CN105441552A - Chicken B subgroup avian leukosis resistantmolecular marker tvb<3731-3732insA> and molecular diagnosis method thereof - Google Patents

Chicken B subgroup avian leukosis resistantmolecular marker tvb<3731-3732insA> and molecular diagnosis method thereof Download PDF

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CN105441552A
CN105441552A CN201511005987.7A CN201511005987A CN105441552A CN 105441552 A CN105441552 A CN 105441552A CN 201511005987 A CN201511005987 A CN 201511005987A CN 105441552 A CN105441552 A CN 105441552A
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tvb
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alv
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谢青梅
陈伟国
蔺文成
舒鼎铭
李昕键
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South China Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The invention belongs to the technical field of resistant variety breeding, and in particular discloses a chicken B subgroup avian leukosis resistantmolecular marker tvb<3731-3732insA> and a molecular diagnosis method thereof. The invention analyzes genetic variation of a Chinese chicken breed tvb receptor gene for the first time and discovers that the 3731st and the 3732nd sites of the Chinese chicken breed tvb receptor gene have A insertion mutation, and the inventor proves, from in vivo and in vitro experiments two aspects, that the natural mutation causes a host to generate genetic resistance to infection of ALV-B; moreover, the inventor establishes a molecular diagnosis method for a B subgroup avian leukosis resistant genetic marker, and the method can be applied to screening breeding materials of ALV-B genetic resistant chicken varieties (strains) so as to carry out breeding of the ALV-B genetic resistant chicken varieties (strains).

