CN109797230A - One breeder A subgroup avian leucosis resistance molecule marks tva507A>GAnd its application - Google Patents
One breeder A subgroup avian leucosis resistance molecule marks tva507A>GAnd its application Download PDFInfo
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- CN109797230A CN109797230A CN201910240248.8A CN201910240248A CN109797230A CN 109797230 A CN109797230 A CN 109797230A CN 201910240248 A CN201910240248 A CN 201910240248A CN 109797230 A CN109797230 A CN 109797230A
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Abstract
The invention discloses a breeder A subgroup avian leucosis resistance molecules to mark tva507A > GAnd its application.The present invention analyzes the hereditary variation of Chinese chicken kind tva gene, it was found that China's chicken kind tva gene DNA sequence (GenBank accession number: AY531262.1) at the 507th there are A > G base mutation, inventor, which further studies, confirms that the natural mutation of the tva gene can cause host to generate genetic resistance to the infection of ALV-A.The present invention is prepared for the detection kit of screening A subgroup avian leucosis resistance chicken using the molecular labeling, it can accurately, quickly determine that detecting sample is A subgroup avian leucosis resistance chicken or susceptible chicken, and it can be applied to the breeding material of screening A subgroup avian leucosis genetic resistance chicken breed (being), to carry out the breeding of A subgroup avian leucosis genetic resistance chicken breed (being), there is application and promotional value well.
Description
Technical field
The invention belongs to disease resistance breeding technology fields, anti-more particularly, to a breeder A subgroup avian leucosis
The relevant tva of property507A > GMolecular labeling and its application.
Background technique
A subgroup avian leucosis be by A subgroup avian leucosis virus (Avian Leukosis Virus subgroup A,
ALV-A a kind of chicken immune inhibition tumour sexually transmitted disease caused by).ALV-A mainly encroaches on the lymphocyte of chicken, and is transferred to
Other organs such as liver,kidney,spleen, lead to lymphocytoma and other malignant tumours.ALV-A can cause chicken infected generate that suppression is immunized
System, production performance decline, or even occur characteristic tumour and it is dead, cause huge economic loss to aviculture.So far,
The disease there is no commercialized vaccine and effective treatment method, mainly be arranged by eliminating positive chicken to purify breeder flock and bio-safety
It applies and is prevented.However, in recent years from commercial broiler, laying hen, Local chicken breeds, wild birds and external import vaccine and ancestral
For isolating ALV-A in chicken.As it can be seen that existing measure can not fully control generation and stream of the A subgroup avian leucosis in China
Row, the disease have become one of the major disease for threatening China's poultry husbandry (especially breeder industry) sustainable and healthy development.Therefore, it grinds
The new strategy that hair is more suitable for prevention and control China avian leukosis is extremely urgent.External existing research confirms, from host genetic resistance side
The breeding for disease resistance of face research avian leukosis has become the available strategy of the prevention and control disease.
ALV-A mediates intrusion host cell by the cell surface specific receptor Tva that tva acceptor gene encodes, and then sends out
Raw infection determines host cell to the ALV-A neurological susceptibility infected or resistance.The genetic mutation of tva acceptor gene will lead to receptor
One deficiency receptor protein being not suitable for as ALV-A receptor of complete missing or expression of albumen, so as to cause host cell
Genetic resistance is generated to the infection of ALV-A.
Therefore, identification A subgroup avian leucosis resistance molecule label, and it is applied to screening A subgroup avian leucosis genetic resistance
Distinguished germ plasm is the key that realize avian leukosis breeding for disease resistance to cultivate the anti-A subgroup avian leucosis chicken breed (being) of heredity.
Summary of the invention
The purpose of the invention is to overcome the prior art that can not fully control A subgroup avian leucosis in the generation of China
With popular deficiency, breeder A subgroup avian leucosis resistance molecule label tva is provided507A > GAnd its application.
The first purpose of the invention is to provide the molecular labelings of a breeder A subgroup avian leucosis genetic resistance.
A second object of the present invention is to provide the molecular labeling answering in screening A subgroup avian leucosis resistance chicken
With.
Third object of the present invention is to provide the molecular labelings in screening A subgroup avian leucosis resistance chicken detection reagent
Application in box.
Fourth object of the present invention is to provide the molecular labeling answering in chicken A subgroup avian leucosis breeding for disease resistance
With.
