DNASTAR software to be analyzed the COI genetic fragment of carpocapsa pononella and other several insects, find carpocapsa pononella
COI gene nucleotide uniformity with Holotrichia parallela is 81.6%, and the COI gene nucleotide uniformity with beet armyworm is
87.9%, the COI gene nucleotide uniformity with cabbage looper is 83.9%, and result shows the COI gene nucleosides of carpocapsa pononella
There is obvious difference with the COI gene nucleotide series of other several insects in acid sequence.
According to the sequencing results, applicant devises following primer:
Forward primer LCO1490:5 '-GGTCAACAAATCATAAAGATATTGG-3 ' (SEQ ID NO:1),
Reverse primer HCO2198:5 '-TAAACTTCAGGGTGACCAAAAAATCA-3 ' (SEQ ID NO:2).
Utilize above-mentioned primer pair, the COI genetic fragment to carpocapsa pononella and other several insects for PRIMER 5.0 software
Sequence is analyzed, and first ensures that this primer can not only expand in carpocapsa pononella clone and carpocapsa pononella ovum genomic DNA
COI Gene, moreover it is possible to expand the line of other several insects (such as Holotrichia parallela, beet armyworm and cabbage looper)
Plastochondria COI genetic fragment；And, the product of amplification guaranteeing, the amplified fragments of carpocapsa pononella COI can be by restriction enzyme
Hinf I is digested (restriction enzyme site is GANTC), and the COI of other insects (such as Holotrichia parallela, beet armyworm and cabbage looper)
Genetic fragment can not be digested by restriction enzyme Hinf I.It is thus possible to effectively distinguish carpocapsa pononella clone, and determine
The clone whether carpocapsa pononella clone is approximated by other genetic backgrounds is polluted.
Utilize the primer of design to detecting, specifically comprise the following steps that
(1) extraction of carpocapsa pononella and Holotrichia parallela, beet armyworm and Trichopllusia ni cell line genomic DNA
To carpocapsa pononella clone Cp-E-10 and Holotrichia parallela clone Hp-E-1, beet armyworm clone Se-1 and
The cell suspension MagExtractor-Genome kit of Trichopllusia ni cell line Tn9-4s carries out the extraction of genomic DNA,
Obtain the Genomic DNA solution of several insect cell line.
(2) the PCR amplification of carpocapsa pononella and Holotrichia parallela, beet armyworm and Trichopllusia ni cell line COI genetic fragment
Respectively with the Genomic DNA solution of carpocapsa pononella, Holotrichia parallela, beet armyworm and Trichopllusia ni cell line as mould
Plate, enters performing PCR amplification with the primer before optimizing, prepares pcr amplification product；
PCR amplification system is:
The Genomic DNA solution of clone: 1 μ L, the forward primer of 10 μm of ol/L: solution 1 μ L, 10 μm of ol/L reversely draw
Thing solution: 1 μ L, 5U/ μ L Taq enzyme: 0.5 μ L, 10 × Taq buffer solution: 5 μ L, 2.5mmol/L dNTP:4 μ L, sterilize pure water:
37.5μL。
PCR amplification condition is:
94 DEG C of denaturations 5 minutes；94 DEG C of denaturation 1 minute, anneal 1 minute for 54 DEG C, and 72 DEG C extend 2 minutes, carry out 35 and follow
Ring；72 DEG C extend 5 minutes.
The pcr amplification product that step (2) is prepared by the agarose gel electrophoresis with 1.0% detects.
Though the mitochondrial COI gene piece of carpocapsa pononella and other several insect cell lines can be expanded with the primer before optimization
Section, but the phenomenon that amplified band difference in brightness is relatively big, band is fuzzy occurs, sometimes do not have amplified production to occur, there is amplification
The problem that effect and repeatability are not sufficiently stable.
Therefore, being optimized above-mentioned primer, the primer sequence information after optimization is as follows:
Forward primer MU1:5 '-GGTCAACAAATCATAAAGATACTGG-3 '；
Reverse primer MD2:5 '-TAAACTTCAGGGTGACCAAATAATA-3 '；
Owing to two primer annealing temperature after optimizing are close, significantly reduce with primer interdimers between primer, draw with this
Thing can not only expand carpocapsa pononella clone and the COI Gene in carpocapsa pononella ovum genomic DNA, moreover it is possible to expands
Increase the COI Gene of other several insects (such as Holotrichia parallela, beet armyworm and cabbage looper), electrophoresis result table
Bright, all detect the band (as shown in Figure 1) at about 700bp for the length, primer amplification band is clear, and brightness is homogeneous, expands
Synergy fruit is good, and repeatability and stability significantly improve.
