CN116411087A - Avian leukosis genetic resistance molecular marker tvb for chicken B subgroup 3215-3216insCC And applications thereof - Google Patents
Avian leukosis genetic resistance molecular marker tvb for chicken B subgroup 3215-3216insCC And applications thereof Download PDFInfo
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Abstract
The invention discloses a avian leukosis genetic resistance molecular marker tvb of chicken B subgroup 3215‑3216insCC And the application thereof, the molecular marker is tvb receptor gene, and CC base is inserted between 3215 th base and 3216 th base. The invention proves that the tvb receptor gene natural mutation causes the infection of host anti-ALV-B from two levels of in vitro and in vivo experiments, and is also based on B subgroup avian leukosis genetic resistance molecular marker tvb 3215‑3216insCC The identification method for the avian leukosis genetic resistance chickens of subgroup B is established, and the method can rapidly and accurately judge whether the detection sample is the ALV-B resistant chickens or the susceptible chickens, can screen breeding materials for breeding ALV-B genetic resistance chickens from Chinese chickens (including local chickens and commercial broiler chickens), and provides technical support for breeding the ALV-B genetic resistance chickens.
Description
Technical Field
The invention relates to the technical field of breeding of poultry genetic resistance varieties, in particular to a molecular marker tvb for avian leukosis genetic resistance of chicken B subgroup 3215-3216insCC And applications thereof.
Background
Avian leukosis of subgroup B of chickens is a chicken immunosuppressive neoplastic infectious disease caused by avian leukosis virus of subgroup B (Avian Leukosis Virus subgroup B, ALV-B). ALV-B can cause immunosuppression of infected chicken groups, is extremely easy to cause secondary avian influenza, newcastle disease and other viral diseases and bacterial diseases such as escherichia coli, salmonella and the like, causes characteristic tumors of infected breeding hens and broilers to die, causes the reduction of the production performance of the infected chicken groups and the quality of meat and eggs, and causes huge economic loss for the poultry industry in the world. At present, no commercial vaccine and no effective treatment method for avian leukosis exist, and prevention and control are mainly carried out through population purification measures, but the population purification period is long (at least 8-10 years), the cost is high (the purification cost of each breeding hen is about 500 yuan), and the purified negative chicken flock still faces the risk of re-infection of ALV, so that the prevention and control effect of avian leukosis is difficult to maintain, and the disease is in continuous fight of pathogen-animal-detection personnel. ALV-B has been isolated in recent years from different types of chickens such as breeder seeds, yellow-feathered broilers, white-feathered broilers, layer chickens, etc. Therefore, the existing measures can not completely control the occurrence and the epidemic of the B subgroup avian leukosis in China, and the disease becomes one of serious diseases which threatens the sustainable healthy development of the chicken industry (especially the breeding hens industry) in China. ALV-B is mainly transmitted vertically, the infection can be amplified from great ancestor generation (pure line), ancestor generation, parent generation and commodity generation by generation, the infection rate of each generation is increased by about 5% -20%,1 ancestor (pure line) chicken can be infected with ALV-B and then can be transmitted to 24 ten thousand commodity chickens, and the poultry leukemia has become the disease which is the greatest hazard to the poultry industry safety in China. Therefore, developing technical innovation for controlling avian leukosis, researching disease-resistant breeding of avian leukosis from the aspect of host genetic resistance has become an effective strategy for preventing and controlling the disease.
The cell surface specific Tvb receptor protein encoded by the chicken Tvb receptor gene mediates invasion of the host cell, which in turn is infected, determining the susceptibility or resistance of the host cell to infection by ALV-B. Genetic mutations in the Tvb receptor gene may result in complete deletion of the expression of the Tvb receptor protein or expression of a defective Tvb receptor protein that is unsuitable for use as an ALV-B receptor, thereby causing genetic resistance of the host cell to infection by ALV-B. Therefore, the genetic resistance molecular marker of the avian leukosis subgroup B is identified and is applied to screening of the avian leukosis subgroup B genetic resistance breeding materials so as to cultivate the avian species (line) of the avian leukosis subgroup B with genetic resistance, which is the key point for realizing the avian leukosis subgroup B disease resistance breeding, thereby ensuring the poultry breeding industry safety in China.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a avian leukosis genetic resistance molecular marker tvb of chicken B subgroup 3215-3216insCC And the application thereof, so as to solve the problem that the occurrence and popularity of the avian leukosis of subgroup B in chicken flocks in China cannot be completely controlled in the prior art.
The first object of the invention is to provide a avian leukosis genetic resistance molecular marker tvb of chicken B subgroup 3215 -3216insCC The molecular marker is tvb acceptor gene, and CC base is inserted between 3215 th base and 3216 th base.
