CN104911273A - Chicken FABP1 gene molecular genetic marker related to chicken good production traits and application thereof - Google Patents
Chicken FABP1 gene molecular genetic marker related to chicken good production traits and application thereof Download PDFInfo
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Abstract
The invention discloses a chicken FABP1 gene molecular genetic marker related to chicken good production traits, which is a polymorphism site on a chicken FABP1 gene mononucleotide polymorphism sequence. The site is a base polymorphism site of which the 83rd position on the FABP1 gene sequence second exon is C or T, and has three genotypes (CC, CT and TT), wherein the TT type is a good slaughter trait molecular genetic marker for meat chickens. The invention also discloses application of the marker in auxiliary screening or prediction of meat chicken species with good slaughter traits. The experiment proves that the polymorphism and productivity of the specific enzyme digestion site are related, and most measurement indexes of the individuals with the genotype of TT are higher than those of the individuals with the genotype of CC or CT, which indicates that the individuals with the genotype of TT can be used as breeding hens for culturing chicken species with good slaughter traits.
Description
Technical field
The invention belongs to field of molecular biotechnology, relate to a kind of excellent slaughter trait molecular genetic marker of table poultry and possess the application in the table poultry kind of excellent slaughter trait at assisting sifting or prediction.
Background technology
At present for the description that molecular genetic marker is comparatively complete, refer to be easy to identify, follow Mendelian inheritance pattern, there is a certain class table feature or genetic material that individual specificity or the regularity of distribution have kind of matter feature, its scope comprises: the variation etc. of the variation features of gene or genetic material product, chromosomal morphological variant, gene or genetic material itself as gene or genetic material carrier.DNA molecular Genetic Markers is a kind of emerging molecular marking technique, is particularly widely used in molecular genetics at molecular biology at present.Because Eukaryotic genetic information is all stored in the DNA sequence dna of karyomit(e) and organelle gene group, therefore theoretically, the molecule marker on DNA level is the most stable the most reliable in all genetic markers.The development of modern molecular biology technique makes directly to utilize the variation of DNA sequence dna nucleotide to become possibility as genetic marker.
Single nucleotide polymorphism (single nucleotide polymorphism, SNP) refer to the DNA sequence polymorphism that the variation of single core thuja acid in chromogene group level causes, and wherein the frequency of minimum a kind of allelotrope in colony is not less than 1%.It comprises the conversion of base, transversion, the form such as insertion and disappearance.Because SNP has the advantage such as automatization of high-density, typical, genetic stability and easy Realization analysis, therefore as a class genetic marker, SNP is widely used in genetic analysis.
Fatty acid binding protein (Fatty acid binding protein, FABP) is extensively present in vertebrates and the vertebrate tenuigenin of expense, belongs to lipid binding protein superfamily member.FABP albumen is albumen in gang's small molecular cell, has very high avidity to longer chain fatty acid, can lipid acid be transferred in cell from cytolemma, play an important role in the metabolism of longer chain fatty acid.Had been found that the FABP albumen of 9 types up to now, the FABP albumen of every type is at different tissues and cells.
FABP1 is liver type fatty acid binding protein, belongs to a member in FABP superfamily, is present in liver cell and small intestinal cell kytoplasm, has high affinity to cetylate, oleic acid and stearate etc., participates in picked-up and the transhipment of lipid acid.At liver, longer chain fatty acid generally absorbs into born of the same parents by the FABP fatty acid transport protein be positioned on liver plasma membrane, subsequently FABP1 auxiliary under be transported to plastosome, peroxysome, endoplasmic reticulum etc. and carry out β-oxidation, thus the fatty acid metabolism in regulating cell, finally make body fat acid metabolic keep relative equilibrium.As molecule marker, FABP1 shows the polymorphism of high heredity.Fatty character is the important indicator evaluating chicken meat quality, and the growth of this type of proterties can directly affect its commodity performance.Therefore, the sign for the SNPs of regulation and control fatty character development gene has a very important role for improving its production performance by the molecular breeding of broiler chicken and assisted Selection.Relate in the colony of chicken in applicant's related experiment, its meat productivity of the individuality that FABP1 mononucleotide does not suddenly change and meat are all better than that homozygous mutation is individual and heterozygous mutant is individual, embody FABP1 gene SNP s intuitively to the impact of the chicken production traits data.If this discovery is applied in the research work such as the breeding of high-quality poultry, the improved seeds be expected to for cultivating high yield and high quality meat proterties are offered help.Through retrieval, current FABP1 gene SNP s is as genetic marker, and the excellent slaughter trait molecular genetic marker particularly as table poultry is applied to screening or predicts that the table poultry kind possessing excellent slaughter trait have not been reported.