Description

Chicken B subgroup avian leucosis resistance molecule mark tvb 3731-3732insAand molecular diagnosis method
Technical field
The present invention relates to resistant variety breeding technique field, more specifically, relate to chicken B subgroup avian leucosis resistance molecule mark tvb 3731-3732insAand molecular diagnosis method.
Background technology
Avian leukosis is a kind of inhibitive ability of immunity tumprigenicity transmissible disease caused by avian leukosis virus (AvianLeukosisVirus, ALV), and this disease can cause and multiplely has communicable optimum and malignant tumour.The avian leukosis virus of infected chicken comprises ALVA-E and J totally 6 subgroups.B subgroup avian leucosis virus (ALV-B) is one of main pathogen of China's avian leukosis, can make chicken infected generation immunosuppression, production performance declines and characteristic tumour and dead, cause huge financial loss to world's aviculture.At present, this disease there is no commercialized vaccine and effective methods for the treatment of, purifies breeder flock and strengthens bio-security prevent mainly through superseded positive chicken.But, existing measure can not control AVL-B at the Occurrence & epidemic of China.In recent years epidemiology survey result display, AVL-B is ubiquity in Chinese commodity broiler chicken, laying hen, regional strain chicken and wild birds, has become one of major disease threatening China's poultry husbandry (especially planting chicken industry) sustainable and healthy development.Therefore, the avian leukosis prevention and control New Policy that research and development are more suitable for China's truth is badly in need of.External existing research confirms, has become the available strategy of this disease of prevention and control from the hereditary breeding for disease resistance of host genetic resistance aspect research avian leukosis.
Excavate avian leukosis genetic resistance functional gene or gene locus, and to be applied to screening avian leukosis genetic resistance distinguished germ plasm to cultivate avian leukosis resistance chicken breed (being) be the key realizing the hereditary breeding for disease resistance of avian leukosis.
Summary of the invention
The present invention, in order to overcome the above-mentioned deficiency of prior art, provides a kind of chicken B subgroup avian leucosis resistance molecule to mark.
Another object of the present invention is to provide a kind of method diagnosing B subgroup avian leucosis resistance chicken.
To achieve these goals, the present invention is achieved by following scheme:
A chicken B subgroup avian leucosis resistance molecule mark, described molecule marker is the A insertion mutation of tvb acceptor gene the 3731st and 3732.This site is referred to as tvb 3731-3732insAmutational site.
The present invention analyzes the heritable variation of Chinese chicken kind tvb acceptor gene first, to find in Chinese chicken kind that tvb acceptor gene the 3731st and 3732 exist A insertion mutation, from external, experiment in vivo two aspects, contriver confirms that this spontaneous mutation causes host to produce genetic resistance to the infection of ALV-B.Concrete reason is that tvb acceptor gene the 3731st and 3732 exist A insertion mutation and are positioned at tvb acceptor gene the 4th exon, thus cause tvb acceptor gene that displacement coding occurs, infer that it may cause tvb Receptor Gene Expression deficient protein be not suitable for as virus receptor, thus cause host to produce genetic resistance to the infection of ALV-B.
Therefore, the present invention protects the application of molecule marker described above in diagnosis B subgroup avian leucosis resistance chicken.
According to the A insertion mutation of tvb acceptor gene the 3731st and 3732, the present invention establishes a kind of method diagnosing B subgroup avian leucosis resistance chicken, specifically comprises the steps:
S1. extract chicken blood DNA to be measured, comprise tvb with the primer amplification of P4 shown in SEQIDNO:7 ~ 8 3731-3732insAthe tvb acceptor gene fragment in mutational site, judges the genotype of chicken to be measured after order-checking;
S2. the genotype as chicken to be measured is tvb insA/insA, be then B subgroup avian leucosis resistance chicken, the genotype as chicken to be measured is tvb s/insAor tvb s/S, be then B subgroup avian leucosis susceptibility chicken.
The present invention is when analyzing the heritable variation of Chinese chicken kind tvb acceptor gene, except finding the A insertion mutation of tvb acceptor gene the 3731st and 3732, also find to there is AG insertion mutation in tvb acceptor gene the 3667th and 3668 nucleotide position, these two spontaneous mutations are all positioned at tvb acceptor gene the 4th exon, thus all cause tvb acceptor gene that displacement coding occurs, infer that it may cause tvb Receptor Gene Expression deficient protein be not suitable for as virus receptor, thus cause host to produce genetic resistance to the infection of ALV-B.Therefore, the present invention can set up a kind of method diagnosing B subgroup avian leucosis resistance chicken according to these two spontaneous mutation sites, specifically comprises the steps:
S1. extract chicken blood DNA to be measured, comprise tvb with the primer amplification of P4 shown in SEQIDNO:7 ~ 8 3667-3668insAGand tvb 3731-3732insAthe tvb acceptor gene fragment in mutational site, judges the genotype of chicken to be measured after order-checking;