A second object of the present invention is to provide a method for detecting the above molecular marker tvb 3215-3216insCC The nucleotide sequence of the primer is shown as SEQ ID NO:1 and SEQ ID NO: 2.
A third object of the present invention is to provide an application of the molecular marker tvb 3215-3216insCC And/or the primer is used for screening/identifying the avian leukosis genetic resistance chicken of subgroup B.
Further, the above application comprises the steps of:
extracting genome DNA of a sample to be detected, amplifying tvb receptor gene fragments, sequencing, and if CC homozygous insertion mutation exists at the 3215 th to 3216 th base positions of the tvb receptor gene sequence (tvb) insCC/insCC ) The sample to be tested is avian leukosis genetic resistance chicken of subgroup B, that is, C is presentC homozygous mutation, has a phenotype of genetic resistance to infection by ALV-B, i.e.the genotype of the chicken to be tested is tvb insCC/insCC The individual is a subgroup B avian leukosis genetic resistance chicken;
if no CC homozygote insertion mutation occurs at the position between the 3215 th and 3216 th bases of the tvb receptor gene sequence, the sample to be tested is a B subgroup avian leukosis susceptible chicken, that is to say, if no CC insertion mutation occurs, the sample is wild type, the sample is susceptible to infection of ALV-B (has no resistance), that is to say, if the genotype of the sample to be tested is wild type tvb s1/s1 The individual is a subgroup B avian leukosis susceptible chicken;
if there is a heterozygous insertion mutation at the base positions 3215 to 3216 of the tvb receptor gene sequence (tvb) s1/insCC ) The individual is avian leukosis susceptibility chicken of subgroup B, but the genotypes are tvb s1/insCC The next generation generated after breeding the breeder cock and the breeder hen can generate the genotype of tvb insCC/insCC Is a subgroup B avian leukemia resistant chicken.
Further, the amplification system at the time of amplification includes: 1. Mu.L of DNA template, 2.5. Mu.L of 10 Xbuffer, 2. Mu.L of dNTPs, 1. Mu.L of upstream and downstream detection primer, 0.5. Mu.L of KOD-FX, and finally ddH 2 O was made up to 25. Mu.L.
Further, the amplification procedure at the time of amplification is: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 30s, for a total of 35 cycles; extending at 72deg.C for 5min, and preserving at 4deg.C.
The fourth object of the invention is to provide a kit for detecting/screening avian leukosis subgroup B genetically resistant chickens, which comprises the primers.
The fifth object of the invention is to use the above kit for screening breeding materials for breeding the genetic anti-B subgroup avian leukosis chicken variety/strain.
The invention has the following beneficial effects:
the invention discovers that CC insertion mutation exists at the 3215 th to 3216 th base of the tvb DNA sequence (GenBank accession number: NC-052553.1) of the ALV-B receptor gene in chicken species in China for the first time (tvb) 3215-3216insCC ) Further studies confirm that the natural mutation of the tvb receptor gene can cause the host chicken pairInfection with ALV-B results in genetic resistance. Therefore, the mutation site is very accurate as a molecular marker for identifying the avian leukosis genetic resistance of chicken subgroup B.
The invention further establishes a B subgroup avian leukosis genetic resistance marker tvb 3215-3216insCC The molecular diagnosis and genotyping method establishes a method for identifying the avian leukosis genetic resistance chicken of subgroup B, can rapidly and accurately judge whether a detection sample is the avian leukosis genetic resistance chicken of subgroup B or a susceptible chicken, can be applied to screening breeding materials for breeding avian leukosis genetic resistance chicken varieties (strains) of subgroup B in chicken breeds (including local chicken breeds and commercial chicken strains) in China, and further develops breeding of the avian leukemia genetic resistance chicken varieties (strains) of subgroup B, and has good application value and popularization prospect.
Drawings
FIG. 1 shows the results of PCR amplification of 5 fragments of tvb receptor gene; wherein M is 2000marker, NC is negative control; 1-5 show the PCR amplification results of the tvb gene 1, 2, 3, 4, 5 gene fragments, respectively.
FIG. 2 is a schematic structural diagram of tvb receptor gene.
FIG. 3 is tvb 3215-3216insCC Sequencing map of different genotypes of mutation sites.
FIG. 4 shows the result of RT-PCR amplification of the sequence of the full-length coding region of the tvb receptor gene; wherein M is 2000Marker.
FIG. 5 shows the results of sequencing analysis of RT-PCR amplification products of the sequence of the full-length coding region of the tvb receptor gene.
