Molecular markers, screening / identification methods and their applications for genetic resistance to subgroup B avian leukosis virus infection in chickens

Abstract

The present invention discloses a molecular marker, screening / identification method and application of chicken genetic resistance to subgroup B avian leukosis virus infection, the method comprising: extracting genomic DNA of a sample to be tested, amplifying a TVB receptor gene fragment, and then sequencing to determine whether there is a deletion of base G at position 3747 of the TVB receptor gene. The method can quickly and accurately determine whether the test sample is an ALV-B resistant chicken or a susceptible chicken, and can screen breeding materials for breeding chicken varieties / lines resistant to ALV-B infection from Chinese chicken breeds (including local chicken breeds and commercial broiler strains), providing technical support for breeding chicken varieties / lines resistant to ALV-B infection.

Description

Technical Field

[0001] The invention relates to the technical field of poultry disease resistance breeding, and in particular to a molecular marker for chicken genetic resistance to subgroup B avian leukosis virus infection, a screening / identification method and an application thereof. Background Art

[0002] Avian leukosis is a type of immunosuppressive tumor infectious disease caused by avian leukosis virus (ALV). Avian leukosis virus of subgroup B (ALV-B) is the main pathogen causing avian leukosis in chickens in my country. ALV-B infection can cause characteristic tumors in infected chickens and lead to death, resulting in decreased production performance of infected chickens. It can also cause serious immunosuppression in infected chickens and easily lead to other viral diseases such as avian influenza and Newcastle disease, and bacterial diseases such as Escherichia coli and Salmonella, causing huge economic losses to my country's poultry industry. ALV-B is mainly transmitted vertically, and its infection can be amplified from great-grandparent generation (pure line) → grandparent generation → parent generation → commercial generation, with the infection rate increasing by about 5%-20% each generation. One great-grandparent generation (pure line) breeder chicken infected with ALV-B can be transmitted to 240,000 commercial chickens. Avian leukosis has become the biggest disease threatening the safety of my country's poultry breeding industry. At present, there is no commercial vaccine or effective treatment for the disease, and it is mainly controlled through population purification measures. However, the population purification cycle is long (at least 8-10 years) and the cost is high (the purification cost of each breeder chicken is about 500 yuan). After purification, negative chickens are still at risk of re-infection with ALV. It is difficult to maintain the purification effect of avian leukosis, and it will fall into a continuous struggle between pathogens, animals and testers. In recent years, epidemiological surveys and research and development of avian leukosis have found that ALV-B occurs and spreads in different types of chickens such as local chicken breeds, yellow-feathered broilers, white-feathered broilers, and laying hens in my country. The disease has become one of the major diseases threatening the sustainable and healthy development of my country's poultry industry (especially the breeder industry). Therefore, it is urgent to carry out technological innovations to control avian leukosis and develop new strategies that are more suitable for the prevention and control of avian leukosis in my country. Foreign studies have confirmed that studying disease-resistant breeding for avian leukosis from the perspective of host genetic resistance has become an effective strategy for the prevention and control of the disease.

[0003] The chicken tvb receptor gene encodes the Tvb receptor protein, which mediates ALV-B invasion into host cells and subsequent infection. The tvb receptor gene determines the susceptibility or resistance of host cells to ALV-B infection. Its genetic mutation may lead to the complete loss of Tvb receptor protein expression or the expression of a defective Tvb receptor protein that is not suitable as an ALV-B receptor, thereby causing chickens to resist ALV-B infection. Therefore, discovering and identifying molecular markers for chickens to resist ALV-B infection and applying them to screen chicken breeding materials that resist ALV-B infection to cultivate chicken varieties (lines) resistant to B subgroup avian leukosis is the key to achieving disease-resistant breeding for B subgroup avian leukosis. Summary of the invention

[0004] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a molecular marker, screening / identification method and application of chicken resistance to subgroup B avian leukosis virus infection, which can overcome the deficiency that the prior art measures cannot completely control the occurrence and prevalence of subgroup B avian leukosis virus in chickens in my country.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: a method for screening / identifying chickens with genetic resistance to avian leukosis of subgroup B is provided, comprising: extracting genomic DNA of the sample to be tested, amplifying the TVB receptor gene fragment, and then sequencing to determine whether there is a deletion of base G at position 3747 of the TVB receptor gene.