S2. if chicken to be measured is at tvb 3667-3668insAGthe genotype in mutational site is tvb insAG/insAG, then ALV-B is infected and produces genetic resistance, if chicken to be measured is at tvb 3731-3732insAthe genotype in mutational site is tvb insA/insA, then ALV-B is infected and produces genetic resistance, if chicken to be measured is at tvb 3667-3668insAGand tvb 3731-3732insAthe genotype in mutational site is respectively tvb insAG/insAGand tvb insA/insA, then ALV-B is infected and produces genetic resistance.
Compared with prior art, the present invention has following beneficial effect:
There is A insertion mutation in Late Cambrian of the present invention tvb acceptor gene the 3731st and 3732 in Chinese chicken kind, and the present invention verifies that this sudden change causes host to produce genetic resistance to the infection of ALV-B first.Therefore, be very accurately using this mutational site as the molecule marker of qualification chicken B subgroup avian leucosis resistance.The molecular diagnosis method of the anti-B subgroup avian leucosis genetic marker set up with this mutational site, can be applicable to the breeding material screening ALV-B genetic resistance chicken breed (being), thus carries out the seed selection of ALV-B genetic resistance chicken breed (being).
The present invention is when analyzing the heritable variation of Chinese chicken kind tvb acceptor gene, except finding the A insertion mutation of tvb acceptor gene the 3731st and 3732, also find to there is AG insertion mutation in tvb acceptor gene the 3667th and 3668 nucleotide position, the method of the diagnosis B subgroup avian leucosis resistance chicken of setting up with these two spontaneous mutation sites, can be applicable to the breeding material screening ALV-B genetic resistance chicken breed (being), thus carry out the seed selection of ALV-B genetic resistance chicken breed (being).
Accompanying drawing explanation
Fig. 1 is Research Thinking of the present invention.
Fig. 2 is RCASBP (B) virus infection tvb 3667-3668insAGsite different genotype CEF cell.
Fig. 3 is RCASBP (B) virus infection tvb 3731-3732insAsite different genotype CEF cell.
Embodiment
To make the present invention below in conjunction with Figure of description and specific embodiment and elaborating further, described embodiment, only for explaining the present invention, is not intended to limit scope of the present invention.The test method used in following embodiment if no special instructions, is ordinary method; The material used, reagent etc. if no special instructions, are the reagent that can obtain from commercial channels and material.
The Genetic Variation Analysis of embodiment 1tvb acceptor gene
According to tvb acceptor gene known dna sequence (GenBank accession number: NC_006109.3), design 5 couples of primer amplification tvb full length gene sequence 5425bp, primer sequence, position and pcr amplified fragment size are as shown in table 1.
Table 1 is tvb acceptor gene full length sequence pcr amplification information
Extract the genomic dna of Chinese chicken kind blood sample, with these 5 pairs of primer amplification tvb acceptor gene full length sequences.PCR reaction system forms: template 1 μ L, 10 × buffer2.5 μ L, dNTPs2 μ L, each 1 μ L, the KOD-FX0.25 μ L of upstream and downstream primer, and sterilized water is mended to 25 μ L.PCR response procedures: 94 DEG C of denaturation 3min, 1 circulation; 94 DEG C of 45s, 58 ~ 65 DEG C of (different primers annealing temperature) 90s, 72 DEG C of 60s, 35 circulations; 10min is extended after 72 DEG C.PCR primer can be observed the specific band that P1 ~ P5 primer increases separately when 2% agarose gel electrophoresis detects, pcr amplification product is carried out cloning and sequencing, analyzes the heritable variation of tvb acceptor gene.Contriver's stochastic analysis heritable variation of multiple Chinese chicken breed (being) tvb acceptor gene, finds that Chinese chicken kind tvb acceptor gene exists 2 kinds of spontaneous mutations: in tvb acceptor gene the 3667th and 3668 nucleotide position, insert AG(tvb 3667-3668insAG), in tvb acceptor gene the 3731st and 3732 nucleotide position, insert A (tvb 3731 -3732insA).
Embodiment 2tvb 3667-3668insAG, tvb 3731-3672insAsudden change causes the functional verification that the anti-ALV-B of host infects.
1, the functional verification of cell in vitro: build RCASBP (B) EGFP expression plasmid, transfection DF-I cell is after 7 days, (supernatant liquor is containing RCASBP (B) virus of carrying EGFP fluorescin for collecting cell supernatant liquor, i.e. ALV-B virus, can with postoperative infection DF-I and CEF cell), after measuring virus infection unit (IU), packing is stored in-80 DEG C.RCASBP (B) virus infects tvb respectively 3667 -3668insAG, tvb 3731-3732insAmutational site different genotype CEF, comprises wild-type tvb s/ScEF(is to ALV-B susceptible), heterozygous mutant tvb s/insAGand tvb s/insAcEF and homozygous mutant tvb insAG/insAGand tvb insA/insAcEF, infects latter 1,2,4,7 day, utilizes Flow Cytometry to detect different time points tvb 3667-3668insAG, tvb 3731-3732insAsudden change different genotype CEF infects the positive cell rate after RCASBP (B) virus, determines tvb 3667-3668insAG, tvb 3731-3732insAthe trend of sudden change different genotype CEF Infection in Vitro RCASBP (B) EGFP virus.In vitro cell experiment result shows: wild-type tvb s/ScEF and heterozygous mutant tvb s/insAGand tvb s/insAcEF is to ALV-B susceptible, and homozygous mutant tvb insAG/insAGand tvb insA/ insAthe infection of the anti-ALV-B of CEF, confirms these 2 kinds of spontaneous mutation tvb of tvb acceptor gene 3667-3668insAGand tvb 3731-3732insAcause the infection of the anti-ALV-B of host, see Fig. 2 and Fig. 3 respectively.
