CN111269968A - PCR-RFLP-based method for rapidly identifying Spodoptera frugiperda - Google Patents
PCR-RFLP-based method for rapidly identifying Spodoptera frugiperda Download PDFInfo
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Abstract
The invention discloses a PCR-RFLP-based method for rapidly identifying Spodoptera frugiperda. According to the invention, through analyzing the differences of spodoptera frugiperda and other related species on the COI sequence and the differences of two genotypes of spodoptera frugiperda on the Tpi sequence, specific SNP sites of spodoptera frugiperda and the genotypes of the spodoptera frugiperda are obtained, and corresponding restriction endonucleases are designed aiming at the SNP sites. The rapid identification of spodoptera frugiperda and the biotype thereof is realized by adopting a polymerase chain reaction-based restriction map diversity analysis technology (PCR-RFLP). Provides a new convenient method for grassland spodoptera littoralis identification, prediction and prevention and control for grassland spodoptera littoralis technicians and related agricultural researchers.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a PCR-RFLP-based method for rapidly identifying Spodoptera frugiperda.
Background
Spodoptera frugiperda (Spodoptera frugiperda) is a thrilling agricultural pest native to south America and is listed as an important inspection and quarantine object in China. It has strong migratory flight ability and diffusion ability. The method is firstly found in Yunnan of China in 2019 in 1 month. Less than a year, the pest has spread to 29 provinces. At present, Spodoptera frugiperda is mainly harmful to corn in China, and can cause 15-73% of yield reduction of crops. Furthermore, it has been found that the pest can also be harmful to wheat, soybean, sugar cane, and the like. Spodoptera frugiperda can also be a serious rice hazard in the United states and Africa. Therefore, the pest also brings potential serious threat to rice production in China.
Spodoptera frugiperda is seriously harmful to corn in China at present, is highly similar to other lepidoptera pests such as prodenia litura, beet armyworm, armyworm and the like frequently occurring to corn, and brings difficulty to the measurement and the prevention of basic agricultural technicians. In addition, spodoptera frugiperda has two distinct biotypes, the maize (Corn strain, type C) and the rice (ricitrain, type R). The former is mainly harmful to crops such as corn, cotton and sorghum, and the latter is mainly harmful to rice, alfalfa, bermudagrass and the like. The two biotypes can hardly be distinguished from each other, but they are differentiated greatly in terms of physiology, ecology, behavior, and the like. For example, corn type has higher resistance to diazinon, carbaryl and other insecticides, while rice type has higher resistance to carbofuran. In addition, maize-type transgenic Bt-transgenic cotton has higher tolerance. Therefore, the need is increasing for accurately identifying the spodoptera frugiperda biotype and carrying out the forecasting and prevention work in a targeted manner.
In the past, DNA barcoding (bar code) method is generally adopted for identifying closely related pests. Namely, the mitochondrial Cytochrome Oxidase (COI) is cloned, then sequencing is carried out, a phylogenetic tree is constructed for cluster analysis, and finally identification is carried out. However, this method relies on extensive sequencing and is not only time consuming but also economical. Furthermore, this method cannot be used for the identification of spodoptera frugiperda biotypes. COI is a mitochondrial gene marker (molecular marker) and belongs to maternal inheritance. Thus, such genetic markers do not allow for accurate identification of progeny of a cross of two biotypes. For this reason, the biotype identification is currently performed mainly using the nuclear gene, triose phosphate isomerase (Tpi). Nagoshi R.N, (2010) developed 10 SNPs on TPi, which were used to distinguish between rice and maize spodoptera frugiperda. This approach was later widely adopted. However, this method is also highly dependent on DNA sequencing, and is also time consuming and uneconomical.
Disclosure of Invention
The first technical problem to be solved by the invention is as follows: how to quickly identify Spodoptera frugiperda and related species thereof.
The second technical problem to be solved by the invention is: how to rapidly identify two biotypes of spodoptera frugiperda.
The technical scheme of the invention is as follows: a PCR-RFLP-based method for rapidly distinguishing Spodoptera frugiperda and its kindreds comprises the following steps:
(1) extracting the genomic DNA of the material to be identified;
(2) performing PCR amplification by taking P1 and P2 as primers and the genomic DNA obtained in the step (1) as a template to obtain a COI gene fragment with the size of 697 bp; the nucleotide sequence of P1 is shown as SEQ ID No.1, and the nucleotide sequence of P2 is shown as SEQ ID No. 2;
(3) carrying out two rounds of enzyme digestion on the COI gene fragment obtained in the step (2):
1) in the first round, double enzyme digestion is carried out on the COI gene fragment obtained in the step (2) by adopting Tail and AlWN I, agarose gel electrophoresis is carried out, and the electrophoresis result is photographed and analyzed: meanwhile, the DNA fragment has 347bp, 263bp and 87bp bands and is spodoptera frugiperda and prodenia litura, the 350bp band is spodoptera exigua, and the 697bp band is mythimna separata;
2) and (2) in the second round, carrying out enzyme digestion and agarose gel electrophoresis on the COI gene fragment obtained in the step (2) by adopting BstY I, and carrying out photographing and analysis on an electrophoresis result: the spodoptera frugiperda has 587bp and 110bp bands, the armyworm has 300bp and 110bp bands, and the spodoptera litura has 697bp bands.
