Primer pair for identifying single female lines of bemisia tabaci of C-strains and C + strains and method for identifying single female lines of bemisia tabaci of C-strains and C + strains

Abstract

The invention discloses a primer pair for identifying single female lines of bemisia tabaci of C-and C + strains and a method for identifying single female lines of bemisia tabaci of C-and C + strains, and belongs to the technical field of agricultural biological detection. The sequence of the primer pair for identifying the single female line of the bemisia tabaci of the C-line and the C + line is as shown in SEQ ID NO.1 and SEQ ID NO.2. According to the invention, a primer pair for amplifying a differential base sequence is developed according to the differential base of the mitochondrial rrnL gene in the mtDNA of the bemisia tabaci of C-and C + strains, and then a method for identifying the single female line of the bemisia tabaci based on PCR-RFLP is developed, so that different strains of the bemisia tabaci can be rapidly, accurately and intuitively identified, and the identification efficiency of the single female line of the bemisia tabaci is improved. And a foundation is laid for the identification of the heat-resistant single female line of the bemisia tabaci, the population dynamic identification of the bemisia tabaci and the research on biology and invasion mechanisms.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural biological detection, and particularly relates to a primer pair for identifying single female lines of C- and C+ strains of Bemisia tabaci and a method for identifying single female lines of C- and C+ strains of Bemisia tabaci. Background Art

[0002] Bemisia tabaci is a global agricultural pest, and there are significant differences in biological characteristics, host adaptability, and drug resistance between its C- and C+ strains. Accurately identifying these two strains is of great significance for the prevention and control of Bemisia tabaci, drug resistance management, and ecological research. However, traditional morphological methods are difficult to distinguish between C- and C+ strains, so molecular biology and genetics techniques need to be used for accurate identification.

[0003] Currently, the methods for identifying C- and C+ strains of Bemisia tabaci mainly include PCR, AFLP, DNA sequencing, SDS-PAGE, Western Blot, etc. Among them, although the PCR method is simple to operate and low in cost, it depends on the design of specific primers and may have false positive or false negative results; AFLP has high resolution, but is complex to operate and high in cost; the results of DNA sequencing technology are accurate and reliable, but the data analysis is complex and the cost is high; although SDS-PAGE can visually display the detection results, its resolution is limited and it is difficult to distinguish highly similar proteins; Western Blot depends on the quality and specificity of antibodies.

[0004] In summary, the existing methods for identifying C- and C+ strains of Bemisia tabaci have defects such as complex operation, high cost, insufficient result stability, and insufficient specificity, and there is an urgent need to develop a method for identifying C- and C+ strains of Bemisia tabaci that is simple to operate, low in cost, stable in results, and high in specificity. Summary of the Invention

[0005] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a primer pair for identifying single female lines of C- and C+ strains of Bemisia tabaci and a method for identifying single female lines of C- and C+ strains of Bemisia tabaci.

[0006] To achieve the above purpose, the present invention adopts the following technical scheme:

[0007] A primer pair for identifying single female lines of C- and C+ strains of Bemisia tabaci, and the sequences of the primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2.

[0008] On the basis of the above scheme, the Bemisia tabaci is the Q-type Bemisia tabaci.

[0009] A method for identifying single female lines of Bemisia tabaci C- and C+ strains. Extract the genomic DNA of the tested Bemisia tabaci as a template, perform PCR amplification using the above-mentioned primer pair, digest the PCR amplification product with a restriction endonuclease, and perform agarose gel electrophoresis on the digested product to detect the length and number of bands of the fragments; the restriction endonuclease is BsmBI.

[0010] On the basis of the above scheme, the Bemisia tabaci is the Q-type Bemisia tabaci.

[0011] On the basis of the above scheme, an agarose gel electrophoresis pattern showing a band with a fragment length of 1278 bp indicates that the tested Bemisia tabaci is the C- strain; an agarose gel electrophoresis pattern showing two bands of 797 bp and 486 bp indicates that the tested Bemisia tabaci is the C+ strain.

[0012] On the basis of the above scheme, the amplification system for the PCR amplification is: 2 μL of genomic DNA solution, 1 μL of each of the 10 μM sense and antisense primers, 12.5 μL of Premix Taq, and made up to 25 μL with DEPC water.

