Primer pair for identifying c- and c+ strain of b. tabaci single female line and method for identifying c- and c+ strain of b. tabaci single female line
By combining PCR amplification and restriction endonuclease digestion with agarose gel electrophoresis, the problem of identifying C- and C+ strains of whiteflies in existing technologies has been solved, enabling rapid, accurate, and low-cost strain identification and supporting population dynamics research of whiteflies.
Patent Information
- Application Number
- CN202510264544.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-07
- Publication Date
- 2025-11-07
- Estimated Expiration
- 2045-03-07
AI Technical Summary
Existing technologies are insufficient for quickly, accurately, and cost-effectively identifying C- and C+ strains of whiteflies, and suffer from problems such as complex operation, high cost, insufficient result stability, and insufficient specificity.
PCR amplification was performed using specific primer pairs, followed by restriction endonuclease digestion with BsmBI and agarose gel electrophoresis to detect the length and number of enzyme-digested fragments, in order to identify mono-female C- and C+ whitefly strains.
This method enables rapid, accurate, and intuitive identification of different strains of whiteflies, laying the foundation for the identification of heat-resistant mono-female strains of whiteflies and the study of population dynamics.
Smart Images
Figure CN120060488B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of agricultural biological detection, and particularly relates to a primer pair for identifying C- and C+ strain Bemisia tabaci monogynous lines and a method for identifying C- and C+ strain Bemisia tabaci monogynous lines. BACKGROUND
[0002] Bemisia tabaci is a global agricultural pest, and the C- and C+ strains of Bemisia tabaci have significant differences in biological characteristics, host adaptability and drug resistance. Accurate identification of the two strains is of great significance for the prevention and control of Bemisia tabaci, drug resistance management and ecological research. However, the traditional morphological method is difficult to distinguish the C- and C+ strains, so it is necessary to use molecular biology and genetic technology for accurate identification.
[0003] At present, the methods for identifying C- and C+ strains of Bemisia tabaci mainly include PCR, AFLP, DNA sequencing, SDS-PAGE and Western Blot. Among them, the PCR method is simple and low in cost, but it depends on the design of specific primers, and may have false positive or false negative results; the AFLP has high resolution, but the operation is complex and the cost is high; the DNA sequencing technology has accurate and reliable results, but the data analysis is complex and the cost is high; the SDS-PAGE can directly show the detection results, but the resolution is limited and it is difficult to distinguish highly similar proteins; the Western Blot depends on the quality and specificity of the antibody.
[0004] In summary, the existing methods for identifying C- and C+ strains of Bemisia tabaci have the defects of complex operation, high cost, insufficient stability of results, insufficient specificity and the like, and it is urgent to develop a method for identifying C- and C+ strains of Bemisia tabaci which is simple in operation, low in cost, stable in results and high in specificity. SUMMARY
[0005] In view of the problems in the prior art, the purpose of the present application is to provide a primer pair for identifying C- and C+ strain Bemisia tabaci monogynous lines and a method for identifying C- and C+ strain Bemisia tabaci monogynous lines.
[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:
[0007] A primer pair for identifying C- and C+ strain Bemisia tabaci monogynous lines, the sequence of the primer pair is shown in SEQ ID NO. 1 and SEQ ID NO. 2.
[0008] On the basis of the above scheme, the Bemisia tabaci is Q type Bemisia tabaci.
[0009] The application discloses a method for identifying C- and C+ strain Bemisia tabaci single gynogenesis, extracts genomic DNA of the Bemisia tabaci to be detected as a template, performs PCR amplification on the template by using the primer pair, performs enzyme cutting on the PCR amplification product by using a restriction endonuclease, and performs agarose gel electrophoresis on the product obtained by the enzyme cutting to detect the length and the band number of the segment; and the restriction endonuclease is BsmBI.
[0010] On the basis of the above scheme, the Bemisia tabaci is Q type Bemisia tabaci.
[0011] On the basis of the above scheme, the agarose gel electrophoresis spectrum shows one band with a segment length of 1278bp, indicating that the Bemisia tabaci to be detected is C- strain; and the agarose gel electrophoresis spectrum shows two bands with segment lengths of 797bp and 486bp, indicating that the Bemisia tabaci to be detected is C+ strain.
[0012] On the basis of the above scheme, the amplification system of the PCR amplification is as follows: 2ul of genomic DNA solution, 1ul of each of 10um positive and negative sense primers, 12.5ul of premix Taq, and DEPC water is added to 25ul.
[0013] On the basis of the above scheme, the reaction condition of the PCR amplification is as follows: 95 DEG C pre-denaturation for 2 minutes; 98 DEG C denaturation for 10 seconds, 60 DEG C annealing for 30 seconds, 72 DEG C extension for 60 seconds, 35 cycles; and 72 DEG C extension for 2 minutes.
