A molecular marker for genetic resistance to chicken K subgroup avian leukosis tva546G>C and its application

Abstract

The invention discloses a chicken K subgroup avian leukosis genetic resistance molecular marker TVA 546G>C And its application. The molecular marker is a G>C base mutation at the 546th base of the tva gene DNA sequence (GenBank accession number is AY531262.1). The present invention analyzes the genetic variation of the tva receptor gene of Chinese chicken breeds, and finds that there is a G>C mutation at the 546th base of the tva receptor gene DNA sequence in Chinese chicken breeds, and confirms from in vitro and in vivo experiments that the natural mutation of the tva gene causes the host to resist ALV-K infection, and establishes a corresponding method that can be used for the breeding of K subgroup avian leukosis-resistant chicken breeds.

Description

Technical Field

[0001] The invention belongs to the technical field of poultry disease resistance variety breeding, and specifically relates to a chicken K subgroup avian leukosis genetic resistance molecular marker tva 546G＞C and its applications. Background Art

[0002] Avian Leukosis subgroup K is an immunosuppressive tumor infectious disease of chickens caused by Avian Leukosis Virus subgroup K (ALV-K). ALV-K mainly infects offspring through vertical transmission. Its infection can cause the infected chickens to expand from "great-grandparent generation-grandparent generation-parent generation" generation by generation, and the infection rate increases by about 5%-20% each generation. ALV-K can cause immunosuppression in infected chickens, reduced production performance, and even characteristic tumors and death, causing huge economic losses to the poultry industry.

[0003] To date, there is no commercial vaccine or effective treatment for K subgroup avian leukosis. The disease is mainly controlled by the traditional method of eliminating positive chickens and purifying the breeder flocks at home and abroad. However, the disadvantages of this method are: (1) long purification time: it takes 3-5 generations, or 5-8 years, to purify a breeder flock; (2) high labor intensity: each breeder flock must be tested 4-6 times using ELISA and virus isolation methods for each generation; (3) high cost: the cost of ALV purification and testing for each chicken is about 500 yuan, and my country spends more than 1 billion yuan on avian leukosis purification each year. (4) After purification, negative chicken flocks are still at risk of reinfection with avian leukosis, and the purification effect is prone to recurrence, which easily leads to a continuous struggle between pathogen (ALV-K)-animal-detection personnel. Epidemiological surveys and studies on avian leukosis in recent years have found that ALV-K is common in local chicken breeds, commercial broilers and laying hens in my country. It can be seen that the existing measures cannot completely control the occurrence and prevalence of K subgroup avian leukosis in Chinese chickens. 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 develop new strategies and new means that are more suitable for the prevention and control of K subgroup avian leukosis in my country. Studying the disease-resistant breeding of avian leukosis from the perspective of host genetic resistance can become an effective strategy for the prevention and control of the disease. Identifying molecular markers of genetic resistance to K subgroup avian leukosis and applying them to screen breeding materials with genetic resistance to K subgroup avian leukosis to cultivate chicken varieties (lines) with genetic resistance to K subgroup avian leukosis is the key to achieving disease-resistant breeding for K subgroup avian leukosis. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a chicken K subgroup avian leukosis genetic resistance molecular marker tva 546G＞C The invention and its application overcome the deficiency of the existing technology that the occurrence and prevalence of K subgroup avian leukosis in Chinese chickens cannot be completely controlled.

[0005] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0006] A molecular marker for genetic resistance to avian leukosis in chickens K subgroup 546G＞C The molecular marker is a G>C base mutation at the 546th base of the tva gene.

[0007] The present invention analyzes the genetic variation of the tva receptor gene in Chinese chicken breeds (including 6570 blood samples from 28 local chicken breeds and 57 yellow-feathered broiler strains) and finds that the 546th base of the tva receptor gene DNA sequence in Chinese chicken breeds (GenBank accession number AY531262.1) has a G>C mutation, referred to as tva 546G＞C Mutation site.