[0006] The above method specifically comprises the following steps:

[0007] Extract the DNA of the sample to be tested, amplify the tvb receptor gene fragment, and sequence it to determine whether the chicken is genetically resistant to ALV-B infection;

[0008] If the 3747th base position of the tvb receptor gene in the test sample has a homozygous deletion G mutation tvb delG / delG , indicating that the individual has a phenotype of resistance to ALV-B infection, then the individual is a chicken genetically resistant to ALV-B infection;

[0009] If there is no G deletion mutation at the 3747th base position of the TVB receptor gene in the test sample, it means that the genotype of the individual is wild-type TVB s1 / s1 , then the individual is an ALV-B susceptible chicken;

[0010] If there is a heterozygous insertion G mutation tvb at the 3747th base position of the tvb receptor gene in the test sample s1 / delG , indicating that the individual is ALV-B susceptible chicken, but the genotype is tvb s1 / delG The next generation produced by the mating of breeding roosters and breeding hens can produce the genotype tvb delG / delG individuals, thereby producing chicken individuals resistant to ALV-B infection.

[0011] On the basis of the above technical solution, the present invention can also be improved as follows:

[0012] Furthermore, the amplification system used in the above amplification process includes: 1 μL DNA template, 2.5 μL 10× buffer, 2 μL dNTPs, 1 μL upstream and downstream detection primers, 0.5 μL KOD-FX, and finally ddH2O is used to make up to 25 μL.

[0013] Furthermore, the amplification program used in the above amplification process is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles; post-extension at 72°C for 5 min, and finally storage at 4°C.

[0014] The above method can be used to breed chicken varieties / lines that are genetically resistant to subgroup B avian leukosis.

[0015] The invention also provides a molecular marker for chicken genetic resistance to infection by subgroup B avian leukosis virus, wherein the molecular marker is a deletion of the base G at position 3747 of the TVB receptor gene.

[0016] The DNA sequence of the tvb receptor gene can be found in GenBank with the accession number NC_052553.1. The base G at position 3747 is missing and is referred to as tvb 3747delG Mutation site.

[0017] The present invention also provides primers for detecting the above molecular markers, and the nucleotide sequence of the primers is as follows:

[0018] Forward primer F: 5'-GAGCCCGAGCAGAGCTGTGT-3' (SEQ ID NO: 1)

[0019] Reverse primer R: 5'-CAGCTTCCAACTCACTCGG-3' (SEQ ID NO: 2).

[0020] The application of the above molecular markers and / or primers in screening / identifying chickens genetically resistant to subgroup B avian leukosis virus infection.

[0021] A kit for detecting / screening chickens with genetic resistance to subgroup B avian leukosis, the kit comprising the above primers.

[0022] The kit is used in screening and breeding chicken breeds / lines that are genetically resistant to subgroup B avian leukosis.

[0023] The present invention has the following beneficial effects:

[0024] The present invention analyzes the genetic variation of the TVB receptor gene in Chinese chicken breeds (including 1998 blood samples from 15 local chicken breeds and 15 yellow-feathered broiler strains) and finds that a natural mutation of the base G deletion at position 3747 of the TVB receptor gene DNA sequence (GenBank accession number: NC_052553.1, see SEQ ID NO: 19 for details) in Chinese chicken breeds, referred to as TVB 3747delG Mutation site.

[0025] Based on the above research, TVB was confirmed from in vitro and in vivo experiments. 3747delGNatural mutations cause chickens to resist infection with ALV-B. The specific reason is that TVB 3747delG The mutation is located in the 4th exon of the tvb receptor gene. The mutation causes a G deletion (tvb c.317delG) at the 317th base of the tvb receptor gene coding sequence CDS (tvb receptor gene mRNA reference sequence NM_204115.3, see SEQ ID NO: 20). The tvb c.317delG mutation causes the encoded Tvb receptor protein sequence (Tvb receptor protein reference sequence NP_989446.2, see SEQ ID NO: 21) to shift the frame starting from the 106th amino acid residue and terminate the coding prematurely at the 130th amino acid residue, resulting in a change in the structure of the cysteine-rich region (CRD) of the Tvb receptor protein (CRD of the Tvb receptor protein is a key structural domain that mediates ALV-B infection and entry into host cells), and expresses a truncated Tvb delG Receptor protein, thereby eliciting host resistance to ALV-B infection.