[0014] On the basis of the above scheme, the enzyme cutting condition of the restriction endonuclease is as follows: 55 DEG C, 60 minutes.
[0015] Advantages of the technical scheme of the application
[0016] According to the different bases of the mitochondrial rrnL gene in the C- and C+ strain Bemisia tabaci mtDNA, the application develops a primer pair containing the different base sequences, takes the genomic DNA of the Bemisia tabaci to be detected as a template, performs PCR amplification on the template by using the primer pair, performs enzyme cutting on the amplification product by using a restriction endonuclease, and performs agarose gel electrophoresis on the enzyme cutting segment to detect the length and the band number of the segment, so that the purpose of identifying the C- and C+ strain Bemisia tabaci single gynogenesis is achieved.
[0017] The method for identifying the Bemisia tabaci single gynogenesis based on PCR-RFLP can quickly, accurately and intuitively identify different strains of the Bemisia tabaci, and lays a foundation for identification of the heat-resistant Bemisia tabaci single gynogenesis, population dynamic identification of the Bemisia tabaci, biological research and research on an invasion mechanism. BRIEF DESCRIPTION OF DRAWINGS
[0018] Figure 1is an agarose gel electrophoresis chart of PCR products before and after BsmBI enzyme digestion in Example 3 (wherein M: Marker 2000, from top to bottom, 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp);
[0019] Figure 2 is an agarose gel electrophoresis chart of PCR products after BsmBI enzyme digestion in Example 4 (wherein sample wells 1-5 are Bemisia tabaci C- strain in Shouguang region of Shandong Province, and 6-10 are Bemisia tabaci C+ strain in Shouguang region of Shandong Province);
[0020] Figure 3 is an agarose gel electrophoresis chart of PCR products after BsmBI enzyme digestion in Example 5 (wherein sample wells 1-5 are Bemisia tabaci C- strain in Lingshui region of Hainan Province, and 6-10 are Bemisia tabaci C+ strain in Lingshui region of Hainan Province). DETAILED DESCRIPTION
[0021] The terms used in the present application have the meanings generally understood by those of ordinary skill in the art, unless otherwise specified. The present application is described in further detail below in conjunction with specific examples and with reference to the data. The following examples are merely intended to illustrate the present application, and in no way limit the scope of the present application.
[0022] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The experimental materials, reagents, and drugs used in the following examples can be purchased through general channels, unless otherwise specified.
[0023] In the following examples, the BsmBI endonuclease (also known as Esp3I) was purchased from Yugong Bio, the Premix Taq (ExTaq) was purchased from Takara, the autoclaved STE (TNE) buffer (pH 8.0) was purchased from Solarbio, the Proteinase K was purchased from Aikangrui, and other reagents and consumables were all commercially available products.
[0024] The whitefly in the following examples is Q-type whitefly (MED cryptic species).
[0025] Example 1
[0026] Obtaining of different strain (C- and C+) Bemisia tabaci rrnL gene difference sites
[0027] (1) Whiteflies were collected from Shouguang (SG) in Shandong Province and Lingshui (LS) in Hainan Province, respectively, and detected according to the method described in [Li H, Wei X, Ding T, Chu D. Genome-Wide Profiling of Cardinium-Responsive MicroRNAs in the Exotic Whitefly, Bemisia tabaci (Gennadius) Biotype Q. Front Physiol. 2018 Nov 12; 9: 1580. doi: 10.3389 / fphys.2018.01580. PMID: 30483149; PMCID: PMC6241202.], and divided into C- and C+ strains, respectively. According to the method described therein, single female lines C+ and C- with the same genetic background were constructed from whiteflies collected in Shouguang (SG) in Shandong Province and Lingshui (LS) in Hainan Province, respectively. These two strains have significant differences in biological characteristics, such as high temperature tolerance.
[0028] (2) Based on the two strains (C- and C+) of whitefly mitochondrial genome established by the above method, the sequences of the mitochondrial genomes of the two strains were aligned using molecular software SnapGene 5.2, and it was found that there was a restriction enzyme recognition site in the difference sequence of the rrnL gene of the two strains. In the C+ strain, it is the recognition site of restriction enzyme BsmBI (enzyme cleavage site 5'-CGTCTCN-3', 3'-GCAGAGNNNNN-5').
[0029] Example 2
[0030] A primer pair for identifying C- and C+ strain whitefly single female lines, the sequence is as follows:
[0031] Forward primer: 5'-TCGCCCGTCATTCTTACA-3' (SEQ ID NO. 1);
[0032] Reverse primer: 5'-GCGCTGTTATCCCTTAGGTAACT-3' (SEQ ID NO. 2).