[0013] On the basis of the above scheme, the reaction conditions for the PCR amplification are: pre-denaturation at 95 °C for 2 minutes; denaturation at 98 °C for 10 seconds, annealing at 60 °C for 30 seconds, extension at 72 °C for 60 seconds, for 35 cycles; extension at 72 °C for 2 minutes.

[0014] On the basis of the above scheme, the digestion conditions for the restriction endonuclease are: 55 °C, 60 minutes.

[0015] Advantages of the technical solution of the present invention

[0016] The present invention developed a primer pair for amplifying a sequence containing the differential bases based on the differential bases of the mitochondrial rrnL gene in the mtDNA of Bemisia tabaci C- and C+ strains. Using the genomic DNA of the tested Bemisia tabaci as a template, perform PCR amplification with this primer pair, digest the amplification product with a restriction endonuclease, and perform agarose gel electrophoresis to detect the length and number of bands of the digested fragments; thus achieving the purpose of identifying single female lines of Bemisia tabaci C- and C+ strains.

[0017] The method for identifying single female lines of Bemisia tabaci based on PCR-RFLP of the present invention can quickly, accurately and intuitively identify different strains of Bemisia tabaci, laying a foundation for the identification of single female lines with heat tolerance of Bemisia tabaci, the identification of population dynamics of Bemisia tabaci, and the research on biology and invasion mechanism. Description of the drawings

[0018] Figure 1It is the agarose gel electrophoresis diagram of the PCR product before digestion with enzymes and after digestion with BsmBI in Example 3 (where M: Marker 2000, and from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp in turn);

[0019] Figure 2 It is the agarose gel electrophoresis diagram of the PCR product after digestion with BsmBI in Example 4 (where the sample loading wells 1-5 are the C-strain of Bemisia tabaci in Shouguang area, Shandong Province, and 6-10 are the C+ strain of Bemisia tabaci in Shouguang area, Shandong Province);

[0020] Figure 3 It is the agarose gel electrophoresis diagram of the PCR product after digestion with BsmBI in Example 5 (where the sample loading wells 1-5 are the C-strain of Bemisia tabaci in Lingshui area, Hainan Province, and 6-10 are the C+ strain of Bemisia tabaci in Lingshui area, Hainan Province). Detailed implementation mode

[0021] The terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified. The present invention will be further described in detail below with reference to specific examples and data. The following examples are only for illustrating the present invention and do not limit the scope of the present invention in any way.

[0022] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The test materials, reagents, drugs, etc. used in the following examples can be obtained through general channels without special instructions.

[0023] In the following examples, the BsmBI endonuclease (also known as Esp3I) is purchased from Yugong Biotech Co., Ltd., Premix Taq (ExTaq) is purchased from Takara Co., Ltd., the autoclaved STE (TNE) buffer solution (pH 8.0) is purchased from Solarbio Co., Ltd., Proteinase K is purchased from Aikery Biotech Co., Ltd., and other reagent consumables are all ordinary commercially available products.

[0024] The Bemisia tabaci in the following examples is the Q-type Bemisia tabaci (MED cryptic species).

[0025] Example 1

[0026] Obtaining of differential sites of the rrnL gene of different strains (C- and C+) of Bemisia tabaci

[0027] (1) Bemisia tabaci were collected from Shouguang (SG) in Shandong Province and Lingshui (LS) in Hainan Province respectively. 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.], the above-mentioned Bemisia tabaci were detected respectively, divided into Bemisia tabaci C-strain and C+ strain, and according to the method described therein, single-female lines C+ and C- with the same genetic background were constructed for the Bemisia tabaci collected from 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 mitochondrial genomes of the two strains (C- and C+) of Bemisia tabaci established by the above method, the molecular software SnapGene 5.2 was used to align the mitochondrial genome sequences of the two strains. It was found that there is a recognition site of a restriction endonuclease in the differential sequence of the rrnL gene of the two strains. In the C+ strain, it is the recognition site of the restriction endonuclease BsmBI (restriction site 5’-CGTCTCN^-3’, 3’-GCAGAGNNNNN^-5’).