FIG. 6 is a recombinant plasmid pmCherry-C1-tvb s1/s1 And pEGFPC1-tvb insCC/insCC Is subjected to enzyme digestion identification; wherein M is 5000 markers.
FIG. 7 shows the results of 24h fluorescent expression of recombinant plasmid transfected 293FT cells.
FIG. 8 shows the expression results of the fusion protein in 293-FT cells.
FIG. 9 is a schematic diagram of construction of RCASBP (B) -EGFP recombinant expression plasmid.
FIG. 10 shows the results of the digestion and identification of RCASBP (B) -EGFP recombinant expression plasmid.
FIG. 11 shows the result of RCASBP (B) -EGFP virus rescue.
FIG. 12 is RCASBP (B) -EGFP infection tvb 3215-3216insCC The process of mutating the site to different genotypes.
Detailed Description
The invention firstly analyzes the genetic variation condition of tvb receptor genes of chicken breeds (comprising 20 local chicken breeds and 20 yellow feather broiler breeds) in China, and discovers that CC insertion mutation exists at the 3215 th to 3216 th base positions of tvb receptor gene DNA sequences (GenBank accession number: NC_052553.1, specifically SEQ ID NO: 19) in the chicken breeds in China, which is called tvb for short 3215-3216insCC Mutation sites.
Secondly, experiments in vitro and in vivo prove that the tvb receptor gene natural mutation causes the infection of the host against ALV-B. Of particular reason is tvb 3215-3216insCC The mutation is located in the 3 rd exon region of tvb receptor gene, and is inserted into CC (tvb c.195_196 insCC) at the 195-196 th base position of tvb gene coding sequence CDS (tvb gene mRNA reference sequence NM_204115.3, see SEQ ID NO:20 for details). Tvb c.195_196insCC mutation causes the Tvb receptor protein sequence encoded thereby (Tvb receptor protein reference sequence NP_989446.2, see SEQ ID NO:21 in particular) to shift from amino acid residue 66 and terminate encoding prematurely at amino acid residue 91, resulting in a change in the structure of the cysteine rich region 2 (CRD 2) of the Tvb receptor protein (CRD of the Tvb receptor protein is the critical domain mediating ALV-B infection and entering the host cell) and expression of a truncated Tvb insCC The receptor protein, thereby causing genetic resistance to infection by ALV-B in the host.
Finally, avian leukosis genetic resistance molecular marker tvb based on subgroup B 3215-3216insCC The identification method for the avian leukosis genetic resistance chickens of subgroup B is established, and the method can rapidly and accurately judge whether the detection sample is the ALV-B resistant chickens or the susceptible chickens, can screen breeding materials for breeding the ALV-B genetic resistance chickens from chicken breeds (including local chicken breeds and commercial broiler chickens) in China, and provides technical support for breeding the ALV-B genetic resistance chickens.
The examples given below are only intended to illustrate the invention and are not intended to limit the scope thereof. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: development of mutation sites
1. Primer design
Referring to the DNA sequence of the chicken tvb receptor gene in NCBI database (GenBank accession number: NC-052553.1), 5 pairs of primers were designed to amplify the full-length sequence 6596bp (1 segment, 2 segments, 3 segments, 4 segments and 5 segments) of the tvb receptor gene by PCR with 5 segments, and the primer sequences, positions and PCR amplified segment sizes are shown in Table 1.
TABLE 1 full-length sequence PCR amplification primer information for tvb receptor genes
2. PCR amplification of tvb receptor genes
Referring to the instruction book of the Tiangen blood DNA extraction kit, the genome DNA of 2636 blood samples of different chicken species (including 20 different local chicken species and 20 yellow-feather broiler strains) in China is extracted, and the 5 pairs of primers are used for PCR amplification of tvb receptor gene full-length sequences.
The PCR reaction system comprises: 1. Mu.L of DNA template, 2.5. Mu.L of 10 Xbuffer, 2. Mu.L of dNTPs, 1. Mu.L of each of the upstream and downstream primers, 0.5. Mu.L of KOD-FX, and ddH 2 O was made up to 25. Mu.L.
PCR reaction procedure: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing (1 stage 62 ℃,2, 3 stage 58 ℃,4, 5 stage 60 ℃), annealing for 30s,72 ℃ extension for 90s, total 35 cycles; extending at 72deg.C for 10min, and preserving at 4deg.C. The PCR products were detected by 2% agarose gel electrophoresis, and the detection results are shown in FIG. 1.
As can be seen from FIG. 1, after PCR amplification, 1079bp, 1254bp, 1530bp, 1239bp and 1451bp 5 fragments can be amplified, and the size of the band is consistent with that of the target fragment.