[0033] Example 3
[0034] A method for identifying C- and C+ strain whitefly single female lines, the steps are as follows:
[0035] (1) Extract whitefly genomic DNA
[0036] Single female whitefly was placed in a 0.2 mL centrifuge tube containing 30 μL alkaline lysis solution, which was autoclaved STE (TNE) buffer (pH 8.0): Proteinase K = 35:2 (volume ratio). After the homogenate was ground thoroughly with a sealing gun head, it was placed in a PCR instrument at 65°C for 15 min and at 95°C for 10 min to obtain a whitefly genomic DNA solution.
[0037] (2) PCR amplification of a partial fragment of the whitefly rrnL gene
[0038] The genomic DNA solution of the Hainan Lingshui C- strain (LSC-), the Shandong Shouguang C- strain (SGC-), the Hainan Lingshui C+ strain (LSC+), and the Shandong Shouguang C+ strain (SGC+) of whitefly was used as a template for PCR amplification to obtain a PCR amplification product.
[0039] Forward primer: 5'-TCGCCCGTCATTCTTACA-3' (SEQ ID NO. 1);
[0040] Reverse primer: 5'-GCGCTGTTATCCCTTAGGTAACT-3' (SEQ ID NO. 2).
[0041] The PCR amplification system was as follows: 2 μL of the genomic DNA solution, 1 μL of each of the 10 μM forward and reverse primers, 12.5 μL of Premix Taq (Ex Taq), and DEPC water to make up to 25 μL.
[0042] The PCR amplification conditions were as follows: pre-denaturation at 95°C for 2 min; denaturation at 98°C for 10 s, annealing at 60°C for 30 s, extension at 72°C for 60 s, for 35 cycles; extension at 72°C for 2 min; and 4°C, ∞.
[0043] (3) Detection of the PCR amplification product obtained in step (2) by agarose gel electrophoresis (as shown in lanes 1-4). Figure 1
[0044] The above PCR amplification product was sequenced to obtain the PCR amplification product sequences of the Hainan Lingshui C- strain and the Shandong Shouguang C- strain as shown in SEQ ID NO. 3, and the PCR amplification product sequences of the Hainan Lingshui C+ strain and the Shandong Shouguang C+ strain as shown in SEQ ID NO. 4.
[0045] SEQ ID NO. 3 (5'→3')
[0046]
[0047] SEQ ID NO. 4 (5'→ 3')
[0048]
[0049] (4) Using restriction enzyme BsmBI to cut the PCR product prepared in step (3) to obtain a cut product;
[0050] Cutting system: 10XHN Buffer 2 μL, PCR product 5 μL, BsmBI endonuclease 1 μL, DEPC water to 20 μL.
[0051] Reaction condition: 55°C for 60 minutes in PCR instrument.
[0052] (5) Using agarose gel electrophoresis to separate the cut product prepared in step (4), imaging on ultraviolet gel imaging instrument, and observing polymorphism.
[0053] The results show that the cut result of restriction enzyme BsmBI is that there is a band with a length of 1278 bp on the imaging film of Hainan Lingshui C- strain (LSC-) and Shandong Shouguang C- strain (SGC-), and the electrophoretogram of Hainan Lingshui C+ strain (LSC+) and Shandong Shouguang C+ strain (SGC+) shows that the measured sample has two bands with lengths of 797 bp and 486 bp (including sticky end base) (as shown in lanes 6-9). Figure 1
[0054] Example 4
[0055] A method for identifying C- and C+ strains of Bemisia tabaci single female line, the steps are as follows:
[0056] (1) Extracting Bemisia tabaci genomic DNA
[0057] In the two single female lines of Shandong Shouguang C- and C+ established in the laboratory, 5 female Bemisia tabaci were randomly taken and placed in 0.2 mL centrifuge tubes containing 30 μL alkaline lysis solution, and the alkaline lysis solution was autoclaved STE (TNE) buffer (pH 8.0): Proteinase K = 35:2 (volume ratio). After grinding and homogenizing with a sealing gun head, the solution was placed in a PCR instrument at 65°C for 15 min and at 95°C for 10 min to prepare a Bemisia tabaci genomic DNA solution.
[0058] (2) PCR amplification of part of the Bemisia tabaci rrnL gene
[0059] The genomic DNA solution of Shandong Shouguang C- strain (SGC-) and Shandong Shouguang C+ strain (SGC+) was used as a template for PCR amplification to prepare a PCR product;
[0060] Forward primer: 5'-TCGCCCGTCATTCTTACA-3' (SEQ ID NO. 1);
[0061] Antisense primer: 5'-GCGCTGTTATCCCTTAGGTAACT-3' (SEQ ID NO. 2).