Fifth object of the present invention is to provide a pair of primers for detecting the molecular labeling.
Sixth object of the present invention is to provide detect the primer of the molecular labeling in screening A subgroup avian leucosis resistance
Application in chicken detection kit.
7th purpose of the invention is to provide application of the kit in chicken A subgroup avian leucosis breeding for disease resistance.
To achieve the goals above, the present invention is achieved by the following technical programs:
The present invention analyzes the hereditary variation situation of Chinese chicken kind tva acceptor gene, finds tva gene in Chinese chicken kind
There are the mutation of A > G, inventor passes through external real DNA sequence dna (GenBank accession number is AY531262.1) the 507th bit base
The infection that the tva gene natural mutation causes the anti-ALV-A of host is illustrated in verifying.Concrete reason is tva507A > GMutation leads to tva
1 breakdown point signal conserved sequence CTC of gene intronAThe A base mutation of C is G, is risen in tva gene mRNA cleavage reaction
Key effect.Speculate tva507A > GMutation may interfere with the shearing of tva mRNA precursor (pre-mRNA), and tva gene is caused to include
Son 1 remaines in tva mRNA transcript, leads to the tva gene expression one deficiency Tva receptor being not suitable for as ALV-A receptor
Albumen, so that the infection to ALV-A generates genetic resistance.
The present invention also further establishes the method and detection of screening A subgroup avian leucosis resistance chicken using the molecular labeling
Kit, testing result and external ALV-A attack malicious consistent.
Therefore claimed breeder A subgroup avian leucosis resistance molecule label, the molecular labeling are located at chicken
A > G base mutation at tva gene DNA sequence the 507th, is denoted as tva507A > GMutation.
If the 507th bit base of tva gene DNA sequence is wild type A/A, susceptible to the infection of ALV-A (non-resistant)；I.e.
Such as the tva of chicken to be measured507A > GResistance locus genotype is tva507A/A, then the individual is A subgroup avian leucosis susceptible chicken；
If the heterozygous mutation (A/G) that A sports G occurs for the 507th bit base of tva gene DNA sequence, which is A sub-
Group's avian leukosis susceptible chicken, but stealthy genetic resistance is generated to the infection of ALV-A.
The next generation of the male and female chicken post-coitum of heterozygous mutation (A/G), which can filter out to infect ALV-A, generates resistance
Phenotype；I.e. if the genotype of stock cock to be measured, Breeding hens is tva507A/G, there may be genotype by the next generation of post-coitum
For tva507G/GIndividual；
If the homozygous mutant (G/G) that A sports G occurs for the 507th bit base of tva gene order, have to ALV-A's
Infection generates the phenotype of resistance；I.e. if the genotype of chicken to be measured is tva507G/G, then the individual is A subgroup avian leucosis resistance chicken.
The following contents is also claimed in the present invention simultaneously:
Application of the molecular labeling in screening A subgroup avian leucosis resistance chicken detection kit.
Application of the molecular labeling in chicken A subgroup avian leucosis breeding for disease resistance.
Further, a pair of primer for detecting the molecular labeling, nucleotide sequence such as SEQ is also claimed in the present invention
Shown in ID NO:1.
Forward primer F:5 '-GGTTCAGCAGATCCTCATC-3 '
Reverse primer R:5 '-AGCACTACAAAGCCGTTC-3 '
Application of the reagent of the molecular labeling in screening A subgroup avian leucosis resistance chicken detection kit is detected,
It belongs to the scope of protection of the present invention.
Preferably, the reagent is primer described above.
Further, a kind of detection kit for screening A subgroup avian leucosis resistance chicken, including institute is also claimed in the present invention
The reagent stated.
Preferably, the reagent is primer described above.
Most preferably, the kit includes: detection primer and PCR amplification reagent；
Wherein, its nucleotide sequence of detection primer is as shown in SEQ ID NO:1；
PCR amplification reagent includes: 10 × buffer, dNTPs, KOD-FX, ddH2O.
Its application method the following steps are included:
S1, sample gene to be tested group DNA is extracted；
S2, PCR detection
PCR reaction system composition: 1 2.5 2 μ L of μ L, dNTPs of μ L, 10 × buffer of DNA profiling, upstream and downstream detection primer
(its nucleotide sequence is as shown in SEQ ID NO:1) 1 μ L, KOD-FX 0.5 μ L, ddH2O are mended to 25 μ L.