Further, in step (2), the PCR amplification program parameters are set as follows: 95 ℃ for 5 min; followed by 35 cycles of amplification (95 ℃, 30 s; 54 ℃, 30 s; 72 ℃, 40 s); and finally, supplementary extension is carried out for 10min at 72 ℃.
Further, in the step (3), the method for performing double enzyme digestion on the COI gene fragment obtained in the step (2) by adopting Tail and AlWN I comprises the following steps: firstly adding AlWN I, and reacting for 1 hour at 37 ℃; then, Tail was added thereto, and the reaction was carried out at 65 ℃ for 1 hour.
By the method, prodenia litura, spodoptera exigua, armyworm and spodoptera frugiperda can be distinguished, but the biotype of spodoptera frugiperda cannot be determined.
Therefore, the invention also provides a PCR-RFLP-based method for rapidly distinguishing spodoptera frugiperda biotypes, the previous steps are consistent with the technical scheme of the PCR-RFLP-based method for rapidly distinguishing spodoptera frugiperda and the relatives thereof, after identifying that the material to be identified is spodoptera frugiperda, the Tpi gene fragment is further subjected to PCR amplification, then the enzyme digestion is carried out by Ban I, and the biotype can be analyzed from the band. The method comprises the following specific steps:
(1) extracting the genomic DNA of the material to be identified;
(2) carrying out PCR amplification by taking P3 and P4 as primers to obtain a Tpi gene fragment with the size of 638 bp; the nucleotide sequence of P3 is shown as SEQ ID No.3, and the nucleotide sequence of P4 is shown as SEQ ID No. 4;
(3) performing Ban I enzyme digestion and agarose gel electrophoresis on the Tpi gene fragment obtained in the step (2), and photographing and analyzing an electrophoresis result: the spodoptera frugiperda with the 638bp band is rice spodoptera frugiperda, and the spodoptera frugiperda with the 370bp and 268bp bands is corn spodoptera frugiperda.
Further, in step (2), the PCR amplification program parameters are set as follows: 95 ℃ for 5 min; then 35 cycles of amplification are carried out at 95 ℃ for 30 s; at 54 ℃ for 30 s; 72 ℃ for 40 s; and finally, supplementary extension is carried out for 10min at 72 ℃.
Further, in the step (3), the Tpi gene fragment obtained in the step (2) was digested with Ban I at 60 ℃ for 1 hour.
Compared with the prior art, the invention has the following beneficial effects:
the method can not only rapidly distinguish spodoptera frugiperda and its kindreds (prodenia litura, spodoptera exigua and armyworm), but also distinguish spodoptera frugiperda's biotype (rice spodoptera frugiperda and corn spodoptera frugiperda), has the advantages of time saving and economy, and provides a new convenient method for carrying out spodoptera frugiperda identification, prediction and prevention and control by basic agricultural technicians and relevant agricultural researchers.
Drawings
FIG. 1 PCR amplification of the COI gene;
FIG. 2 shows a cleavage map of Spodoptera frugiperda and its cognate species COI, wherein (A) the first round of cleavage and identification and (B) the second round of cleavage and identification are performed;
FIG. 3 PCR amplification of the Tpi gene;
FIG. 4 shows Tpi gene restriction maps of two biotypes of Spodoptera frugiperda, wherein R type is rice type and C type is maize type.
Detailed Description
Example 1
Genomic DNA of spodoptera frugiperda and three related species (prodenia litura, spodoptera exigua and armyworm) and two biotypes (R type and C type) of spodoptera frugiperda are extracted.