Summary of the invention
For the deficiencies in the prior art, the problem to be solved in the present invention is to provide a kind of chicken FABP1 gene molecule genetic marker relevant to the excellent production traits of chicken and possesses the application in the table poultry kind of excellent slaughter trait at assisting sifting or prediction.
The chicken FABP1 gene molecule genetic marker relevant to the excellent production traits of chicken of the present invention is the pleomorphism site in chicken FABP1 gene mononucleotide polymorphism sequence, it is characterized in that: the pleomorphism site of described chicken FABP1 gene mononucleotide polymorphism sequence is the nucleotide polymorphisms site of C or T the 83rd of FABP1 gene order Second Exon, show as CC, CT or TT tri-kinds of genotype, wherein TT type is the excellent slaughter trait molecular genetic marker of table poultry.
The chicken FABP1 gene molecule genetic marker relevant to the excellent production traits of chicken of the present invention possesses the application in the table poultry kind of excellent slaughter trait at assisting sifting or prediction.
The concrete grammar of above-mentioned application is: with chicken FABPl genomic dna to be detected for template, pcr amplification is carried out with primer pair FABP1 E2F/E2R, increased laggard row agarose gel electrophoresis, then uses specific restriction endonuclease to carry out enzyme to the object fragment amplified and cut; Afterwards again agarose gel electrophoresis is carried out to digestion products, detect the clip size of digestion products, analyze the nucleotide polymorphisms site determining chicken FABP1 gene, when the 83rd of FABP1 gene order Second Exon is C or T, and genotype is when being TT type, can judge that chicken breed to be detected is as the kind chicken with excellent slaughter trait;
Wherein:
The nucleotides sequence of described primer pair FABP1 E2F/E2R is classified as:
FABP1 E2F：CTTGAGAGGCTCTTCCACAATAG；
FABP1 E2R：GTTGGACTGGATAGCCTTTAAGTTC；
The response procedures of described pcr amplification is: 95 DEG C of denaturation 4-5min; 94 DEG C of sex change 30-35s, 55-61 DEG C of annealing 30-35s, 72 DEG C extend 1min, 28-35 circulation, and last 72 DEG C extend 10min, and are placed in 4 DEG C of preservations;
Described specific restriction endonuclease is: ASP MDI, Bsp AI, MboI, CacI and HacI, and it is the following sequence amplified in goal gene that its enzyme cuts action site: 5 '
↓gATC ... 3 ' or 3 ' ... CTAG
↑5 '.
Aforesaid method is preferred embodiment: for FABP1 E2F/E2R primer pair, pcr amplification program is: 95 DEG C of denaturation 4min; 94 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C extend 1min, 35 circulations, and last 72 DEG C extend 10min, and are placed in 4 DEG C of preservations.Described specific restriction endonuclease is: MboI.
The single nucleotide polymorphism of the generation on the method rapid screening chicken FABP1 gene Second Exon site that the present invention utilizes restriction nuclease endoenzyme to cut detects, by carrying out gene type and gene frequency analysis to the SNP of chicken breed, enzyme is cut between polymorphic site and chicken growth traits simultaneously and carry out association analysis, find the SNPs relevant to chicken growth traits as molecule marker, and the excellent slaughter trait molecular genetic marker using this function SNPs as table poultry, widespread use in the table poultry kind of excellent slaughter trait is possessed at assisting sifting or prediction, accelerate the seed selection speed of breeding chicken and improve population quality.
The present invention, by being restriction enzyme site with SNP site, selects suitable specific endonucleases, and the DNA fragmentation then cut out according to enzyme after SNP sudden change varies in size and determines chicken FABP1 gene mononucleotide polymorphism feature.