[0008] Then, in vitro and in vivo experiments confirmed that the natural mutation of the tva gene caused the host to resist ALV-K infection. The specific reason is that the Tva receptor is a member of the low-density lipoprotein receptor (LDLR) family. There is an LDL-A repeating motif composed of 40 amino acid residues between the 11th to 50th amino acid residues in the extracellular region of the Tva receptor protein, which is a key region for mediating ALV-K infection of host cells. The LDL-A repeating motif is rich in 6 cysteines, and 3 essential cysteine ​​disulfide bonds are formed between these 6 cysteine ​​residues, which is crucial for stabilizing and maintaining the structure and integrity of the Tva receptor protein. tva 546G＞C The mutation causes a G>C mutation at the 95th base of the tva gene coding sequence CDS (tva gene mRNA reference sequence NM_001044645.1), causing the 11th amino acid in the extracellular region of the Tva receptor protein to be replaced by cysteine ​​(Cys) to serine (Ser). The Cys11Ser replacement leads to the breakage of the cysteine ​​disulfide bond, thereby changing the structural stability and integrity of the LDL-A region of the Tva receptor protein, causing the tva gene to express a defective Tva receptor protein, causing the host to develop genetic resistance to ALV-K infection.

[0009] A primer for detecting the above molecular marker, the nucleotide sequence of the primer is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0010] The application of the above molecular markers or primers in screening / identifying K subgroup avian leukosis-resistant chickens.

[0011] Furthermore, the above application includes the following steps:

[0012] (1) Extract genomic DNA from the sample to be tested, and amplify the tva gene by PCR using the primers shown in SEQ ID NO.1 and SEQ ID NO.2. 546G＞C The tva gene fragment at the mutation site is sequenced to determine whether it is a resistant chicken;

[0013] (2) If the 546th base of the tva gene DNA sequence (GenBank accession number AY531262.1) undergoes a homozygous mutation from G to C (tva 546C / C ), then it has a phenotype of genetic resistance to ALV-K infection, that is, if the genotype of the chicken to be tested is tva 546C / C , then the individual is a K subgroup avian leukosis resistant chicken;

[0014] If the 546th base of the tva gene DNA sequence does not undergo a G to C mutation, it is wild type (tva 546G / G ), then the chicken is susceptible to ALV-K infection (no resistance); that is, if the genotype of the chicken to be tested is wild-type tva 546G / G , then the individual is a chicken susceptible to avian leukosis of subgroup K;

[0015] If the 546th base of the tva gene DNA sequence undergoes a heterozygous mutation from G to C (tva 546G / C ), then the individual is a chicken susceptible to avian leukosis of subgroup K, but the genotype is tva 546G / C The next generation produced by the mating of breeding roosters and breeding hens can produce the genotype tva 546C / C If an individual is an avian leukosis-resistant chicken of subgroup K, then the individual is a K subgroup avian leukosis-resistant chicken.

[0016] Furthermore, the PCR amplification system includes: 1 μL of DNA template, 2.5 μL of 10× buffer, 2 μL of dNTPs, 1 μL of upstream and downstream detection primers, 0.5 μL of KOD-FX, and finally supplemented to 25 μL with ddH2O.

[0017] Furthermore, the PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and 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.

[0018] The application of the above molecular markers or primers in the breeding of K subgroup avian leukosis-resistant chickens.

[0019] A kit for detecting / screening K subgroup avian leukosis-resistant chickens, comprising the above primers.

[0020] The application of the above kit in breeding of K subgroup avian leukosis-resistant chickens.

[0021] Beneficial effects of the present invention:

[0022] The present invention is the first to find that the 546th base of the ALV-K receptor gene tva DNA sequence (GenBank accession number AY531262.1) in Chinese chicken breeds has a G>C mutation (tva 546G＞C ), the inventors further confirmed that the natural mutation of the tva gene can cause the host chicken to produce genetic resistance to ALV-K infection. Therefore, it is very accurate to use this mutation site as a molecular marker to identify the resistance of chicken K subgroup avian leukosis.