[0026] The present invention is also based on the molecular marker TVB of chicken genetic resistance to B subgroup avian leukosis virus infection 3747delG The established method for identifying chickens resistant to ALV-B infection can quickly and accurately determine whether the test sample is a chicken genetically resistant to ALV-B or susceptible to ALV-B. It can be used to screen breeding materials for breeding chicken varieties / lines genetically resistant to ALV-B infection from Chinese chicken breeds (including local chicken breeds and commercial broiler strains), providing technical support for the breeding of chicken varieties / lines genetically resistant to ALV-B infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 For our chicken breed TVB 3747delG The results of discovery and identification of mutation sites; among them, Figure 1 A is the PCR amplification result of the complete gene sequence of the tvb receptor gene of Chinese chicken breeds, M: 2000 marker, NC is the negative control; 1-5 represent the PCR amplification results of the gene fragments of tvb gene 1, 2, 3, 4, 5 respectively; Figure 1 B is a schematic diagram of the structure of the tvb receptor gene; Figure 1 C red circle is tvb 3747delG At the mutation position of the TVB gene DNA sequence, it was shown that a G deletion mutation occurred at base 3747 of exon 4 of the TVB receptor gene.

[0028] Figure 2 For TVB 3747delG Results of sequence analysis of the coding region of the tvb gene at the mutation site.

[0029] Figure 3 The expression results of mCherry-tvb fusion protein in 293-FT cells.

[0030] Figure 4 This is the construction of RCASBP(B)-EGFP recombinant expression plasmid and the virus rescue results.

[0031] Figure 5 Infection of tvb with RCASBP(B)-EGFP 3747delG The process of CEF of different genotypes at the mutation site. DETAILED DESCRIPTION

[0032] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0033] Example 1: tvb 3747delG Discovery and identification of mutation sites

[0034] 1. Design of primers for amplification of TVB receptor gene

[0035] With reference to the DNA sequence of the chicken tvb receptor gene in the NCBI database (GenBank accession number: NC_052553.1, see SEQ ID NO: 19 for details), 5 pairs of primers were designed to PCR amplify the full-length sequence of the tvb gene 6596 bp (segment 1, segment 2, segment 3, segment 4 and segment 5) in 5 segments. The primer sequences, positions and PCR amplification fragment sizes are shown in Table 1.

[0036] Table 1 Primer information for PCR amplification of the full-length sequence of the tvb receptor gene

[0037]

[0038] 2. PCR amplification

[0039] Referring to the instructions of the Tiangen Blood DNA Extraction Kit, genomic DNA from 1998 blood samples of different chicken breeds in my country (including 15 local chicken breeds and 15 yellow-feathered broiler strains) was extracted, and the full-length sequence of the TVB receptor gene was amplified by PCR using the five pairs of primers. The PCR amplification system and amplification procedure are shown in Tables 2 and 3, respectively.

[0040] Table 2 PCR amplification system

[0041]

[0042] Table 3 PCR amplification system

[0043]

[0044] The PCR amplification products were detected by 2% agarose gel electrophoresis. Figure 1 A, by Figure 1 A shows that PCR amplified five fragments of 1079bp, 1254bp, 1530bp, 1239bp and 1451bp, and the band sizes were consistent with the target fragment sizes.

[0045] 3. Sequencing analysis

[0046] The tvb receptor gene amplification product was sent to Shanghai Bioengineering Co., Ltd. for purification and sequencing. DNAstar and Mutation Surveyor gene sequence analysis software were used to compare the sequences and analyze the genetic variation of the tvb receptor gene in Chinese chicken breeds to screen candidate genetic resistance sites for ALV-B.

[0047] By analyzing the genetic variation of the TVB receptor gene in 15 local chicken breeds and 15 yellow-feathered broiler strains (a total of 1998 blood samples), we screened and found that the G deletion at base 3747 of exon 4 of the TVB gene in Chinese chicken breeds (TVB 3747delG )'s natural mutation ( Figure 1 B) Figure 1 As shown in C, the sequence sequencing maps are tvb from top to bottom. 3747delG Mutation site wild type tvb s1 / s1 Individual, heterozygous mutant tvb s1 / delG Individuals and homozygous mutant tvb delG / delG Individual sequence, the red line indicates the G deletion mutation at base 3747 of the tvb receptor gene sequence.