2, the functional verification of challenge test in body: carry tvb 3667-3668insAG, tvb 3731-3732insAthe chicken random packet of mutational site different genotype, raises in shield retaining, and 1 age in days and 5 ages in days are the wild poison of abdominal injection equivalent ALV-B respectively.The wild poison of body inside fire attack ALV-B, after 1 month, gathers tvb 3667-3668insAG, tvb 3731-3732insAthe blood sample of mutational site different genotype chicken, utilizes TRIZOL test kit extracting blood sample total serum IgE.The env gene coded sequence of RT-PCR amplification ALV-B, RT-PCR amplification upstream primer (5'-CCTGGAAAGGTGAGCAAG-3') of design ALV-B-env and downstream primer (5'-TGGAGGGAGAATCGTGAA-3'), RT-PCR expanding fragment length is 966bp.PrimeScriptROneStepRT-PCRKitVer.2 test kit is utilized to carry out RT-PCR amplification, PCR response procedures: 50 DEG C of reverse transcription 30min; 94 DEG C of 30s, 56 DEG C of 60s, 72 DEG C of 60s, 30 circulations; 10min is extended after 72 DEG C.PCR primer detects at 2% agarose gel electrophoresis, and as observed the object band of 966bp, then this sample generation viremia (ALV-B is positive), as the amplification without object band, then viremia (ALV-B is negative) does not occur this sample.Body inside fire attack poison test-results shows: tvb 3667-3668insAGmutational site wild-type tvb s/Schicken (16) and tvb 3731-3732insAmutational site wild-type tvb s/Schicken (12) is the ALV-B positive, heterozygous mutant tvb after attacking the wild poison of ALV-B s/insAGchicken (26) and tvb s/insAchicken (33) is also the ALV-B positive after attacking the wild poison of ALV-B, and homozygous mutant tvb insAG/insAGchicken (18) and tvb insA/insAchicken (15) is ALV-B feminine gender, in table 2 after attacking the wild poison of ALV-B.
Table 2 is tvb 3667-3668insAG, tvb 3731-3732insAmutational site different genotype 1 day-old chicks
Attack the wild poison of ALV-B ALV-B positive rate after 1 month
Remarks: ALV-B is B subgroup avian leucosis virus (lower same)
Embodiment 3 sets up the standard of perfection of ALV-B genetic resistance chicken
Utilize the P4 primer PCR in table 1 to increase and comprise tvb 3667-3668insAGand tvb 3731-3732insAthe tvb acceptor gene region in 2 kinds of mutational sites, according to P4 primer PCR amplified production sequencing result, formulates the method standard (see table 3) judging B subgroup avian leucosis genetic resistance chicken.
The standard of perfection of table 3B subgroup avian leucosis genetic resistance chicken
Remarks: if detected chicken is with A or B or any one situation simultaneously with A and B, then this chicken is judged to produce genetic resistance to the infection of ALV-B.
The standard of perfection of the B subgroup avian leucosis genetic resistance chicken of setting up is: if 1 certain chicken is at tvb 3667-3668insAGthe genotype in mutational site is tvb insAG/insAG, then this chicken infects ALV-B and produces genetic resistance; If 2 certain chickens are at tvb 3731-3732insAthe genotype in mutational site is tvb insA/insA, then this chicken infects ALV-B and produces genetic resistance; If 3 certain chickens are at tvb 3667-3668insAGand tvb 3731-3732insAthe genotype in mutational site is tvb insAG/insAGand tvb insA/insA, then this chicken also infects ALV-B and produces genetic resistance.
The screening of embodiment 4ALV-B genetic resistance chicken and utilization
Assess the genetic improvement pot ential of 10 Local chicken breeds and 8 commercial meat chickens strains totally 989 anti-ALV-B of sample with the B subgroup avian leucosis genetic resistance chicken authentication method set up, the results are shown in Table 4.Table 4 result shows that the commercial meat chickens such as Local chicken breeds and C01, C03 and C08 strains such as health and happiness chicken, blue-shelled egg layer, Ningchang decoction and Jining one hundred days chicken have good anti-ALV-B genetic improvement pot ential, the breeding material cultivated anti-ALV-B and infect can be filtered out from these kinds (being), and apply to the seed selection of ALV-B genetic resistance chicken breed (being), with prevention and control B subgroup avian leucosis.
The Chinese chicken kind tvb of table 4 3667-3668insAGand tvb 3731-3732insAthe genotype frequency in mutational site
Remarks: CB01-CB08 etc. represent 8 commercial meat chickens strains.
The result explanation of table 4, the A insertion mutation existed in AG insertion mutation and the 3731st and 3732 nucleotide positions is there is in tvb acceptor gene the 3667th and 3668 nucleotide position, only exist in part chicken breed (being), and, even same chicken breed (being), neither all undergo mutation by all chickens.52 health and happiness chickens such as detecting, wherein 26 just do not suddenly change, and in remaining 26, have some to be homozygous mutations, and some are heterozygous mutants.