PCR response procedures: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 53 DEG C of annealing 60s, 72 DEG C of extension 90s, totally 35
Circulation；Extend 10min, last 4 DEG C of preservations after 72 DEG C.
Pcr amplification product send Sangon Biotech (Shanghai) Co., Ltd. after S3, the detection of 2% agarose gel electrophoresis
It purifies and is sequenced.
S4, result interpretation
If chicken tva to be measured507A > GThe genotype of resistance locus is wild type tva507A/A, then the individual is the white blood of A subgroup fowl
Sick susceptible chicken；
If chicken tva to be measured507A > GThe genotype of resistance locus is heterozygous mutant tva507A/G, then the individual does not have ALV-
The infection resistant phenotype of A is still A subgroup avian leucosis susceptible chicken, but carries the recessive gene to ALV-A infection resistance；
If chicken tva to be measured507A > GThe genotype of resistance locus is homozygous mutant tva507G/G, then the individual is A subgroup fowl
Anti-leukemic chicken.
The following contents is also claimed in the present invention simultaneously:
Detect application of the primer of the molecular labeling in screening A subgroup avian leucosis resistance chicken detection kit.
Application of the kit in chicken A subgroup avian leucosis breeding for disease resistance.
Compared with prior art, the invention has the following beneficial effects:
Present invention firstly discovers that the 507th bit base of tva gene DNA sequence is there are the mutation of A > G, inventor is further ground
Studying carefully confirms that the natural mutation of the tva gene can cause host to generate genetic resistance to the infection of ALV-A.The present invention utilizes this point
Son label is prepared for the detection kit of screening A subgroup avian leucosis resistance chicken, can accurately, quickly determine that detecting sample is A
Subgroup avian leucosis resistance chicken or susceptible chicken, and can be applied to screening A subgroup avian leucosis genetic resistance chicken breed (being)
Breeding material has application and promotion price well to carry out the breeding of A subgroup avian leucosis genetic resistance chicken breed (being)
Value.
Detailed description of the invention
Fig. 1 is 3 segment PCR amplification results of tva gene；M:DL2000marker；A-C: the PCR amplification of primer A, B, C
Product.
Fig. 2 is tva507A > GSite different genotype (wild type, heterozygous mutant and homozygous mutant) sequence figure
Spectrum.
Fig. 3 is RCASBP (A)-EGFP expression plasmid structural schematic diagram.
Fig. 4 is RCASBP (A)-GFP virus infection tva507A > GSite different genotype CEF cell.
Specific embodiment
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.
Test method as used in the following examples is conventional method unless otherwise specified；Used material, examination
Agent etc., unless otherwise specified, for the reagent and material commercially obtained.
Embodiment 1
The Genetic Variation Analysis of tva acceptor gene
One, experimental method
1, the design of primers of tva acceptor gene
It is reference, design with the DNA sequence dna (GenBank accession number: AY531262.1) of chicken tva gene in ncbi database
3 pairs of primers point, 3 segment PCR amplification tva full length gene sequence 3607bp (A sections, B sections and C sections), primer sequence, position and PCR
Amplified fragments size is as shown in table 1.
1 tva acceptor gene full length sequence PCR amplification information of table:
2, PCR reacts
The genomic DNA for extracting different Chinese chicken kind (including Local chicken breeds and Commercial lines) blood samples, is drawn with this 3 Duis
Object PCR amplification tva full length gene sequence.
PCR reaction system composition: DNA profiling 1 μ L, 10 × buffer 2.5 μ L, dNTPs 2 μ L, each 1 μ of upstream and downstream primer
L, KOD-FX 0.5 μ L, ddH2O is mended to 25 μ L.
PCR response procedures: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, annealing temperature (67 DEG C of A section, 60 DEG C of B, C section) are moved back
Fiery 30s, 72 DEG C of extension 90s, totally 35 recycle；Extend 10min, last 4 DEG C of preservations after 72 DEG C.
PCR product is detected through 2% agarose gel electrophoresis.
And send Sangon Biotech (Shanghai) Co., Ltd. to purify and be sequenced pcr amplification product, it utilizes
SeqMan functional module carries out splicing assembling to sequencing gained sequence in DNAstar, and carries out sequence alignment screening variant sites.
Two, experimental result
As a result as shown in Figure 1, PCR amplification goes out the purpose band of A, B and C segment of tva gene, clip size and expected knot
Fruit is consistent.