[0048] Example 2: tvb 3747delG Sequence analysis of the coding region of the tvb gene at the mutation site

[0049] 1. RNA extraction and reverse transcription

[0050] Collection TVB 3747delG Mutation site wild type tvb s1 / s1 and homozygous mutant tvb delG / delG200μL of blood from the breeder chicken was mixed with 800μL TRIZOL (RNase Free EP tube) by oscillation, fully lysed, and allowed to stand at room temperature for 5 minutes. Then, 300μL of chloroform was added, mixed by oscillation, and centrifuged at 4℃, 12,000×g for 15 minutes. The colorless and transparent supernatant on the upper layer was aspirated to a new EP tube, and an equal volume of isopropanol solution was added to precipitate RNA. The tube was allowed to stand for 10 minutes; the tube was centrifuged at 4℃, 12,000×g for 30 minutes, the supernatant was discarded, and the precipitate was washed once with 75% ethanol, the supernatant was discarded, and the tube was allowed to stand at room temperature. After all the ethanol evaporated, 50μL DEPC water was added to dissolve the RNA. 3747delG Mutation site wild type tvb s1 / s1 and homozygous mutant tvb delG / delG The RNA from the blood of the breeder chicken was used as a template and reverse transcribed into cDNA according to the instructions of the reverse transcription kit (Takara). The reverse transcribed cDNA was stored at -20°C for future use.

[0051] 2. RT-PCR amplification of the full-length coding region sequence of the tvb gene

[0052] (1) Primer design

[0053] Referring to the mRNA sequence of the tvb receptor gene published by NCBI (NM_204115.3), primers for amplifying the entire coding region sequence (CDS) of tvb were designed. The primer sequences and the sizes of the RT-PCR amplified fragments are shown in Table 4.

[0054] Table 4 Primer information for RT-PCR amplification of the full-length coding region sequence of the tvb receptor gene

[0055]

[0056] (2) RT-PCR amplification

[0057] TVB 3747delG Mutation site wild type tvb s1 / s1 and homozygous mutant tvb delG / delG The cDNA of the breeder chicken blood sample was used as a template, and RT-PCR amplification was performed according to the instructions of the high-fidelity enzyme KOD-FX produced by Toyobo Company. The amplification system and amplification program are shown in Tables 5 and 6, respectively.

[0058] Table 5 RT-PCR amplification system

[0059]

[0060] Table 6 RT-PCR amplification system

[0061]

[0062] After the above RT-PCR amplification was completed, the amplified product was detected by 1% agarose gel electrophoresis.

[0063] 3. Cloning and sequencing analysis of RT-PCR amplification products

[0064] Refer to the instructions of Axygen's gel recovery and purification kit to recover and purify the RT-PCR product. Refer to the instructions of pMD19-T Vector (Takara) to connect the RT-PCR purified product into the pMD19-T vector. Refer to the instructions of competent cells DH5α to transform the ligation product into DH5α competent cells. Screen positive colonies and perform PCR identification. The bacterial liquid identified as positive by PCR was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Lasergene7.0 software was used to compare and analyze tvb 3747delG Mutation site wild type tvb s1 / s1 and homozygous mutant tvb delG / delG The results of sequencing the full-length coding region of the tvb gene are shown in Figure 2 . Figure 2 A is wild type tvb s1 / s1 , homozygous mutant genotype tvb delG / delG RT-PCR amplification of the full-length coding region sequence of the tvb gene from chicken blood samples, M: 2000Marker; Figure 2 B is the partial nucleotide and deduced amino acid sequence of exon 4 around the G single nucleotide deletion. The G single nucleotide deletion replaces tvb r7 Different from TVB s1 (Numbering starts from the first methionine codon). The red line marks tvb 3747delG The G nucleotide deletion and altered amino acid sequence at the mutation site; Figure 2 C for TVb S1 and TVB delG Schematic representation of the receptor protein. S1 The protein numbering includes the signal peptide (SP), cysteine-rich domains (CRD1-3), transmembrane domain (MSD), and cytoplasmic death domain. delG The position of the frameshift mutation is highlighted with an asterisk, whereas in Tvb delG The premature stop codon TGA is indicated by a triangle. Figure 2 D is the deduced amino acid sequence compared with the known Tvb receptor CRD. S1 Disulfide bonds in CRDs, Tvb delG The putative cysteine ​​substitutions in Tvb are circled. delG The amino acid residues of the frameshift peptide are shown in bold.