3. Sequencing analysis of tvb receptor Gene PCR amplified products
The PCR amplified product of tvb acceptor gene is purified and sequenced by biological engineering (Shanghai) Co., ltd, DNAstar and Mutation Surveyor gene sequence analysis software is used for sequence comparison, analysis of genetic variation of chicken tvb acceptor gene in China, and screening of ALV-B candidate genetic resistance sites.
By analyzing the genetic variation of tvb receptor genes of 20 local chicken species and 20 yellow-feather broiler strains (2636 blood samples), screening and finding that insertion CC 2 bases exist at the position of 3215-3216 bases of exon 3 of tvb receptor genes of Chinese chicken species (tvb) 3215-3216insCC ) Natural mutations of (1) (see FIG. 2). Sequence sequencing patterns of different genotypes at mutation sites are shown in FIG. 3, and the sequences are reference sequences (wild type tvb) s1/s1 Individual), heterozygous mutant tvb s1/insCC Individual and homozygous mutant tvb insCC/insCC The individual sequence, red line marked as CC insertion mutation at the 3215-3216 base positions of tvb gene sequence.
Example 2: tvb receptor gene coding region sequence analysis
1. Extraction and reverse transcription of RNA
Acquisition tvb 3215-3216insCC Mutation site wild type tvb s1/s1 And homozygous mutant tvb insCC/insCC 200 mu L of blood of the breeding hens is uniformly mixed in 800 mu L of TRIZOL (RNase Free EP tube) in an oscillating way, fully cracked, kept stand at room temperature for 5min, added with 300 mu L of chloroform, uniformly mixed in an oscillating way, centrifuged for 15min at 4 ℃ at 12,000Xg, the upper colorless transparent supernatant is sucked into a new EP tube, added with an equal volume of isopropanol solution to precipitate RNA, and kept stand for 10min; centrifuging at 4deg.C for 30min at 12,000Xg, discarding supernatant, washing precipitate with 75% ethanol once, discarding supernatant, standing at room temperature, and adding 50 μLDEPC water to dissolve RNA after ethanol is completely volatilized. To extract tvb 3215-3216insCC Mutation site wild type tvb s1/s1 And homozygous mutant tvb insCC/insCC The blood RNA of the breeder was used as a template and was reverse transcribed into cDNA according to the instructions of the reverse transcription kit (Takara). Reverse transcribed cDNA in preserving at-20 ℃ for standby.
2. RT-PCR amplification of tvb receptor Gene full-Length coding region sequences
(1) Primer design
Referring to NCBI published tvb receptor gene mRNA sequence (NM_ 204115.3), primers were designed to amplify the entire coding region sequence (CDS) of tvb, and the primer sequences and RT-PCR amplified fragment sizes are shown in Table 2.
Table 2 RT-PCR amplification primer information for full-length coding region sequence of tvb receptor gene
(2) RT-PCR amplification
Tvb by 3215-3216insCC Mutation site wild type tvb s1/s1 And homozygous mutant tvb insCC/insCC cDNA of the chicken blood sample was used as a template, and RT-PCR amplification was performed by referring to the instructions of Toyobo Co high-fidelity enzyme KOD-FX.
RT-PCR reaction system is composed of: cDNA template 1. Mu.L, 10 Xbuffer 2.5. Mu.L, dNTPs 2. Mu.L, upstream and downstream primers 1. Mu.L each, KOD-FX 0.5. Mu.L, ddH 2 O was made up to 25. Mu.L.
RT-PCR reaction procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 62℃for 30s, elongation at 72℃for 75s,35 cycles; extending at 72deg.C for 10min, and preserving at 4deg.C. After the reaction, 1% agarose gel electrophoresis was performed.
Wild type tvb s1/s1 Homozygous mutant genotype tvb insCC/insCC The results of RT-PCR amplification of the sequence of the full-length coding region of the tvb receptor gene of the chicken blood sample are shown in FIG. 4. As can be seen from FIG. 4, the amplified band size matches the size of the fragment of interest.
3. Clone sequencing analysis of RT-PCR products
The recovery and purification of RT-PCR products were performed by reference to the procedures of the gel recovery and purification kit instruction of Axygen company. The RT-PCR purified product was ligated into pMD19-T Vector (Takara Co.) with reference to pMD19-T Vector. Referring to competent cell DH 5. Alpha. Instructions, ligation products were transformed into DH 5. Alpha. Competent cells. Screening positive colony, PCR identification and PCR identificationThe positive bacterial liquid was sent to the Shanghai engineering (Shanghai) Co., ltd for sequencing. Tvb was analyzed using Lasergene 7.0 software alignment 3215-3216insCC Sequencing results of the full-length coding region sequences of the wild-type and homozygous mutant tvb genes at the mutation sites are shown in fig. 5.