According to chicken FABP1 gene mononucleotide polymorphism feature, it is the nucleotide polymorphisms site of C or T the 83rd of FABP1 gene order Second Exon that the present invention determines that the pleomorphism site of chicken FABP1 gene mononucleotide polymorphism sequence is, show as CC, CT or TT tri-kinds of genotype, wherein TT type is the excellent slaughter trait molecular genetic marker target of table poultry.Experiment finds: CC, CT or TT tri-kinds of genotypic chickens have very big difference in meat productivity and fleshy optimization etc., TT type chicken slaughter trait (meat productivity and meat) is obviously better than CC, CT two type, so this genotypic chick can be cultivated as the kind chicken possessing excellent slaughter trait, this has also affirmed that genotype is the chicken of TT can cultivate the kind chicken with excellent slaughter trait exploitativeness as kind of chicken.Application method of the present invention is simple, fast, low cost, easy to utilize, in the assisted Selection that can be widely used in chicken and molecular breeding, the improved seeds for cultivating high yield and high quality meat proterties are provided support.
Accompanying drawing explanation
Fig. 1 is the practical application schematic flow sheet of the excellent slaughter trait molecular genetic marker of table poultry of the present invention.
Fig. 2 is FABP1 encoding gene structural representation and the Second Exon sequence of the chicken that the present invention increases.
Wherein, 83 Nucleotide underscores of Second Exon and black matrix amplify letter signal.In wild gene, the 83rd Nucleotide is T, is C in mutator gene.
Fig. 3 is the schematic diagram that different genotype FABP1 gene restriction enzyme site of the present invention and enzyme cut rear gained clip size.
Fig. 4 is that in the present invention, part sample P CR product cuts through specific enzymes the chicken FABP1 gene order agarose gel electrophoresis result figure that examination arrives.Wherein, TT is non-saltant type, and CT is heterozygous mutant, and CC is homozygous mutant.
Embodiment
First the present invention according to the conserved sequence design primer of chicken FABP1 gene, with different varieties chicken genomic dna for template, carries out pcr amplification, order-checking.Then, carry out specific enzymes and cut, and enzyme is cut the agarose gel electrophoresis figure of result and sequence alignment examination goes out different varieties chicken FABP1 gene SNP site.Then, according to the genotype detected in colony, carry out the association analysis of population genetic statistical study and growth traits, filter out molecule marker closely-related with chicken growth traits.Finally, utilize the molecular genetic marker obtained to possess at assisting sifting or prediction in the table poultry kind of excellent slaughter trait to apply, the chicken having the excellent slaughter trait related molecular marker with chicken screened is cultivated as kind of a chicken, to breeding more high-quality chicken.Elaborate to the present invention below, described content is explanation of the invention instead of restriction.
Embodiment 1: the clone of different varieties chicken FABP1 Gene Partial DNA sequence dna
1, the sampling and processing of chicken blood: randomly draw anosis healthy chicken, comprise 670 Type 3 Luqin chickens and 60 thinkling sound Ya chickens, by venous blood collection under wing, EDTA does anti-freezing process.
2, the extraction of genomic dna: utilize Tiangen blood DNA to extract test kit and extract DNA.Use 200 μ L fresh, freezing or add the blood of various antithrombotics, add 20 μ L Proteinase K Solution, mixing; Then add 200 μ L damping fluid GB, mix latter 70 DEG C and place 10 minutes; Add 200 μ L dehydrated alcohols, mixing of fully vibrating, now there will be flocks; Gained solution and flocks are all transferred to the adsorption column CB3 putting into collection tube, centrifugal 30 seconds of 12000rpm also abandons waste liquid; Add 500 μ L damping fluid GD, centrifugal 30 seconds of 12000rpm also abandons waste liquid; Add 700 μ L rinsing liquids PW (adding dehydrated alcohol before use), centrifugal 30 seconds of 12000rpm also abandons waste liquid, and repeats this step twice; After thoroughly eliminating the rinsing liquid in sorbing material, water or elutriant TE is used to be eluted as much as possible by gained DNA product.
3, amplimer design: according to the GenBank accession number of (http://www.ncbi.nlm.nih.gov/) chicken in ncbi database be: the exon sequence of the FABP1 gene of reflNP_989523.1, with this gene order conserved regions sequence for reference design PCR primer pair, its primer pair sequence is as follows:
Upstream primer (FABP1 E2F): CTTGAGAGGCTCTTCCACAATAG;
Downstream primer (FABP1 E2R): GTTGGACTGGATAGCCTTTAAGTTC;
This primer amplification FABP1 gene Second Exon 447bp sequence.