[0029] Example 2

[0030] A primer pair for identifying single-female lines of C- and C+ strains of Bemisia tabaci has the following sequences:

[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 single-female lines of C- and C+ strains of Bemisia tabaci comprises the following steps:

[0035] (1) Extract the genomic DNA of Bemisia tabaci

[0036] Place a single-headed female Bemisia tabaci in a 0.2 mL centrifuge tube containing 30 μL of alkaline lysis solution. The alkaline lysis solution is a high-pressure sterilized STE (TNE) buffer (pH 8.0): Proteinase K = 35:2 (volume ratio). After thoroughly grinding and homogenizing with a sealed pipette tip, place it in a PCR instrument at 65 °C for 15 min; after 95 °C for 10 min, a Bemisia tabaci genomic DNA solution is prepared.

[0037] (2) PCR amplification of a partial fragment of the Bemisia tabaci rrnL gene

[0038] Using the genomic DNA solutions of Bemisia tabaci from Lingshui, Hainan C-strain (LSC-), Shouguang, Shandong C-strain (SGC-), Lingshui, Hainan C+ strain (LSC+), and Shouguang, Shandong C+ strain (SGC+) as templates for PCR amplification to obtain PCR amplification products;

[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 is: 2 μL of genomic DNA solution, 1 μL each of 10 μM forward and reverse primers, 12.5 μL of Premix Taq (Ex Taq), and made up to 25 μL with DEPC water;

[0042] PCR amplification conditions: Pre-denaturation at 95 °C for 2 minutes; denaturation at 98 °C for 10 seconds, annealing at 60 °C for 30 seconds, extension at 72 °C for 60 seconds, for 35 cycles; extension at 72 °C for 2 minutes; 4 °C, ∞.

[0043] (3) Detect the PCR amplification products prepared in step (2) by agarose gel electrophoresis (as shown in Figure 1 lanes 1-4).

[0044] Sequence the above PCR amplification products. The sequences of the PCR amplification products of the Lingshui, Hainan C-strain and the Shouguang, Shandong C-strain are as shown in SEQ ID NO.3, and the sequences of the PCR amplification products of the Lingshui, Hainan C+ strain and the Shouguang, Shandong C+ strain are 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) Digest the PCR amplification product obtained in step (3) with the restriction endonuclease BsmBI to obtain a digested product.

[0050] Digestion system: 2 μL of 10X HN Buffer, 5 μL of PCR amplification product, 1 μL of BsmBI endonuclease, and made up to 20 μL with DEPC water.

[0051] Reaction conditions: Place in a PCR instrument at 55 °C for 60 minutes.

[0052] (5) Separate the digested product obtained in step (4) by agarose gel electrophoresis, image it on a UV gel imager, and observe its polymorphism.

[0053] The results showed that the digestion results of the restriction endonuclease BsmBI showed that there was a band with a fragment length of 1278 bp on the imaging films of the Hainan Lingshui C-strain (LSC-) and the Shandong Shouguang C-strain (SGC-). The electrophoresis patterns of the Hainan Lingshui C+ strain (LSC+) and the Shandong Shouguang C+ strain (SGC+) showed two bands of 797 bp and 486 bp (including sticky end bases) for the tested samples (as Figure 1 shown in lanes 6-9).

[0054] Example 4

[0055] A method for identifying single female lines of Bemisia tabaci C- and C+ strains, the steps are as follows:

[0056] (1) Extract the genomic DNA of Bemisia tabaci

[0057] In the two single female lines of Shandong Shouguang C- and C+ established in the laboratory, randomly take 5 female Bemisia tabaci and place them in a 0.2 mL centrifuge tube containing 30 μL of alkaline lysis solution. The alkaline lysis solution is a high-pressure sterilized STE (TNE) buffer (pH 8.0): Proteinase K = 35:2 (volume ratio). After thoroughly grinding and homogenizing with a sealing gun head, place it in a PCR instrument at 65 °C for 15 min; after 95 °C for 10 min, a genomic DNA solution of Bemisia tabaci is obtained.

[0058] (2) PCR amplify a partial fragment of the rrnL gene of Bemisia tabaci

[0059] Respectively use the genomic DNA solutions of Bemisia tabaci of the Shandong Shouguang C-strain (SGC-) and the Shandong Shouguang C+ strain (SGC+) as templates for PCR amplification to obtain PCR amplification products;

[0060] Forward primer: 5’-TCGCCCGTCATTCTTACA-3’ (SEQ ID NO.1);

[0061] Antisense primer: 5'-GCGCTGTTATCCCTTAGGTAACT-3' (SEQ ID NO.2).