The CC dinucleotide access positions and the altered amino acid sequence are shown in FIG. 5, together with Tvb S1 And Tvb insCC Schematic representation of the receptor protein. Tvb (Tvb) S1 Numbering of proteins includes Signal Peptide (SP), cysteine rich domains (CRD 1 to-3), transmembrane domain (MSD) and cytoplasmic death domain. At Tvb insCC The position of the midamble shift mutation is highlighted with an asterisk, and at Tvb insCC The premature stop codon TGA is indicated by a triangle. The deduced amino acid sequences of CRDs of known Tvb receptors were also compared. Presuming Tvb S1 Disulfide bonds in CRDs, tvb insCC The putative cysteine substitutions in (1) are circled, tvb insCC Amino acid residues of the frameshift peptide are shown in bold. As can be seen from fig. 5, tvb insCC/insCC The nucleotide positions 195-196 of the cDNA of (A) were inserted into CC (tvb c.195_196 insCC). tvb insCC/insCC CC base insertion of cDNA sequence, resulting in Tvb encoded thereby insCC The acceptor protein sequence is frame shifted from amino acid residue 66 and premature termination of encoding occurs at amino acid residue 91. Thereby predicting tvb 3215-3216insCC The frame shift mutation caused by the mutation causes the cysteine of the cysteine-rich region 2 (CRD 2) of the Tvb receptor protein to be replaced by other amino acid residues, resulting in the disulfide bond of the CRD2 to be broken, thereby changing the Tvb insCC Structure of the receptor protein.
Example 3: mutations affect protein expression
1、pmCherry-C1-tvb s1/s1 、pmCherry-C1-tvb insCC/insCC Construction of recombinant expression plasmids
(1) RT-PCR amplification of tvb receptor gene mutation site full-length CDS fragment
RT-PCR amplification primers based on the sequence of the full-length coding region of the tvb receptor gene, xhoI and HindIII cleavage sites (underlined sequences) and protecting bases (as in Table 3) were added, respectively, according to the method of example 2RT-PCR amplification tvb 3215-3216insCC Mutation site wild type tvb s1/s1 And homozygous mutant tvb insCC/insCC tvb full-length coding region sequence of the gene.
Table 3 RT-PCR amplification primers for full length CDS of tvb receptor gene
(2) Purification and ligation of PCR products
Reference was made to the Axygen gel recovery purification kit instructions for recovery of the purified RT-PCR amplification product and ligation to the pMD19-T vector.
(3) Ligation product transformation competent cells
Referring to competent cell DH 5. Alpha. Instructions, the ligation product was transformed into DH 5. Alpha. Competent cells.
(4) Double enzyme digestion reaction
Will contain tvb 3215-3216insCC Mutation site wild type tvb s1/s1 And homozygous mutant tvb insCC/insCC the pMD19-T and pmcherry-C1 plasmids of the full-length coding region sequence fragment of the tvb gene were subjected to XhoI/HindIII double digestion respectively.
(5) Purification and ligation of the cleavage products
The target fragment and the pmcherry-C1 vector are recycled and purified according to the specification of the glue recycling and purifying kit of Axygen company, and then the target fragment and the pmcherry-C1 vector are uniformly mixed according to the ratio of 3:1, centrifuged and placed at 16 ℃ for overnight connection.
(6) Screening and identification of Positive colonies
Single white colonies are picked from the cultured plates, inoculated into 500 mu L Kana LB liquid medium, subjected to shaking culture at 37 ℃ for about 5 hours, directly subjected to PCR identification by taking bacterial liquid as a template, and sent to a biological engineering (Shanghai) stock company for sequencing.
(7) Restriction enzyme identification of pmCherry-C1-tvb recombinant plasmid
The recombinant plasmid of the positive bacterial liquid is extracted by referring to the instruction of a plasmid small-scale extraction kit (Edley). The pmCherry-C1-tvb recombinant plasmid was identified by double restriction with the endonucleases XhoI and HindIII, and the recombinant plasmid was sent to sequencing and verification by Shanghai, inc.