4, pcr amplification chicken FABP1 gene extron: with the DNA of chicken for masterplate, carries out pcr amplification with the primer pair of above-mentioned design, and PCR total reaction system is 30 μ L, in table 1; PCR total reaction program, in table 2.
Table 1 PCR reaction system
The PCR response procedures that table 2 is best in the scope of application
Embodiment 2: use MboI enzyme to carry out specific enzymes to PCR primer in embodiment 1 and cut
1. use MboI enzyme to carry out specific enzymes to goal gene to cut.The action site of this kind of specific enzymes is:
5’…
↓GATC…3’
3’…CTAG
↑…5’
2. endonuclease reaction system is in table 3.
Table 3 MboI endonuclease reaction system
According to above-mentioned system by after each for reaction system solution mixing, be positioned in 37 DEG C of waters bath with thermostatic control and react 3-5h, endonuclease reaction is fully carried out, carries out follow-up test afterwards.
3. pair digestion products carries out agarose gel electrophoresis analysis.Then cut result according to different enzymes and all samples are carried out classification summary, summarize different types.
Enzyme cuts result three kinds, is SNP site full mutation respectively, heterozygous mutant and not suddenling change completely.When SNP site full mutation, MboI has three point of contacts, place in object fragment, obtains the band (5bp, 63bp, 146bp and 233bp) of four different sizes; When the non-full mutation of SNP site, MboI has three restriction enzyme sites in object fragment, obtains five bands varied in size (5bp, 63bp, 146bp, 233bp and 296bp); When SNP site is not suddenlyd change, MboI has two restriction enzyme sites in object fragment, and digestion products size is (5bp, 146bp and 296bp).Because 5bp and 64bp fragment is very little, only detect 146bp, 233bp and 296bp tri-bar segment (as shown in Figure 3) when plain agar sugar electrophoresis.
Polymorphic site with the nucleotide site that square frame marks in sequence below, there are C/T two kinds of genotype in this site, when genotype is T, PCR primer can only be cut into three bar segment by restriction endonuclease, but electrophoresis can detect wherein two (146bp and 296bp); When genotype is C, PCR primer can be cut into more shorter fragments by restriction endonuclease.
TTCAAGAT
ACTGTGACTA
Embodiment 3: molecular genetic marker prepared by the present invention diagnostic use in different chicken colonies polymorphism
1, the diagnosis in colony's polymorphism: utilize above-mentioned SNP pleiomorphism detecting method to carry out specific enzymes to 670 parts of Type 3 Luqin chickens and 60 parts of thinkling sound Ya chicken FABP1 genes and cut detection.
2, the frequency statistics analysis of SNP site:
Genotype frequency refers to that the number of certain specific gene type individuality in a colony accounts for the ratio of individual overall number.Paa=Aaa/N, wherein Paa represents the AA genotype frequency in a certain site, and Aaa represents in colony to have the genotypic number of individuals of AA, and N is the total quantity detecting colony.
Gene frequency refers to that in a colony, a certain gene number is to the relative ratios of its allelotrope sum.The formula calculated can be write as: PA=(2NM+NA
al+ NA
a2+ ...+NA
an)/2N.In formula, PA represents allelotrope A frequency, and NM represents in colony to have the genotypic individual amount of AA, A
al-anthe individual different multiple allelomorphos of n for allelotrope A.Statistics is in table 6 and table 7.
The genotype in table 6 Type 3 Luqin chicken FABP1 gene polymorphic site and gene frequency
The genotype in table 7 thinkling sound Ya chicken FABP1 gene polymorphic site and gene frequency
3, the association analysis of genetic effect
Adopt PASW (18.0) statistical software, to Type 3 Luqin chicken and Shiqiza different genotype, individual and body footage is according to having carried out test of significance.
(1) major traits measured comprises: Slaughter weight, slaughter traits, dressing percentage, heavy, the complete clean thorax of half clean thorax are heavy, half clean thorax rate, complete clean thorax rate, chest muscle weight, chest muscle rate, leg flesh weight, leg flesh rate, abdomen fat weight, abdominal fat, sebum is thick, flesh fat is wide.