[0023] The present invention further establishes a K subgroup avian leukosis genetic resistance marker tva 546G＞C A molecular diagnosis and gene typing method was used to establish an identification method for diagnosing K subgroup avian leukosis-resistant chickens, which can quickly and accurately determine whether the test sample is a K subgroup avian leukosis-resistant chicken or a susceptible chicken. It can be used to screen breeding materials for K subgroup avian leukosis-genetically resistant chicken varieties (lines) in Chinese chicken breeds (including local chicken breeds and commercial chicken lines), thereby carrying out the selection and breeding of K subgroup avian leukosis-genetically resistant chicken varieties (lines), which has great application and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The PCR amplification results of three fragments of the tva gene; M: DL2000 marker; 1-3: PCR amplification products of primers 1, 2, and 3;

[0025] Figure 2 for tva 546G＞C Sequencing maps of different genotypes at the locus;

[0026] Figure 3 The figure is a schematic diagram of the construction of RCASBP(K)-EGFP expression plasmid and its fluorescent reporter virus rescue; A: schematic diagram of the construction of RCASBP(K)-EGFP expression plasmid; B: restriction enzyme digestion identification of RCASBP(K)-EGFP plasmid; C: RCASBP(K)-EGFP virus rescue;

[0027] Figure 4 RCASBP(K)-GFP was used to infect tva 546G＞C The situation of CEF with different genotypes at the mutation site. DETAILED DESCRIPTION

[0028] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0029] Example 1 Molecular marker screening

[0030] 1. Primer design for PCR amplification of tva receptor gene

[0031] With reference to the DNA sequence of chicken tva gene in NCBI database (GenBank accession number: AY531262.1), three pairs of primers were designed to PCR amplify the full-length sequence of tva gene 3607 bp (segment 1, segment 2 and segment 3) in three segments. The primer sequences, positions and PCR amplification fragment sizes are shown in Table 1.

[0032] Table 1 PCR amplification information of the full-length sequence of tva receptor gene

[0033]

[0034] 2. PCR amplification of tva receptor gene

[0035] (1) Genomic DNA from 6570 blood samples of different Chinese chicken breeds (including 28 local chicken breeds and 57 yellow-feathered broiler strains) was extracted, and the full-length sequence of the tva gene was amplified by PCR using the three pairs of primers.

[0036] The PCR reaction system consisted of: 1 μL DNA template, 2.5 μL 10× buffer, 2 μL dNTPs, 1 μL each of upstream and downstream primers, 0.5 μL KOD-FX, and ddH2O to 25 μL.

[0037] PCR reaction program: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing (1st stage at 62°C, 2nd and 3rd stages at 60°C) for 30 s, extension at 72°C for 90 s, for a total of 35 cycles; post-extension at 72°C for 10 min, and finally storage at 4°C.

[0038] (2) The PCR products were detected by 2% agarose gel electrophoresis, and the PCR amplification products were sent to Sangon Biotech (Shanghai) 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 tva receptor gene in Chinese chicken breeds to screen for candidate genetic resistance loci for ALV-K. The results are shown in Figure 1 and Figure 2 , Figure 2 The sequences in the middle are, from top to bottom, the reference sequence (wild-type individual), the sequences of the heterozygous mutant individual and the sequences of the homozygous mutant individual. The box indicates the G>C mutation site at the 546th base of the tva gene sequence.

[0039] like Figure 1As shown, PCR amplified the target bands of fragments 1, 2 and 3 of the tva gene, and the fragment sizes were consistent with the expected results.

[0040] like Figure 2 As shown in the figure, by analyzing the genetic variation of the tva receptor gene in 28 local chicken breeds and 57 yellow-feathered broiler strains (a total of 6570 blood samples), it was screened and found that there was a SNP site (tva) in which the 546th base of the tva receptor gene sequence of Chinese chicken breeds mutated from G to C. 546G＞C ).

[0041] Example 2 tva 546G＞C Effects of mutations on host resistance

[0042] 1. In vitro cell experiments

[0043] (1) The RCASBP(K)-EGFP expression plasmid was constructed and transfected into DF-1 cells. Seven days after transfection, the RCASBP(K)-EGFP virus (i.e., ALV-K reporter virus carrying EGFP fluorescent protein) in the supernatant of DF-1 cells was rescued and collected ( Figure 3 ), and after determining the virus infection unit (IU), the cells were aliquoted and stored at -80°C.