The Genetic Variation Analysis of tva acceptor gene finds that there are A mutation for tva gene DNA sequence the 507th in Chinese chicken kind
For the SNP site (tva of G507A > G), sequence map is as shown in Figure 2.Sequence is successively from top to bottom reference sequences in Fig. 2
The sequence of (wild type individual), heterozygous mutant individual and homozygous mutant individual, box show tva gene order 507
The mutational site A > G existing at bit base.
Embodiment 2
tva507A > GMutation causes the anti-ALV-A infection of host
One, experimental method
RCASBP (A)-EGFP expression plasmid (ALV-A report carrier) is successfully constructed, sees Fig. 3.By RCASBP (A)-
EGFP expression plasmid is transfected into DF-1 cell, 7 days after transfection, collects RCASBP (A)-GFP virus of cell supernatant (i.e.
ALV-A reporter virus, the virus can infect chicken fibroblasts CEF).Utilize the tva of RCASBP (A)-GFP virus infection507A > G
Mutational site wild type tva507A/A, heterozygous mutant tva507A/GWith homozygous mutant tva507G/GCEF in embodiment 1 (using examining
It is prepared by the chicken of survey), 1 after infection, 2,4,7 days, Flow Cytometry is utilized to detect RCASBP (A)-GFP virus infection tva507A > G
The case where mutational site different genotype CEF.
Two, experimental result
As a result as shown in figure 3, wild type tva507A/ACEF and heterozygous mutant tva507A/GCEF is susceptible to ALV-A, and pure
Close saltant type tva507G/GThe infection of the anti-ALV-A of CEF, it was demonstrated that tva507A > GNatural mutation leads to the infection of the anti-ALV-A of host.
Embodiment 3
A kind of kit of the infection resistance for the ALV-A detecting chicken
One, it forms
Detection primer and PCR amplification reagent,
Wherein, its nucleotide sequence of detection primer is as shown in SEQ ID NO:1,
PCR amplification reagent includes: 10 × buffer, dNTPs, KOD-FX, ddH2O。
Two, application method
1, sample gene to be tested group DNA is extracted；
2, PCR is detected
PCR reaction system composition: 1 2.5 2 μ L of μ L, dNTPs of μ L, 10 × buffer of DNA profiling, upstream and downstream detection primer
(its nucleotide sequence is as shown in SEQ ID NO:1) 1 μ L, KOD-FX 0.5 μ L, ddH2O is mended to 25 μ L.
PCR response procedures: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 53 DEG C of annealing 30s, 72 DEG C of extension 90s, totally 35
Circulation；Extend 10min, last 4 DEG C of preservations after 72 DEG C.
3, pcr amplification product send Sangon Biotech (Shanghai) Co., Ltd. after the detection of 2% agarose gel electrophoresis
It purifies and is sequenced.
4, result interpretation
The standard of perfection of 2 A subgroup avian leucosis genetic resistance chicken of table
If tva507A > GResistance locus genotype is wild type tva507A/A, then, should to the non-resistant (susceptible) of ALV-A infection
Individual is A subgroup avian leucosis susceptible chicken；
If tva507A > GResistance locus genotype is tva507A/G, then susceptible to the infection of ALV-A, which is A subgroup fowl
Leukaemia susceptible chicken, but carry A subgroup avian leucosis recessive inheritance resistant gene；
If tva507A > GResistance locus genotype is tva507G/G, then resistance is generated to the infection of ALV-A, which is A sub-
Group avian leukosis resistance chicken.
Embodiment 4
The Resistance detecting of chicken In vivo infection ALV-A
One, experimental method
The resistance situation that the kit detection chicken established with embodiment 3 infects ALV-A, to same this progress of lot sample ALV-
The test of A In vivo infection host, concrete operations are as follows:
By tva507A > GMutation wild type, heterozygous mutant and homozygous mutant 1 day-old chicks be grouped at random, raising in
Equivalent ALV-A (GD08 plants) are injected intraperitoneally in isolator, 1 age in days and 5 ages in days respectively.ALV-A attacked poison after 1 month, acquired chicken
Blood sample extracts blood sample total serum IgE using TRIZOL kit.
Design the RT-PCR amplification upstream primer and downstream primer of ALV-A-env:
Env-F:5 '-GGATGAGGTGACTAAGAAAG-3 '；
Env-R:5 '-AGAGAAAGAGGGGTGTCTAAGGAGA-3 '.