[0065] Depend on Figure 2 A: It can be seen that with tvb 3747delG Mutation site wild type tvb s1 / s1 and homozygous mutant tvb delG / delG The cDNA of breeder chickens was used as template to amplify tvb by RT-PCR. 3747delG Mutation site wild type tvb s1 / s1 and homozygous mutant tvb delG / delG The full-length coding region sequence (CDS) band of the tvb gene was found, and the size of the band was consistent with the size of the target fragment.

[0066] The results of cloning and sequencing of RT-PCR amplification products showed that: delG / delG The cDNA has a G deletion at position 317 (tvb c.317delG) ( Figure 2 B) tvb delG / delG The G base of the cDNA sequence is missing, resulting in the Tvb delG The receptor protein sequence starts frameshifting at position 106 AA and terminates prematurely at position 130 AA ( Figure 2 C) Predict TVB 3747delG The frameshift mutation caused by the mutation causes the cysteine ​​in the cysteine-rich region 3 (CRD3) of the Tvb receptor protein to be replaced by other amino acid residues, resulting in the breakage of the disulfide bond of CRD3, thereby changing the Tvb delG The structure of the receptor protein Figure 2 D).

[0067] Example 3: tvb 3747delG Mutation causes tvb gene to express truncated Tvb delG Receptor protein

[0068] 1.pmCherry-C1-tvb s1 / s1 、pmCherry-C1-tvb delG / delG Construction of recombinant expression plasmid

[0069] (1) RT-PCR amplification of the full-length CDS fragment of the tvb receptor gene mutation site

[0070] According to the RT-PCR amplification primers of the full-length coding region sequence of the tvb receptor gene, XhoI and HindIII restriction sites (underlined sequences) and protective bases (as shown in Table 7) were added respectively, and RT-PCR amplification of tvb was performed according to the method of Example 2. 3215-3216insCC Mutation site wild type tvb s1 / s1 and homozygous mutant tvb delG / delG The full-length coding region sequence of the TVB receptor gene.

[0071] Table 7 RT-PCR primers for the full-length CDS of the tvb receptor gene

[0072]

[0073] (2) Purification and ligation of PCR products

[0074] Refer to the instructions of Axygen's gel recovery and purification kit to recover and purify the RT-PCR amplification product and connect it to the pMD19-T vector.

[0075] (3) Transformation of ligation products into competent cells

[0076] Refer to the instructions of the competent cell DH5α and transform the ligation product into DH5α competent cells.

[0077] (4) Double enzyme digestion reaction

[0078] Will contain tvb 3747delG Mutation site wild type tvb s1 / s1 and homozygous mutant tvb delG / delG The pMD19-T and pmcherry-C1 plasmids containing the full-length coding region sequence fragment of the tvb gene were double-digested with XhoI / HindIII, respectively.

[0079] (5) Purification and ligation of enzyme digestion products

[0080] After the target fragment and pmcherry-C1 vector were recovered and purified according to the instructions of Axygen's gel recovery and purification kit, the target fragment and pmcherry-C1 vector were mixed at a ratio of 3:1, centrifuged, and placed at 16°C for connection overnight.

[0081] (6) Screening and identification of positive colonies

[0082] A single white colony was picked from the culture plate and inoculated into 500 μL Kana LB liquid culture medium. After shaking culture at 37°C for about 5 h, PCR identification was performed directly using the bacterial liquid as a template and the colony was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0083] (7) Enzyme digestion identification of pmCherry-C1-tvb recombinant plasmid

[0084] Refer to the instructions of the plasmid small-scale extraction kit (Adlai) to extract the recombinant plasmid of the positive bacterial solution. The pmCherry-C1-tvb recombinant plasmid was double-digested with endonucleases XhoI and HindIII for identification, and the recombinant plasmid was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. The results are shown in Figure 3 .Depend on Figure 3 A shows that the recombinant expression plasmid was successfully constructed. The constructed recombinant plasmids were named pmCherry-C1-tvbs1 / s1 and pEGFPC1-tvb delG / delG .