1) Two biotype individuals, namely spodoptera frugiperda, prodenia litura, spodoptera exigua and armyworm, and spodoptera frugiperda, are adopted from fields in different places and soaked in 100% ethanol for storage;
2) an appropriate amount of tissue mass was taken and 1ml of 2% CTAB extract (2% CTAB formulation: 100mmol/L of LTris-HClpH7.0, 20mmol/L of EDTA, 1.4mol/L of NaCl) and then transferring 700. mu.L of the homogenate to a new centrifuge tube, and carrying out water bath at 65 ℃ for 45 min;
3) add 700. mu.L of chloroform: shaking the isoamyl alcohol (volume is 24: 1) up and down for 2-3 min;
4) centrifuging at 12000 Xg for 10 min;
5) taking about 500 mu L of supernatant, transferring the supernatant into a new centrifuge tube, adding 1/10 volumes of NaAc (3mol/L, pH5.2), and turning upside down to mix evenly;
6) adding 2 times of ice-cold absolute ethyl alcohol, and turning upside down and mixing uniformly; standing at-20 deg.C for more than 1 h;
7) centrifuging at 12000 Xg for 10min, and discarding the supernatant; washing the precipitate with 70% ethanol twice;
8) after ethanol volatilizes, adding 50-100 mu of LTE buffer solution (containing 50 mu g/ml RNase) and fully dissolving;
9) electrophoresis is carried out to detect the integrity of the genome DNA;
10) detecting the concentration of the genome DNA by using Nano drop;
11) storing at-20 deg.C or-80 deg.C.
Secondly, PCR amplification of COI gene fragments;
1) diluting genome DNA of Spodoptera frugiperda and related species by 10 times, and using the diluted genome DNA as a PCR template;
2) with P1 (nucleic acid sequence: 5'-ATTCAACAAATCATAAAGATATTGG-3' SEQ ID No.1) and P2 (nucleic acid sequence: 5'-TGTCCAAAAAATCAAAATAAATG-3' SEQ ID No.2) as a primer, and carrying out PCR amplification on the COI gene segment. The PCR program parameters were set as follows: 95 ℃ for 5 min; followed by 35 cycles of amplification (95 ℃, 30 s; 54 ℃, 30 s; 72 ℃, 40 s); and finally, supplementary extension is carried out for 10min at 72 ℃. The PCR product size was 697bp (FIG. 1);
and (III) adopting Tail, AlWN I and BstY I to carry out enzyme digestion on the COI fragment, thereby identifying the Spodoptera frugiperda and the related species thereof.
1) The PCR product of COI was digested in two rounds. The first round of double digestion with Tail and AlWN I: firstly adding AlWN I into a PCR product, and reacting for 1 hour at 37 ℃; then, Tail was added thereto, and the reaction was carried out at 65 ℃ for 1 hour.
2) And (5) carrying out agarose gel electrophoresis, and photographing and analyzing the electrophoresis result. In the first round of enzyme digestion experiments, Spodoptera frugiperda and Spodoptera litura have the same enzyme digestion patterns (347,263 and 87bp), Spodoptera exigua has an electrophoresis band (about 350bp), armyworm cannot be digested, and the electrophoresis band is 697 bp. This round of cleavage identified spodoptera exigua (a in fig. 2);
3) and carrying out second round enzyme digestion on the PCR product of the COI by adopting BstY I.
4) And (5) carrying out agarose gel electrophoresis, and photographing and analyzing the electrophoresis result. In the second round of the restriction enzyme digestion experiment, Spodoptera frugiperda has two electrophoresis bands (587 and 110 bp); myxoplasma has 2 electrophoretic bands (about 300bp and 110 bp); the prodenia litura can not be cut by BstY I, and the electrophoresis band is 697bp (B in figure 2). This round of enzyme digestion allowed identification of Spodoptera frugiperda, Spodoptera litura and armyworm.
(IV) PCR amplification of Tpi Gene fragments
1) Diluting 10 times of genome DNA of two types of spodoptera frugiperda and using the diluted genome DNA as a PCR template;
2) primer P3 (nucleic acid sequence: 5'-GTGAAATCTCCCCTGCTATG-3' SEQ ID No.3) and P4 (nucleic acid sequence: 5'-GCATGTACGTCTTGAGCCTG-3' SEQ ID No.4) amplified Tpi fragments of both spodoptera frugiperda genotypes. The PCR program parameters were set as follows: 95 ℃ for 5 min; followed by 35 cycles of amplification (95 ℃, 30 s; 54 ℃, 30 s; 72 ℃, 40 s); and finally, supplementary extension is carried out for 10min at 72 ℃. The product size was 638bp (FIG. 3);
(V) adopting Ban I to carry out enzyme digestion on the Tpi fragment, and identifying two biological types of spodoptera frugiperda according to an enzyme digestion map
1) And (3) digesting the PCR product of the Tpi by using Ban I under the reaction conditions: 1 hour at 60 ℃;
2) and (5) carrying out agarose gel electrophoresis, and photographing and analyzing the electrophoresis result. The rice spodoptera frugiperda has 1 electrophoretic band (638bp), and the maize spodoptera frugiperda has 2 electrophoretic bands (370 and 268bp) (FIG. 4).