[0044] (2) Using ALV-K fluorescent reporter virus RCASBP(K)-EGFP to infect tva 546G＞C Mutation site wild type tva 546G / G , heterozygous mutation tva 546G / C and homozygous mutant tva 546C / C CEF (chicken fibroblasts CEF were prepared from 9-day-old chicken embryos hatched from the breeder chickens detected in Example 1) and RCASBP(K)-EGFP virus infection was detected by flow cytometry 1, 2, 4, and 7 days after infection. 546G＞C The GPF positive cell rate (%) indicates the virus infection rate. The results are shown in Figure 4 .

[0045] like Figure 4 As shown, wild-type tva 546G / G CEF and heterozygous mutant tva 546G / C CEF cells are susceptible to RCASBP(K)-EGFP virus, while homozygous mutant tva 546C / C CEF cells are resistant to infection with RCASBP(K)-EGFP virus, indicating that tva 546G ＞C Natural mutations lead to host resistance to ALV-K infection.

[0046] 2. In vivo experiments

[0047] (1) TVA 546G＞C One-day-old chicks of mutant wild type, heterozygous mutant and homozygous mutant were randomly divided into groups and raised in isolators. The chicks were intraperitoneally injected with equal amounts of ALV-K (GDFX0601 strain) at 1 day and 5 days of age. One month after the challenge, blood samples were collected from the chicks and total RNA was extracted using the TRIZOL kit.

[0048] Design upstream and downstream primers for RT-PCR amplification of ALV-K-env:

[0049] env-F: 5'-GCACCACCTTGGGAACTGACC-3';

[0050] env-R: 5'-GGCGTGGATCGACAGCACAC-3'.

[0051] (3) RT-PCR amplified the env gene coding sequence of ALV-K. The length of the RT-PCR amplified fragment was 633 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, 60°C for 30 s, 72°C for 60 s, 35 cycles; 72°C post-extension for 10 min. The PCR product was detected by 2% agarose gel electrophoresis. If a 633 bp target band was observed, the sample had viremia (ALV-K positive). If no target band was amplified, the sample did not have viremia (ALV-K negative). The results are shown in Table 2.

[0052] Table 2 tva 546G＞C The incidence of ALV-K infection in one-day-old chickens with different genotypes at the mutation site after infection with wild-type ALV-K virus for one month

[0053]

[0054] As shown in Table 2, tva 546G＞C Mutation site wild type tva 546G / G All chickens (27) were ALV-K positive after being challenged with wild-type ALV-K virus. 546G / C All chickens (25) were positive for ALV-K after being challenged with wild-type ALV-K virus, while the homozygous mutant tva 546C / C All 33 chickens were ALV-K negative after being challenged with wild-type ALV-K virus. 546G＞C Natural mutations lead to resistance to ALV-K infection in the host. The results of the ALV-K challenge test were consistent with the results of the in vitro infection test, confirming that tva 546G＞C Natural mutation is a molecular marker for genetic resistance to avian leukosis of subgroup K in host chickens.

[0055] Example 3 Screening of ALV-K genetically resistant chickens

[0056] 1. Referring to the DNA sequence of tva gene (GenBank accession number AY531262.1), PCR primers (forward primer F: 5'-CGGCCCGCTTTATAGGCGTTG-3' (SEQ ID NO.1); reverse primer R: 5'-CCCACTCGTCCCGTCCATCG-3' (SEQ ID NO.2)) were designed to amplify the tva gene. 546G＞C The region of the TVA receptor gene where the mutation occurs.

[0057] 2. Extract genomic DNA from 1782 samples of 15 local chicken breeds and 15 yellow-feathered broiler strains;

[0058] 3. PCR testing

[0059] The PCR reaction system consisted of: 1 μL DNA template, 2.5 μL 10× buffer, 2 μL dNTPs, 1 μL each of 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.

[0060] PCR reaction program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°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.

[0061] 4. After 2% agarose gel electrophoresis, the PCR amplification product was sent to Shanghai Biotech Co., Ltd. for purification and sequencing to determine the genotype and whether it was a resistant chicken. The determination criteria are shown in Table 3.