[0085] 2. Western Blotting Detection of pmCherry-C1-tvb Recombinant Expression Plasmid

[0086] Refer to the instructions of Lipofectamine 3000 Reagent and use Lipofectamine 3000 transfection reagent to transfect pmCherry-C1 control plasmid and pmCherry-C1-tvb s1 / s1 , pEGFPC1-tvb delG / delG Transfected into 293FT cells (6-well plate) prepared in advance to 70-80% confluent monolayer, and observed red fluorescence after 24 hours. 48 hours after transfection, collected 293FT cell supernatant for Western Blotting detection, the results are shown in Figure 3 .

[0087] Depend on Figure 3 B shows that 24 hours after transfection, the transfected pmCherry-C1 and pmCherry-C1-tvb were detected under an inverted fluorescence microscope. s1 / s1 、pmCherry-C1-tvb delG / delG The 293-FT cells with the recombinant plasmid emitted red fluorescence, indicating that the mCherry-Tvb fusion protein was expressed in 293-FT cells. 48 hours after transfection, the cell supernatant samples were collected for Western Blotting. The results showed that the blank control plasmid pmCherry-C1 expressed a protein of about 27 kD in 293-FT cells, and pmCherry-C1-tvb s1 / s1 mCherry-Tvb expressed in 293-FT cells is about 70 kD s1 Fusion protein, pmCherry-C1-tvb delG / delG The recombinant plasmid expressed mCherry-Tvb of approximately 41 kD in 293-FT cells delG Fusion protein ( Figure 3 C), the results show that tvb 3747delG Mutation causes the tvb receptor gene to express a truncated Tvb delG Receptor protein.

[0088] Example 4: tvb 3747delG Identification of mutations that confer host resistance to ALV-B infection

[0089] 1. In vitro cell infection verification

[0090] (1) Constructing RCASBP(B)-EGFP recombinant expression plasmid, transfecting it into DF-1 cells, and 7 days after transfection, rescuing and collecting RCASBP(B)-EGFP virus (i.e., ALV-B reporter virus carrying EGFP fluorescent protein) in the supernatant of DF-1 cells ( Figure 4 , wherein, A: schematic diagram of the construction of RCASBP(B)-EGFP expression plasmid; B: restriction enzyme digestion identification of RCASBP(B)-EGFP plasmid; C: RCASBP(B)-EGFP virus rescue), after determining the virus infection unit (IU), the cells were aliquoted and stored at -80°C.

[0091] (2) Using ALV-B fluorescent reporter virus RCASBP(B)-EGFP to infect tvb 3747delG Mutation site wild type tvb s1 / s1 , heterozygous mutant tvb s1 / delG and homozygous mutant tvb delG / delG CEF (chicken fibroblasts CEF were prepared from 9-day-old chicken embryos hatched from the breeder chickens detected in Example 1) and RCASBP(B)-EGFP virus infection was detected by flow cytometry 1, 2, 3, and 7 days after infection. 3747delG The situation of CEF with different genotypes at the mutation site. The GPF-positive cell rate (%) indicates the infection rate of the virus. The specific results are shown in Figure 5 .

[0092] Figure 5 A: RCASBP (B)-EGFP infected tvb s1 / s1 、tvb s1 / delG 、tvb delG / delG The time course of CEFs; Figure 5 B is flow cytometry detection of RCASBP(B)-EGFP infected tvb 3747delG Representative graph of GFP-positive cell rates in CEFs of different genotypes at the mutation site after 7 days. Figure 5 It can be seen that RCASBP(B)-EGFP can efficiently infect wild-type tvb s1 / s1 CEFs, the infection rate was about 20% on day 1, and almost all cells were infected by day 7 ( Figure 5 A). RCASBP(B)-EGFP is positive for tvb delG / delG The infection efficiency of CEFs was significantly reduced, with about 3% of cells infected on the first day, and the spread was slow, with about 11% of cells infected on the third day and reaching 18% on the seventh day. Figure 5 A). With TVB delG / delG In comparison, TVB s1 / delG CEFs were more sensitive to infection with RCASBP(B)-EGFP and showed infection characteristics similar to those of the wild type ( Figure 5 A). GFP-negative and GFP-positive cells are clearly different, and there are two independent groups of GFP-positive and GFP-negative cells in the FACS scatter plot ( Figure 5 B). The results show that tvb 3747delG The mutation renders host cells resistant to infection with RCASBP(B)-EGFP in vitro.