RT-PCR expands the env gene coded sequence of ALV-A, and RT-PCR expanding fragment length is 692bp.It utilizesOne Step RT-PCR Kit Ver.2 kit carries out RT-PCR amplification, PCR response procedures: 50 DEG C of reversions
Record 30min；94 DEG C of 30s, 56 DEG C of 60s, 72 DEG C of 60s, 35 circulations；Extend 10min after 72 DEG C.PCR product is solidifying in 2% agarose
Gel electrophoresis detection, such as observes the purpose band of 692bp, then viremia virusemia (ALV-A is positive) occurs for the sample, such as without purpose item
The amplification of band, then there is no viremia virusemia for the sample (ALV-A is negative).
Two, experimental result
ALV-A challenge test the result shows that: tva507A > GMutational site wild type tva507A/AChicken (22) attacks the open country ALV-A
It is the ALV-A positive, heterozygous mutant tva after poison507A/GChicken (30) is also the ALV-A positive after attacking the wild poison of ALV-A, and
Homozygous mutant tva507G/GChicken (25) is ALV-A feminine gender after attacking the wild poison of ALV-A, is shown in Table 3.
3 ALV-A infection rate of table
ALV-A challenge test is consistent with the kit test result of embodiment 3.
Embodiment 5
The screening and application of ALV-A genetic resistance chicken
One, experimental method
8 Local chicken breeds and 10 commercial meat chickens strains totally 966 anti-ALV-A of sample are detected with the kit of embodiment 3
Resistance and genetic improvement pot ential.
Two, experimental result
It the results are shown in Table 4.Table 4 the result shows that the Local chicken breeds such as Qingyuan Chicken, apricot bramble finch, health and happiness chicken and blue-shelled egg layer with
And the commercial meat chickens strain such as CB01, CB07 and CB10 has good anti-ALV-A genetic improvement pot ential, it can be from these kinds (being)
In filter out the breeding material for cultivating anti-ALV-A infection, and apply to the breeding of ALV-A genetic resistance chicken breed (being), to prevent
Control A subgroup avian leucosis.
The Chinese chicken kind tva of table 4507A > GThe genotype frequency in mutational site
Remarks: CB01-CB10 etc. represents 10 commercial meat chickens strains.
Claims (9)
1. a breeder A subgroup avian leucosis resistance molecule marks, which is characterized in that the molecular labeling is located at chicken tva gene DNA
Sequence GenBank accession number is A > G base mutation at the 507th of AY531262.1.
2. application of the molecular labeling described in claim 1 in preparation screening A subgroup avian leucosis resistance chicken detection kit.
3. application of the molecular labeling described in claim 1 in chicken A subgroup avian leucosis breeding for disease resistance.
4. the primer of molecular labeling described in a pair of detection claim 1, which is characterized in that its nucleotide sequence such as SEQ ID NO:
Shown in 1.
5. detecting the reagent of molecular labeling described in claim 1 in screening A subgroup avian leucosis resistance chicken detection kit
Using.
6. applying according to claim 5, which is characterized in that the reagent is primer described in claim 4.
7. a kind of kit for detecting chicken A subgroup avian leucosis resistance, which is characterized in that including the examination described in claim 5
Agent.
8. kit according to claim 7, which is characterized in that the reagent is primer described in claim 4.
9. application of the kit described in claim 7 in chicken A subgroup avian leucosis breeding for disease resistance.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114410808A (en) * | 2022-03-30 | 2022-04-29 | 华南农业大学 | Genetic resistance molecular marker for avian A, K subgroup avian leukosis and application thereof |
-
2019
- 2019-03-27 CN CN201910240248.8A patent/CN109797230A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| ELLEDER,D. AND SVOBODA,J: "Gallus gallus Tva (tva) gene, tva-s allele, complete cds, alternatively spliced", 《GENBANK》 * |
| MARKÉTA REINIŠOVÁ等: "Intronic Deletions That Disrupt mRNA Splicing of the tva Receptor Gene Result in Decreased Susceptibility to Infection by Avian Sarcoma and Leukosis Virus Subgroup A", 《JOURNAL OF VIROLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114410808A (en) * | 2022-03-30 | 2022-04-29 | 华南农业大学 | Genetic resistance molecular marker for avian A, K subgroup avian leukosis and application thereof |
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