[0062] Table 3 Identification criteria for chickens with genetic resistance to avian leukosis in subgroup K

[0063]

[0064] If tva 546G＞C The genotype of the resistance locus is wild type tva 546G / G , then it has no resistance to ALV-K infection (susceptible), and the individual is a chicken susceptible to avian leukosis of subgroup K;

[0065] If tva 546G＞C The resistance locus genotype is tva 546G / C , then the individual is susceptible to ALV-K infection, but the individual carries a recessive gene for genetic resistance to K subgroup avian leukosis;

[0066] If tva546G＞C The resistance locus genotype is tva 546C / C , then it develops genetic resistance to ALV-K infection and the individual is a K subgroup avian leukosis-resistant chicken.

[0067] 5. Test results

[0068] Chinese chicken breeds tva 546G＞C The genotyping results of the resistance loci are shown in Table 4. The resistance loci of local chicken breeds such as Beijing Oily Chicken, Silky Chicken, Anyi Gray Chicken, and Yellow-feathered Broiler Line 3, Yellow-feathered Broiler Line 7, Yellow-feathered Broiler Line 13, and Yellow-feathered Broiler Line 15 were found to be tva 546G＞C Resistance genotype tva at resistance locus 546C / C , and their frequencies were 0.08, 0.16, 0.13, 0.22, 0.18, 0.10 and 0.22, respectively, indicating that these Chinese local chicken breeds and independently bred yellow-feathered broiler strains have good potential for genetic improvement of resistance to ALV-K. Breeding materials for breeding resistance to ALV-K infection can be screened from these chicken breeds and used in the selection and breeding of ALV-K genetically resistant chicken breeds (strains) to prevent and control K subgroup avian leukosis.

[0069] Table 4 Chinese chicken breeds tva 546G＞C Genotype frequency distribution of mutation sites

[0070]

[0071] Sequence Listing <110> South China Agricultural University <120> A molecular marker for genetic resistance to avian leukosis in chickens K subgroup <ssupp> 546G>C< / ssupp> And its application <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty one <212> DNA <213> Artificial Sequence <400> 1 cggcccgctt tataggcgtt g 21 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 cccactcgtc ccgtccatcg 20

Claims

1. A molecular marker for detecting genetic resistance to avian leukosis in chicken K subgroups: tva 546G>C Use of a primer pair in preparing a kit for screening / identifying chickens with genetic resistance to K subgroup avian leukosis, characterized in that: The chicken is a Chinese chicken breed, and the base at the 546th position of the chicken tva gene sequence of the molecular marker with GenBank accession number AY531262.1 is G / C. If the tva gene of the Chinese chicken breed to be tested has a homozygous mutation of G>C base at the 546th base, the chicken sample to be tested is a K subgroup avian leukosis resistant chicken; if the tva gene of the Chinese chicken breed to be tested has no G>C base mutation or has a G>C base heterozygous mutation at the 546th base, the chicken sample to be tested is a K subgroup avian leukosis susceptible chicken.

2. The use according to claim 1, characterized in that: For amplifying the molecular marker tva 546G>C The nucleotide sequences of the primers are shown in SEQ ID NO.1 and SEQ ID NO.

2.

3. The use according to claim 1, characterized in that: The screening / identification comprises the following steps: (1) Extract the genomic DNA of the sample to be tested, amplify the genomic DNA by PCR using the primers shown in SEQ ID NO.1 and SEQ ID NO.2, and sequence the tva 546G>C Genotyping of mutation sites; (2) If the tva gene of the Chinese chicken breed to be tested has a homozygous mutation of G>C at the 546th base, it is a K subgroup avian leukosis resistant chicken; if the tva gene of the sample to be tested does not have a G>C base mutation at the 546th base or has a heterozygous mutation of G>C, it is a K subgroup avian leukosis susceptible chicken.

4. The use according to claim 3, characterized in that: The PCR amplification system includes: 1 µL of DNA template, 2.5 µL of 10× buffer, 2 µL of dNTPs, 1 µL of upstream and downstream detection primers, 0.5 µL of KOD-FX, and finally ddH2O is used to make up to 25 µL.

5. The use according to claim 3, characterized in that: The PCR amplification procedure was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°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.

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