The PCR-RFLP collection of illustrative plates of embodiment more than 2 strain carpocapsa pononella clone
(1) extraction of a few strain carpocapsa pononella clone genomic DNAs
7 strain carpocapsa pononella clones Cp-E-4 that laboratory is set up, Cp-E-6, Cp-E-9, Cp-E-10, Cp-E-
11st, the cell suspending liquid MagExtractor-Genome kit of Cp-E-12 and Cp-E-13 carries out carrying of genomic DNA
Take, obtain the Genomic DNA solution of each clone of carpocapsa pononella.
(2) the PCR amplification of a few strain carpocapsa pononella clone COI genetic fragments
Enter performing PCR amplification with the Genomic DNA solution of a few strain carpocapsa pononella clones for template respectively, prepare PCR amplification
Product；
PCR amplification system is:
The Genomic DNA solution of carpocapsa pononella clone: 1 μ L, the forward primer of 10 μm of ol/L: solution 1 μ L, 10 μm of ol/L
Reverse primer solution: 1 μ L, 5U/ μ L Taq enzyme: 0.5 μ L, 10 × Taq buffer solution: 5 μ L, 2.5mmol/L dNTP:4 μ L, go out
Bacterium pure water: 37.5 μ L.
Primer sequence is as follows:
Forward primer MU1:5 '-GGTCAACAAATCATAAAGATACTGG-3 '；
Reverse primer MD2:5 '-TAAACTTCAGGGTGACCAAATAATA-3 '；
PCR amplification condition is:
94 DEG C of denaturations 5 minutes；94 DEG C of denaturation 1 minute, anneal 1 minute for 54 DEG C, and 72 DEG C extend 2 minutes, carry out 35 and follow
Ring；72 DEG C extend 5 minutes.
(3) pcr amplification product utilizing restriction enzyme Hinf I to prepare step (2) is digested, and must be digested product
Thing；
It is digested system as follows:
10 × NEB buffer solution 2 μ L, pcr amplification product 5 μ L, Hinf I restriction endonuclease 1 μ L, sterilize pure water 12 μ L.
Endonuclease reaction condition is: 37 DEG C are digested 1 hour.
(4) digestion products preparing step (3) with the agarose gel electrophoresis of 1.2% separates, after EB dyeing
Imaging on ultraviolet gel imaging instrument, observes its polymorphism.Result shows, the PCR-RFLP product of a few strain carpocapsa pononella clones
All having band at 320bp and 380bp, this several strains are detected sample and are carpocapsa pononella clone (as shown in Figure 3), also illustrate
The repeatability of this discrimination method and stability are preferable.
Embodiment 3 carpocapsa pononella clone and the difference of other several insect cell line PCR-RFLP collection of illustrative plates
(1) extraction of carpocapsa pononella and Holotrichia parallela, beet armyworm and cabbage looper genomic DNA
To carpocapsa pononella clone Cp-E-10 and Holotrichia parallela clone Hp-E-1, beet armyworm clone Se-1 and
The cell suspension MagExtractor-Genome kit of Trichopllusia ni cell line Tn9-4s carries out the extraction of genomic DNA,
Obtain carpocapsa pononella and the Genomic DNA solution of Holotrichia parallela, beet armyworm and Trichopllusia ni cell line.
(2) the PCR amplification of carpocapsa pononella and Holotrichia parallela, beet armyworm and cabbage looper COI genetic fragment
Respectively with the Genomic DNA solution of carpocapsa pononella and Holotrichia parallela, beet armyworm and Trichopllusia ni cell line
Genomic DNA solution is that template enters performing PCR amplification, prepares pcr amplification product.
PCR amplification system is:
The Genomic DNA solution of carpocapsa pononella, Holotrichia parallela, beet armyworm and Trichopllusia ni cell line: 1 μ L, 10 μ
The forward primer of mol/L: the reverse primer solution of solution 1 μ L, 10 μm of ol/L: 1 μ L, 5U/ μ L Taq enzyme: 0.5 μ L, 10 × Taq
Buffer solution: 5 μ L, 2.5mmol/L dNTP:4 μ L, sterilize pure water: 37.5 μ L.