[0062] The PCR amplification system is as follows: 2 μL of genomic DNA solution, 1 μL each of 10 μM sense and antisense primers, 12.5 μL of Premix Taq (Ex Taq), and made up to 25 μL with DEPC water;

[0063] PCR amplification conditions: Pre-denaturation at 95°C for 2 minutes; denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 60 seconds, for 35 cycles; extension at 72°C for 2 minutes; 4°C, ∞.

[0064] (3) Digest the PCR amplification product obtained in step (2) with the restriction endonuclease BsmBI to obtain a digested product;

[0065] Digestion system: 2 μL of 10X HN Buffer, 5 μL of PCR amplification product, 1 μL of BsmBI endonuclease, and made up to 20 μL with DEPC water.

[0066] Reaction conditions: Incubate in a PCR instrument at 55°C for 60 minutes.

[0067] (4) Separate the digested product obtained in step (3) by agarose gel electrophoresis, image it on a UV gel imager, and observe its polymorphism.

[0068] The results showed that there was a band with a fragment length of 1278 bp on the imaging film of sample wells 1 - 5 for the Shandong Shouguang C-strain; the electrophoresis pattern of sample wells 6 - 10 for the Shandong Shouguang C+ strain showed two bands of 797 bp and 486 bp (including sticky end bases) for the tested sample (as Figure 2 ).

[0069] Example 5

[0070] A method for identifying single female lines of Bemisia tabaci C- and C+ strains, the steps are as follows:

[0071] Using two single female lines of Hainan Lingshui C- and C+ established in the laboratory as detection samples, extract their genomic DNA; the detection method is the same as in Example 4.

[0072] Image the agarose gel electrophoresis results on a UV gel imager, showing that there was a band with a fragment length of 1278 bp on the imaging film of sample wells 1 - 5 for the Hainan Lingshui C- strain; the electrophoresis pattern of sample wells 6 - 10 for the Hainan Lingshui C+ strain showed two bands of 797 bp and 486 bp (including sticky end bases) for the tested sample (as Figure 3 ).

[0073] As described above, it is only the preferred embodiment of the present invention, and it is not intended to limit the present invention in other forms. Any person skilled in the art may use the technical content disclosed above to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A primer pair for distinguishing C- and C+ strains of Bemisia tabaci monogyne, characterized in that: The primer pair sequences are shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. Use of the primer pair according to claim 1 in identifying the monogynous line of Bemisia tabaci, characterized in that: The whitefly is a Q-type whitefly.

3. A method for distinguishing C- and C+ strains of Bemisia tabaci monogyne, characterized in that: Extract the genomic DNA of the whitefly to be tested as a template, use the primer pair of claim 1 for PCR amplification, use restriction endonuclease to digest the PCR amplification product, and perform agarose gel electrophoresis to detect the length of the fragment and the number of bands of the product obtained by digestion; the restriction endonuclease is BsmBI.

4. The method for distinguishing C- and C+ strains of Bemisia tabaci monogyne according to claim 3, characterized in that: The whitefly is a Q-type whitefly.

5. The method for distinguishing C- and C+ strains of Bemisia tabaci monogyne according to claim 4, characterized in that: The agarose gel electrophoresis pattern showed a band with a fragment length of 1278 bp, indicating that the tested whitefly was a C- strain; the agarose gel electrophoresis pattern showed two bands of 797 bp and 486 bp, indicating that the tested whitefly was a C+ strain.

6. The method for distinguishing C- and C+ strains of Bemisia tabaci monogyne according to any one of claims 3 to 5, characterized in that: The amplification system of the PCR amplification is: 2 μL of genomic DNA solution, 1 μL of 10 μM forward and antisense primers, 12.5 μL of Premix Taq, and DEPC water to make up to 25 μL.

7. The method for distinguishing C- and C+ strains of Bemisia tabaci monogyne according to claim 6, characterized in that: The reaction conditions of the PCR amplification are: pre-denaturation at 95°C for 2 minutes; denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 60 seconds, for 35 cycles; and extension at 72°C for 2 minutes.

8. The method for distinguishing C- and C+ strains of Bemisia tabaci monogyne according to claim 8, characterized in that: The restriction endonuclease digestion conditions are: 55° C., 60 minutes.

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