By the method, Spodoptera frugiperda and relatives thereof (prodenia litura, spodoptera exigua and armyworm) can be quickly distinguished, biotypes of Spodoptera frugiperda (rice Spodoptera frugiperda and corn Spodoptera frugiperda) can be distinguished, time is saved, economy is achieved, and a novel convenient method is provided for basic-level agricultural technicians and related agricultural researchers to identify, predict, forecast and prevent Spodoptera frugiperda.
Sequence listing
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Claims (6)
1. A PCR-RFLP-based method for rapidly distinguishing Spodoptera frugiperda and the relative thereof is characterized by comprising the following steps:
(1) extracting the genomic DNA of the material to be identified;
(2) performing PCR amplification by taking P1 and P2 as primers and the genomic DNA obtained in the step (1) as a template to obtain a COI gene fragment with the size of 697 bp; the nucleotide sequence of P1 is shown as SEQ ID No.1, and the nucleotide sequence of P2 is shown as SEQ ID No. 2;
(3) carrying out two rounds of enzyme digestion on the COI gene fragment obtained in the step (2):
1) in the first round, double enzyme digestion is carried out on the COI gene fragment obtained in the step (2) by adopting Tail and AlWN I, agarose gel electrophoresis is carried out, and the electrophoresis result is photographed and analyzed: simultaneously, segments with 347bp, 263bp and 87bp are spodoptera frugiperda and prodenia litura, 350bp is spodoptera exigua, and 697bp is armyworm;
2) and (2) in the second round, carrying out enzyme digestion and agarose gel electrophoresis on the COI gene fragment obtained in the step (2) by adopting BstY I, and carrying out photographing and analysis on an electrophoresis result: the spodoptera frugiperda with 587bp and 110bp fragments is spodoptera frugiperda, the mythimna frugiperda with 300bp and 110bp fragments is armyworm, and the prodenia litura with 697bp fragments is spodoptera litura.
2. The method of claim 1, wherein in step (2), the PCR amplification program parameters are set as follows: 95 ℃ for 5 min; then 35 cycles of amplification are carried out at 95 ℃ for 30 s; at 54 ℃, 30; 72 ℃ for 40 s; and finally, supplementary extension is carried out for 10min at 72 ℃.
3. The method of claim 1, wherein in the step (3), the method of double digestion of the COI gene fragment obtained in the step (2) by using Tail and AlWN I comprises the following steps: firstly adding AlWN I, and reacting for 1 hour at 37 ℃; then, Tail was added thereto, and the reaction was carried out at 65 ℃ for 1 hour.
4. A PCR-RFLP-based method for rapidly distinguishing Spodoptera frugiperda biotypes is characterized by comprising the following steps:
(1) extracting the genomic DNA of the material to be identified;
(2) performing PCR amplification by taking P3 and P4 as primers and the genomic DNA obtained in the step (1) as a template to obtain a Tpi gene fragment with the size of 638 bp; the nucleotide sequence of P3 is shown as SEQ ID No.3, and the nucleotide sequence of P4 is shown as SEQ ID No. 4;
(3) performing Ban I enzyme digestion and agarose gel electrophoresis on the Tpi gene fragment obtained in the step (2), and photographing and analyzing an electrophoresis result: the spodoptera frugiperda with the 638bp band is rice spodoptera frugiperda, and the spodoptera frugiperda with the 370bp and 268bp bands is corn spodoptera frugiperda.
5. The method of claim 4, wherein in step (2), the PCR amplification program parameters are set as follows: 95 ℃ for 5 min; then 35 cycles of amplification are carried out at 95 ℃ for 30 s; at 54 ℃ for 30 s; 72 ℃ for 40 s; and finally, supplementary extension is carried out for 10min at 72 ℃.
6. The method according to claim 4, wherein in the step (3), the Tpi gene fragment obtained in the step (2) is digested with BanI under the condition of 60 ℃ for 1 hour.
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| CN117089632A (en) * | 2023-10-19 | 2023-11-21 | 中国热带农业科学院三亚研究院 | Sequence combination for rapidly detecting spodoptera frugiperda based on CRISPR/Cas12a-RPA and application thereof |
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| CN113308552B (en) * | 2021-06-10 | 2023-04-07 | 四川大学 | Spodoptera frugiperda species specific primer pair, kit and identification method |
| CN117089632A (en) * | 2023-10-19 | 2023-11-21 | 中国热带农业科学院三亚研究院 | Sequence combination for rapidly detecting spodoptera frugiperda based on CRISPR/Cas12a-RPA and application thereof |
| CN117089632B (en) * | 2023-10-19 | 2024-05-14 | 中国热带农业科学院三亚研究院 | Sequence combination for rapidly detecting spodoptera frugiperda based on CRISPR/Cas12a-RPA and application thereof |
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