[0093] 2. In vivo challenge test verification

[0094] (1) TVB 3747delG One-day-old chicks with wild-type, heterozygous and homozygous mutations at the mutation site were randomly divided into groups of 25 per group and kept in an isolator. At one day old, each chick was intraperitoneally inoculated with 0.5 mL of ALV-B SDAU09C2 virus solution (S / P value = 2.2), and challenged again at 5 days old. Two weeks after the challenge, anticoagulated blood samples were collected from each chick, and genomic DNA was extracted. The tvb of each chick was sequenced by direct sequencing. 3747delG One month after infection, blood samples were collected from the chickens, and total RNA from the blood samples was extracted using the TRIZOL kit. The ALV-BSDAU09C2 strain-specific detection primers were used to detect the viremia of each chicken by RT-PCR to confirm that the ALV-BSDAU09C2 strain was infected with tvb 3747delG The status of chicks with different genotypes of mutations.

[0095] Design upstream and downstream primers for RT-PCR amplification of ALV-B-env:

[0096] env-F: 5'-GCAGGCATTTCTGACTGGGC-3' (SEQ ID NO: 17);

[0097] env-R: 5'-AGACCTTCCGATAGGTGAGG-3' (SEQ ID NO: 18).

[0098] (2) RT-PCR amplified the coding sequence of the env gene of ALV-B. The length of the RT-PCR amplified fragment was 435 bp. One Step RT-PCR Kit Ver.2 was used for RT-PCR amplification. The PCR reaction procedure was as follows: 50°C reverse transcription for 30 min; 94°C for 30 s, 58°C for 30 s, 72°C for 45 s, 35 cycles; 72°C post-extension for 10 min. The PCR product was detected by 2% agarose gel electrophoresis. If a 435 bp target band was observed, the sample had viremia (ALV-B positive). If no target band was amplified, the sample did not have viremia (ALV-B negative). The results are shown in Table 8.

[0099] Table 8 ALV-B infection tvb 3747delG Positive viremia in chickens with different genotypes at mutation sites

[0100]

[0101] As shown in Table 8, 26 1-day-old wild-type tvb s1 / s1 After the chicks were inoculated with the ALV-B SDAU09C2 strain virus solution, all of them were ALV-B positive. delG / delG After the genotype chicks were inoculated with the ALV-B SDAU09C2 strain virus liquid, the ALV-B positive rate dropped to 7.4%. s1 / delG The ALV-B positive rate of chicks inoculated with ALV-B SDAU09C2 strain virus liquid was 90.9% (Table 8). The results of the challenge test showed that the results of ALV-B infection in vivo and in vitro were consistent, confirming that tvb 3747delG Mutation causes host chickens to resist infection with ALV-B, tvb 3747delG Natural mutations as molecular markers of chicken resistance to ALV-B infection.

[0102] Example 5: Establishment and application of identification criteria for chickens resistant to ALV-B infection

[0103] 1. Establishment of identification standards for chickens resistant to ALV-B infection

[0104] (1) Primer design

[0105] Referring to the DNA sequence of the tvb receptor gene (GenBank accession number: NC_052553.1), PCR primers (forward primer F: 5'-GAGCCCGAGCAGAGCTGTGT-3' (SEQ ID NO: 1); reverse primer R: 5'-CAGCTTCCAACTCACTCGG-3' (SEQ ID NO: 2)) were designed to amplify the tvb receptor gene. 3747delG The genomic region of the TVB receptor gene containing the mutation site.

[0106] (2) TVB 3747delG PCR amplification of the genomic region of the TVB receptor gene at the mutation site

[0107] The PCR reaction system consisted of: 1 μL DNA template, 2.5 μL 10× buffer, 2 μL dNTPs, 1 μL upstream and downstream detection primers (whose nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2), 0.5 μL KOD-FX, and ddH2O to 25 μL.

[0108] PCR reaction program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles; post-extension at 72°C for 5 min, and finally storage at 4°C.

[0109] (3) Sequencing of PCR amplification products

[0110] The PCR amplification products were detected by 2% agarose gel electrophoresis and sent to Shanghai Biotech Co., Ltd. for purification and sequencing. 3747delG The mutation sites were genotyped.

[0111] According to tvb 3747delG The mutation site genotype was used to establish the identification criteria for chickens resistant to ALV-B infection. The criteria are shown in Table 9.