As can be seen from FIG. 6, the recombinant plasmids were successfully constructed, and the constructed recombinant plasmids were designated as pmCherry-C1-tvb, respectively s1/s1 And pEGFPC1-tvb insCC/insCC 。
2. Western Blotting detection of pmCherry-C1-tvb recombinant expression plasmid
The pmCherry-C1 control plasmid and the pmCherry-C1-tvb were transfected with Lipofectamine 3000Reagent, see Lipofectamine 3000Reagent instructions s1/s1 、pEGFPC1-tvb insCC/insCC The red fluorescence was observed 24 hours later by transfecting 293-FT cells (6 well plates) prepared in advance up to 70 to 80% confluence with monolayers, and the results are shown in FIG. 7. As can be seen from FIG. 7, the transfection of pmCherry-C1, pmCherry-C1-tvb was detected under an inverted fluorescence microscope s1/s1 、pmCherry-C1-tvb insCC/insCC The recombinant plasmid 293-FT cells emit red fluorescence, and the result shows that mCherry-Tvb fusion protein is expressed in the 293-FT cells.
After 48h of transfection, 293FT cell supernatants were collected for Western Blotting detection, as shown in FIG. 8. As can be seen from FIG. 8, the blank plasmid pmCherry-C1 expresses a protein of about 27kD in 293-FT cells, pmCherry-C1-tvb s1/s1 mCherry-Tvb expressed at about 70kD in 293-FT cells s1 Fusion protein, and pmCherry-C1-tvb insCC/insCC Recombinant plasmid expresses mCherry-Tvb with the size of about 37kD in 293-FT cells insCC Fusion protein, results showed tvb 3215-3216insCC Mutation results in Tvb receptor gene expression of a truncated Tvb insCC A receptor protein.
Example 4: identification of a mutation to render a host resistant to ALV-B infection
1. In vitro cell infection verification
(1) Constructing RCASBP (B) -EGFP recombinant expression plasmid, transfecting the recombinant expression plasmid into DF-1 cells, saving and collecting the RCASBP (B) -EGFP virus (namely, carrying EGFP fluorescent protein ALV-B report virus) of DF-1 cell supernatant 7 days after transfection, and after measuring virus Infection Units (IU), sub-packaging and storing at-80 ℃.
The construction schematic diagram of the RCASBP (B) -EGFP recombinant expression plasmid is shown in figure 9; the result of the digestion identification of the RCASBP (B) -EGFP recombinant expression plasmid is shown in figure 10; the results of RCASBP (B) -EGFP virus rescue are shown in FIG. 11.
(2) Infection of tvb with ALV-B fluorescence reporter virus RCASBP (B) -EGFP, respectively 3215-3216insCC Mutation site wild type tvb s1/s1 Heterozygous mutant tvb s1/insCC And homozygous mutant tvb insCC/insCC CEF (chicken fibroblast CEF prepared from 9-day-old chick embryo hatched after breeding of the breeder examined in example 1), RCASBP (B) -EGFP virus infection tvb was examined by flow cytometry 1, 2, 3, 7 days after infection 3215-3216insCC The rate (%) of GPF positive cells in the case of CEF with different genotypes at the mutation sites indicates the infection rate of the virus, and the specific results are shown in FIG. 12.
As can be seen from fig. 12, wild type tvb s1/s1 CEFs are susceptible to ALV-B and serve as positive controls. RCASBP (B) -EGFP can efficiently infect tvb s1/s1 CEFs, infection rate was approximately 20% on day 1, and almost all cells infected by day 7 (FIG. 12A). Whereas RCASBP (B) -EGFP pair tvb insCC/insCC CEFs were inefficient in infection, with approximately 2% of the cells infected on day 1, and spread slowly, with approximately 8% of the cells infected on day 3, reaching 16% on day 7 (FIG. 12A). And tvb insCC/insCC Tvb compared with CEFs s1/insCC CEFs are more susceptible to infection by RCASBP (B) -EGFP, and exhibit similar infectious properties to the wild type (FIG. 12A). GFP-negative and GFP-positive cells were significantly different, with two independent peaks in the FACS histogram (fig. 12B). The above results indicate tvb 3215-3216insCC The mutation causes infection of host cells in vitro against RCASBP (B) -EGFP.
2. In vivo toxicity attack test verification
(1) Will tvb 3215-3216insCC Mutation sites wild-type, heterozygous mutant and homozygous mutant 1 day old chickens were randomly grouped, 25 animals per group, and kept in isolators. Each chicken was inoculated intraperitoneally with 0.3mL of ALV-B SDAU09C2 strain virus solution (S/P value=2.2) at 1 day of age, and challenged once again at 5 days of age. 2 weeks after the virus challenge, an anticoagulated blood sample is collected from each chick, genomic DNA is extracted from each chick tvb by a direct sequencing method 3215-3216insCC Genotyping was performed at the mutation site. 1 month after virus challenge, a blood sample of the chicken is collected, TRIZOL kit is used for extracting total RNA of the blood sample, ALV-B SDAU09C2 strain specificity detection primer is used for detecting viremia of each chicken by RT-PCR, and tvb is determined 3215-3216insCC Infection status of chicks of different genotypes on ALV-B SDAU09C2 strain was mutated.