Primer sequence is as follows:
Forward primer MU1:5 '-GGTCAACAAATCATAAAGATACTGG-3 '；
Reverse primer MD2:5 '-TAAACTTCAGGGTGACCAAATAATA-3 '；
PCR amplification condition is:
94 DEG C of denaturations 5 minutes；94 DEG C of denaturation 1 minute, anneal 1 minute for 54 DEG C, and 72 DEG C extend 2 minutes, carry out 35 and follow
Ring；72 DEG C extend 5 minutes.
(3) utilize restriction enzyme Hinf I to step (2) prepare pcr amplification product be digested, be digested product
Thing；
It is digested system as follows:
10 × NEB buffer solution 2 μ L, pcr amplification product 5 μ L, Hinf I restriction endonuclease 1 μ L, sterilize pure water 12 μ L.
Endonuclease reaction condition is: 37 DEG C are digested 1 hour.
(4) digestion products preparing step (3) with the agarose gel electrophoresis of 1.2% separates, after EB dyeing
Imaging on ultraviolet gel imaging instrument, observes its polymorphism.Result shows, imaging gel has at 320bp and 380bp 2
During band, detected sample source is in carpocapsa pononella；When electrophoresis pattern shows the band of only about 700bp, then tested test sample
Product derive from other insect cell lines (such as Holotrichia parallela, beet armyworm and cabbage looper) (as shown in Figure 4).
Claims (6)
1. the PCR primer pair detecting carpocapsa pononella clone, it is characterised in that the forward primer of described PCR primer pair
Nucleotides sequence be classified as SEQ ID NO:3, the nucleotides sequence of reverse primer is classified as SEQ ID NO:4.
2. the PCR primer described in claim 1 is to the application in detection carpocapsa pononella clone.
3. the discrimination method of a carpocapsa pononella clone, it is characterised in that described method comprises the steps:
1) extract the genomic DNA of sample to be identified, obtain DNA extract；
2) utilize the PCR primer described in claim 1 to step 1) in DNA extract enter performing PCR amplification；
3) with restriction enzyme Hinf I to step 2) pcr amplification product be digested；
4) by step 3) digestion products enters row agarose gel electrophoresis analysis, when PCR primer electrophoresis pattern display sample exists
When 320bp and 380bp has band, then sample is defined as carpocapsa pononella clone.
4. method as claimed in claim 3, it is characterised in that described step 2) PCR amplification system as follows:
The reverse primer solution 1 μ L, 5U/ μ of the forward primer solution 1 μ L of Genomic DNA solution 1 μ L, 10 μm of ol/L, 10 μm of ol/L
L Taq enzyme 0.5 μ L, 10 × Taq buffer solution 5 μ L, 2.5mmol/L dNTP4 μ L, sterilize pure water 37.5 μ L.
5. method as claimed in claim 3, it is characterised in that described step 2) PCR amplification condition as follows:
94 DEG C of denaturations 5 minutes；94 DEG C of denaturation 1 minute, anneal 1 minute for 54 DEG C, and 72 DEG C extend 2 minutes, carry out 35 circulations；72
DEG C extend 5 minutes.
6. method as claimed in claim 3, it is characterised in that described step 3) in restriction enzyme Hinf I endonuclease reaction
Condition is 37 DEG C and is digested 1 hour.
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| 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 |
| CN110257528B (en) * | 2018-12-21 | 2021-03-26 | 中国农业科学院植物保护研究所 | Codling moth specific SS-COII primer and application thereof |
| CN111321106B (en) * | 2020-03-03 | 2022-07-26 | 青岛农业大学 | Holotrichia parallela cell line and application thereof |
| CN111235088B (en) * | 2020-03-15 | 2022-07-26 | 青岛农业大学 | Holotrichia parallela embryonic cell line and application thereof |
| CN114891788A (en) * | 2022-06-21 | 2022-08-12 | 青岛农业大学 | LAMP method-based codling moth identification primer group, identification method and identification kit |
| CN117568493B (en) * | 2024-01-16 | 2024-04-12 | 苏州良辰生物医药科技有限公司 | Reagent and kit for identifying insect cell line |
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