[0112] Table 9 Identification criteria for chickens resistant to ALV-B infection

[0113]

[0114] If tvb 3747delG The resistance locus genotype is wild type tvb s1 / s1 , then it is not resistant (susceptible) to ALV-B infection, and the individual is an ALV-B susceptible chicken;

[0115] If tvb 3747delG The resistance locus genotype is tvb s1 / delG , then the individual is susceptible to ALV-B infection, but the individual carries a recessive gene for ALV-B genetic resistance;

[0116] If tvb 3747delG The resistance locus genotype is tvb delG / delG , then it develops genetic resistance to ALV-B infection, and the individual is an ALV-B infection-resistant chicken.

[0117] 2. Application of the identification standard for chickens resistant to ALV-B infection

[0118] (1) Whole genome DNA extraction

[0119] According to the instructions of the Tiangen Blood Genome Extraction Kit, DNA was extracted from a total of 1998 samples of anticoagulated blood from 15 local Chinese chicken breeds and 15 yellow-feathered broiler chicken strains, and stored at -20°C for future use.

[0120] (2) PCR amplification and sequencing

[0121] PCR amplification primers and amplification procedures are as described in Example 5 and 1, and different chicken breeds in my country are tvb 3747delGThe genomic region of the tvb gene at the resistance site was amplified by PCR, and the PCR amplification product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing after being qualified by 2% agarose gel electrophoresis.

[0122] (3) TVB 3747delG Resistance locus genotyping

[0123] Chromas software was used to analyze the sequencing peak graph of the sequencing results and the sequencing samples TVB 3747delG Genotype of the resistance locus.

[0124] my country's local chicken breeds and yellow-feathered broiler strains TVB 3747delG The genotyping results of the resistance loci are shown in Table 10. The local chicken breeds such as Beijing Oil Chicken, Ningdu Yellow Chicken, and Tibetan Chicken have TVB 3747delG The resistance genotype of the resistance locus is tvb delG / delG , with frequencies of 0.17, 0.08 and 0.15 respectively. Yellow-feathered broiler strain 2, yellow-feathered broiler strain 6, yellow-feathered broiler strain 10, yellow-feathered broiler strain 19 and yellow-feathered broiler strain 20 also have tvb 3747delG The resistance genotype of the resistance locus is tvb delG / delG , with frequencies of 0.13, 0.15, 0.13, 0.17 and 0.06, respectively, indicating that these local chicken breeds and yellow-feathered broiler strains have good potential for genetic improvement of resistance to ALV-B. Breeding materials with genetic resistance to ALV-B infection can be screened out from these chicken breeds and used to breed chicken breeds (strains) with genetic resistance to ALV-B infection to prevent and control subgroup B avian leukosis.

[0125] Table 10 my country's chicken breeds TVB 3747delG Genotype frequency distribution of resistance loci

[0126]

[0127]

[0128] All nucleotide sequences in this application are shown in Table 11 (wherein SEQ ID NO: 19 is not recorded here):

[0129] Table 11 Sequence Listing

[0130]

[0131] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of primers for detecting molecular markers of chicken genetic resistance to subgroup B avian leukosis virus infection in the preparation of a reagent for screening or identifying chickens with genetic resistance to subgroup B avian leukosis, characterized in that: The molecular marker is tvb The deletion of the base G at position 3747 of the receptor gene, tvb The receptor gene sequence is shown in SEQ ID NO: 19; If the sample is tested tvb There is a homozygous deletion G mutation at base position 3747 of the receptor gene tvb delG / delG , then the individual is a chicken genetically resistant to subgroup B avian leukosis virus infection; If the sample is tested tvb If there is no G deletion mutation at base position 3747 of the receptor gene, the individual is a chicken susceptible to subgroup B avian leukosis virus infection; If the sample is tested tvb There is a heterozygous insertion G mutation at base position 3747 of the receptor gene tvb s1 / delG , then the individual is a chicken susceptible to infection by subgroup B avian leukosis virus, but it carries a recessive gene for genetic resistance to subgroup B avian leukosis virus.

2. The use according to claim 1, characterized in that: The primer sequences are as follows: Forward primer F: 5′-GAGCCCGAGCAGAGCTGTGT-3′; Reverse primer R: 5′-CAGCTTCCAACTCACTCGG-3′.

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