Claims (5)

1. chicken B subgroup avian leucosis resistance molecule mark, it is characterized in that, described molecule marker is the A insertion mutation of tvb acceptor gene the 3731st and 3732.
2. the application of molecule marker according to claim 1 in diagnosis B subgroup avian leucosis resistance chicken.
3. diagnose a method for B subgroup avian leucosis resistance chicken, it is characterized in that, comprise the steps:
S1. extract chicken blood DNA to be measured, comprise tvb with the primer amplification of P4 shown in SEQIDNO:7 ~ 8 3731-3732insAthe tvb acceptor gene fragment in mutational site, judges the genotype of chicken to be measured after order-checking;
S2. the genotype as chicken to be measured is tvb insA/insA, be then B subgroup avian leucosis resistance chicken, the genotype as chicken to be measured is tvb s/insAor tvb s/S, be then B subgroup avian leucosis susceptibility chicken.
4.tvb 3667-3668insAGand tvb 3731-3732insAthe application suddenlyd change in diagnosis B subgroup avian leucosis resistance chicken, described tvb 3667-3668insAGfor the AG insertion mutation of tvb acceptor gene the 3667th and 3668, tvb 3731-3732insAfor the A insertion mutation of tvb acceptor gene the 3731st and 3732.
5. diagnose a method for B subgroup avian leucosis resistance chicken, it is characterized in that, comprise the steps:
S1. extract chicken blood DNA to be measured, comprise tvb with the primer amplification of P4 shown in SEQIDNO:7 ~ 8 3667-3668insAGand tvb 3731-3732insAthe tvb acceptor gene fragment in mutational site, judges the genotype of chicken to be measured after order-checking;
S2. if chicken to be measured is at tvb 3667-3668insAGthe genotype in mutational site is tvb insAG/insAG, then ALV-B is infected and produces genetic resistance, if chicken to be measured is at tvb 3731-3732insAthe genotype in mutational site is tvb insA/insA, then ALV-B is infected and produces genetic resistance, if chicken to be measured is at tvb 3667-3668insAGand tvb 3731-3732insAthe genotype in mutational site is respectively tvb insAG/insAGand tvb insA/insA, then ALV-B is infected and produces genetic resistance.
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CN109811064A (en) * 2019-04-02 2019-05-28 华南农业大学 One kind molecular labeling relevant to chicken J subgroup avian leucosis resistance and its application

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CN104152445A (en) * 2014-07-30 2014-11-19 华南农业大学 Chicken B,D,E-subgroup avian leukaemia genetic resistance related mononucleotide polymorphism molecular marker and application thereof

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CN109811064B (en) * 2019-04-02 2023-12-05 华南农业大学 Molecular marker related to avian leukosis resistance of chicken J subgroup and application thereof

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