Designing an upstream primer and a downstream primer for RT-PCR amplification of ALV-B-env:
env-F：5’-GCAGGCATTTCTGACTGGGC-3’(SEQ ID NO：17)；
env-R：5’-AGACCTTCCGATAGGTGAGG-3’(SEQ ID NO：18)。
(2) The env gene coding sequence of ALV-B is amplified by RT-PCR, and the length of the amplified fragment of the RT-PCR is 435bp. By means of
RT-PCR amplification was performed on One Step RT-PCR Kit Ver.2 Kit, PCR reaction procedure: reverse transcription is carried out for 30min at 50 ℃;94℃30s,58℃30s,72℃45s,35 cycles; extending for 10min at 72 ℃. The PCR products were detected by 2% agarose gel electrophoresis, if 435bp target band was observed, the sample developed viremia (ALV-B positive), if no target band was amplified, the sample did not develop viremia (ALV-B negative), and the results are shown in Table 4.
TABLE 4ALV-B infection tvb 3215-3216insCC Post-viremia positive condition of chickens with different mutation sites
As shown in Table 4, 22 wild-type tvb strains inoculated with ALV-B SDAU09C2 virus solution s1/s1 In the chicks, all were ALV-B positive. However, 24 are tvb only insCC/insCC After the chicks were inoculated with the virus solution of strain ALV-B SDAU09C2, the ALV-B positive rate was reduced to 12.5% (Table 4), whereas 29 was tvb only s1/insCC The positive rate of ALV-B after the genetic chick is inoculated with the virus liquid of the ALV-B SDAU09C2 strain is 93.1 percent. The toxicity attack test result shows that the infection in vivo and the virus in vitro are feltThe dyeing results were consistent, confirming tvb 3215-3216insCC Mutant host against ALV-B infection, tvb 3215-3216insCC Naturally mutating to obtain the avian leukosis genetic resistance molecular marker of the B subgroup of the host chickens.
Example 5: establishment and application of B subgroup avian leukosis genetic resistance chicken identification standard
1. Establishment of identification standard of avian leukosis genetic resistance chicken of subgroup B
(1) Primer design
With reference to the tvb receptor gene DNA sequence (GenBank accession number: NC-052553.1), PCR primers (forward primer F:5'-GCACAATCAGCTCTTGCTGC-3' (SEQ ID NO: 1) and reverse primer R:5'-GCAGCCTGCAAGCACCGCCA-3' (SEQ ID NO: 2)) were designed to amplify the primer comprising tvb 3215-3216insCC Tvb receptor genomic region of the mutation site.
(2)tvb 3215-3216insCC PCR amplification of the tvb receptor genomic region at the mutation site
The PCR reaction system comprises: 1. Mu.L of DNA template, 2.5. Mu.L of 10 Xbuffer, 2. Mu.L of dNTPs, 1. Mu.L of upstream and downstream detection primer (the nucleotide sequences of which are shown as SEQ ID NO:1 and SEQ ID NO: 2), 0.5. Mu.L of KOD-FX, and 25. Mu.L of ddH2O were supplemented.
PCR reaction procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 30s, for a total of 35 cycles; extending at 72deg.C for 5min, and preserving at 4deg.C.
(3) Sequencing of PCR amplified products
The PCR amplified product was detected by 2% agarose gel electrophoresis, purified and sequenced by Shanghai Bioengineering (Shanghai) Co., ltd, and tvb was obtained according to the sequencing result 3215-3216insCC Genotyping was performed at the mutation site.
According to tvb 3215-3216insCC The mutation site genotype establishes the identification standard of subgroup B avian leukosis genetic resistance chicken, and the judgment standard is shown in Table 5.
Table 5B identification criteria for avian leukosis subgroup genetic resistance chickens
If tvb 3215-3216insCC The genotype of the resistance locus is wild type tvb s1/s1 No resistance (susceptibility) to ALV-B infection, the individual being a subgroup B avian leukemia susceptible chicken;
if tvb 3215-3216insCC The resistance locus genotype was tvb s1/insCC Susceptible to ALV-B infection, but the individual carries avian leukosis subgroup B genetic resistance recessive genes;
if tvb 3215-3216insCC The resistance locus genotype was tvb insCC/insCC Genetic resistance is then developed to infection with ALV-B, and the individual is avian leukemia resistant chicken of subgroup B.
2. Application of B subgroup avian leukosis genetic resistance chicken identification standard
(1) Extraction of whole genome DNA
According to the instruction of the extraction kit of the root blood genome, 2636 parts of DNA of anticoagulated blood of 20 local chicken species in China and 20 yellow-feather broiler strains are extracted and stored at-20 ℃ for standby.