[0062] PCR amplification system: 2 μL of genomic DNA solution, 1 μL of 10 μM sense and antisense primers, 12.5 μL of Premix Taq (Ex Taq), and DEPC water to make up to 25 μL;
[0063] PCR amplification conditions: 2 minutes of pre-denaturation at 95°C; 10 seconds of denaturation at 98°C, 30 seconds of annealing at 60°C, 60 seconds of extension at 72°C, 35 cycles; 2 minutes of extension at 72°C; 4°C, ∞.
[0064] (3) The PCR amplification product obtained in step (2) is digested with restriction enzyme BsmBI to obtain a digested product;
[0065] Digestion system: 2 μL of 10XHN Buffer, 5 μL of PCR amplification product, 1 μL of BsmBI endonuclease, and DEPC water to make up to 20 μL.
[0066] Reaction conditions: 55°C for 60 minutes in a PCR instrument.
[0067] (4) The digested product obtained in step (3) is separated by agarose gel electrophoresis, imaged on an ultraviolet gel imaging instrument, and observed for polymorphism.
[0068] The results show that sample wells 1-5 of the Shouguang C- strain have a band with a length of 1278 bp on the imaged film; sample wells 6-10 of the Shouguang C+ strain have two bands with lengths of 797 bp and 486 bp (including sticky end bases) on the electrophoretogram (as shown in Figure 2 ).
[0069] Example 5
[0070] A method for identifying C- and C+ strains of B. tabaci single females, comprising the following steps:
[0071] The C- and C+ single female strains of B. tabaci established in the laboratory in Lingshui, Hainan are used as detection samples, and their genomic DNA is extracted; the detection method is the same as in Example 4.
[0072] The results of agarose gel electrophoresis imaged on an ultraviolet gel imaging instrument show that sample wells 1-5 of the Lingshui C- strain have a band with a length of 1278 bp on the imaged film; sample wells 6-10 of the Lingshui C+ strain have two bands with lengths of 797 bp and 486 bp (including sticky end bases) on the electrophoretogram (as shown in Figure 3 ).
[0073] The above merely describes preferred embodiments of the present application, but is not intended to limit the present application to other forms, and any person skilled in the art can make changes or modifications to the above disclosed technical contents into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiments without departing from the technical solution content of the present application and according to the technical essence of the present application still belongs to the protection scope of the technical solution of the present application.
Claims
1. Use of primer pairs in discriminating C- and C+ strain Bemisia tabaci monophagous lines, characterized in that, The primer pair sequence is shown as SEQ ID NO. 1 and SEQ ID NO. 2; the primer pair is used for PCR amplification of the sequence containing the C- and C+ strain Bemisia tabaci rrnL gene difference sites, and the C- and C+ strain Bemisia tabaci rrnL gene difference sites are the bases at position 476 of the nucleic acid sequences shown as SEQ ID NO. 3 and SEQ ID NO. 4; and the Bemisia tabaci is Q-type Bemisia tabaci.
2. A method of discriminating C- and C+ strain Bemisia tabaci monophagous lines, characterized in that, Genomic DNA of the to-be-tested Bemisia tabaci is extracted as a template, a primer pair is used for PCR amplification, and a restriction enzyme is used for enzyme digestion of the PCR amplification product; the length and band number of the product obtained by enzyme digestion are detected by agarose gel electrophoresis; the restriction enzyme is BsmBI; the Bemisia tabaci is Q-type Bemisia tabaci; agarose gel electrophoresis shows a band with a length of 1278bp, indicating that the to-be-tested Bemisia tabaci is a C- strain; agarose gel electrophoresis shows two bands with lengths of 797bp and 486bp, indicating that the to-be-tested Bemisia tabaci is a C+ strain; and the primer pair sequence is shown as SEQ ID NO. 1 and SEQ ID NO.
2.
3. The method of claim 2, wherein the C- and C+ strain of B. tabaci monophagous females are identified by, The amplification system of the PCR amplification is: 2µL of genomic DNA solution, 1µL of each of 10µM forward and reverse primers, 12.5µL of Premix Taq, and DEPC water to make up to 25µL.
4. The method of claim 3, wherein the C- and C+ strain of B. tabaci monophagous females are identified by, The reaction conditions of the PCR amplification are: 95℃ pre-denaturation for 2 minutes; 98℃ denaturation for 10 seconds, 60℃ annealing for 30 seconds, 72℃ extension for 60 seconds, 35 cycles; and 72℃ extension for 2 minutes.
5. The method of claim 4, wherein the C- and C+ strain of B. tabaci monophagotrope are identified by, The enzyme digestion conditions of the restriction enzyme are: 55℃, 60 minutes.