(2) PCR amplification and sequencing
The PCR amplification primers and the amplification procedure are as described in examples 5 and 1, for different chicken species tvb in China 3215-3216insCC And (3) carrying out PCR amplification on the tvb genome region of the mutation site, and sending the PCR amplification product to a biological engineering (Shanghai) stock company for sequencing after the PCR amplification product is detected to be qualified by 2% agarose gel electrophoresis.
(3)tvb 3215-3216insCC Resistance locus genotyping
Sequencing peak pattern analysis of sequencing results using Chromas software, sequencing sample tvb was analyzed 3215-3216insCC Genotype of the resistance locus.
Chinese local chicken species and yellow-feather broiler strain tvb 3215-3216insCC The genotyping results of the resistance sites are shown in Table 6 and Table 7, and tvb of local chickens such as Qingyuan Ma chicken, sha-bang chicken, chongren Ma chicken, he-Tian chicken and Wenchang chicken exist 3215-3216insCC Resistance genotype tvb of resistance locus insCC/insCC The frequencies of the yellow feather broiler strain are respectively 0.15, 0.17, 0.06, 0.10 and 0.15, and the yellow feather broiler strain 3 and the yellow feather broiler strainTvb also exist for broiler line 7, yellow-feathered broiler line 11 and yellow-feathered broiler line 20 3215-3216insCC Resistance genotype tvb of resistance locus insCC/insCC The frequencies of the breeding materials are respectively 0.07, 0.20, 0.13 and 0.17, which shows that the local chicken species and yellow feather broiler chickens cultivated independently in China have good ALV-B resistant genetic improvement potential, and the breeding materials resistant to ALV-B infection can be screened from the chicken species and applied to the cultivation of chicken varieties (strains) resistant to ALV-B infection, so as to prevent and control B subgroup avian leukosis.
TABLE 6 Chinese local chicken species tvb 3215-3216insCC Genotype frequency distribution of genetic resistance sites
Table 7 yellow-feathered broiler strain tvb of our country 3215-3216insCC Allele frequency distribution of genetic resistance sites
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (9)
1. Avian leukosis genetic resistance molecular marker tvb for chicken B subgroup 3215-3216insCC Characterized in that the molecular marker is tvb receptor gene, and CC bases are inserted between the 3215 th base and the 3216 th base.
2. Molecular marker tvb for detecting avian leukosis genetic resistance of subgroup B chickens according to claim 1 3215-3216insCC Is specific to the primer of (2)Characterized in that the nucleotide sequence of the primer is shown as SEQ ID NO:1 and SEQ ID NO: 2.
3. The avian leukosis genetic resistance molecular marker tvb of subgroup B chickens of claim 1 3215-3216insCC And/or the use of the primers of claim 2 for screening/identifying avian leukosis subgroup B genetically resistant chickens.
4. The use according to claim 3, characterized by the following steps:
extracting genome DNA of a sample to be detected, amplifying tvb receptor gene fragments, sequencing, and if CC homozygous insertion mutation exists at the 3215 th to 3216 th base positions of the tvb receptor gene sequence, obtaining the sample to be detected as subgroup B avian leukosis genetic resistance chicken;
if there is heterozygous insertion mutation tvb at the position of 3215-3216 base in tvb acceptor gene sequence s1/insCC The sample to be tested is a recessive gene carrying B subgroup avian leukosis genetic resistance;
if no insertion mutation occurs at the position between the 3215 th and 3216 th bases of the tvb receptor gene sequence, the sample to be tested is the avian leukosis susceptibility chicken of subgroup B.
5. The use according to claim 3, wherein the amplification system comprises: 1. Mu.L of DNA template, 2.5. Mu.L of 10 Xbuffer, 2. Mu.L of dNTPs, 1. Mu.L of upstream and downstream detection primer, 0.5. Mu.L of KOD-FX, and finally ddH 2 O was made up to 25. Mu.L.
6. The use according to claim 3, wherein the amplification program at the time of amplification is: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 30s, for a total of 35 cycles; extending at 72deg.C for 5min, and preserving at 4deg.C.
7. A kit for detecting/screening avian leukosis subgroup B genetically resistant chickens, comprising the primer of claim 2.
8. The use of the kit of claim 7 for screening breeding materials of a variety/strain of a avian leukosis-resistant subgroup B genetic subgroup B chicken.
9. Use of the molecular marker of claim 1 and/or the primer of claim 2 for screening breeding materials of a genetic anti-B subgroup avian leukosis chicken variety/strain.
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