(2) colony measured: randomly draw healthy anosis Type 3 Luqin chicken 670 and only reach thinkling sound Ya chicken 60 and only analyze respectively.
Statistics is in table 8 and table 9.
The association analysis of table 8 Type 3 Luqin chicken FABP1 E2 polymorphism and slaughter trait
The association analysis of table 9 thinkling sound Ya chicken FABP1 E2 polymorphism and slaughter trait
| FABP1SNP site | C/C(3)Mean±SD | C/T(12)Mean±SD | T/T(45)Mean±SD |
| Slaughter weight | 1332.67±300.55 | 1389.03±76.32 | 1335.87±39.12 |
| Slaughter traits | 1194.57±274.12 | 1260.40±66.14 | 1196.66±34.84 |
| Carcass rate | 89.39±0.10 | 90.90±0.68 | 89.67±0.52 |
| Half clean thorax weight | 1100.23±254.94 | 1140.49±61.12 | 1100.22±31.95 |
| Half clean thorax rate | 82.29±0.62 | 82.26±0.80 | 82.49±3.72 |
| Complete clean thorax weight | 1002.33±233.23 | 1039.56±57.43 | 996.60±30.35 |
| Complete clean thorax rate | 74.95±1.03 | 74.89±3.02 | 74.61±3.59 |
| Chest muscle weight | 144.75±26.74 | 139.84±6.36 | 158.83±114.48 |
| Chest muscle rate | 15.14±0.88 | 13.56±0.26 | 16.53±2.32 |
| Leg flesh weight | 177.53±47.88 | 190.42±11.43 | 182.25±6.14 |
| Leg flesh rate | 17.42±0.66 | 18.29±17.63 | 18.28±0.23 |
| Abdomen fat weight | 17.96±9.92 | 25.36±5.17 | 17.91±1.70 |
| Abdominal fat | 1.51±0.53 | 2.13±0.42 | 1.86±0.19 |
| Sebum is thick | 3.24±0.66 | 4.02±0.37 | 4.68±0.25 |
| Flesh fat is wide | 5.43±0.55 | 6.02±0.45 | 5.67±0.28 |
| Wing weight | 103.59±36.72 | 73.00±10.79 | 87.10±5.50 |
| Wing rate | 11.47±0.35 | 10.90±0.22 | 11.17±0.20 |
| Leg weight | 279.51±103.60 | 264.75±76.22 | 236.30±15.51 |
| Leg ratio | 30.53±0.89 | 46.54±19.26 | 29.91±0.30 |
Note: the difference between the letter display different genotype in genotype subscript, there is same letter and represent that between two kinds of genotype, difference is not significantly (P>0.05), letter is different represents significant difference (P<0.05) between two kinds of genotype, markdly not show do not have significant difference between this genotype and other genotype.
Analyze display, in Type 3 Luqin chicken, genotype is its Slaughter weight of individuality of TT, slaughter traits, heavy, the complete clean thorax of half clean thorax is heavy, chest muscle is heavy and leg flesh heavily etc. measurement index all with CC type and CT type individual difference significantly, and TT type measurement numerical value is maximum.Same, in thinkling sound Ya chicken, individual relative to CC, the take off data of the individual production traits of TT type is even more ideal, and namely TT type chicken is more suitable for screening and the breeding of carrying out excellent kind chicken as kind of chicken.
Claims (4)
1. a chicken FABP1 gene molecule genetic marker relevant to the excellent production traits of chicken, this mark is the pleomorphism site in chicken FABP1 gene mononucleotide polymorphism sequence, it is characterized in that: the pleomorphism site of described chicken FABP1 gene mononucleotide polymorphism sequence is the nucleotide polymorphisms site of C or T the 83rd of FABP1 gene order Second Exon, show as CC, CT or TT tri-kinds of genotype, wherein TT type is the excellent slaughter trait molecular genetic marker of table poultry.
2. the chicken FABP1 gene molecule genetic marker that the production traits excellent in chicken described in claim 1 is relevant possesses the application in the table poultry kind of excellent slaughter trait at assisting sifting or prediction.
3. application according to claim 2, method is: with chicken FABPl genomic dna to be detected for template, pcr amplification is carried out with primer pair FABP1 E2F/E2R, increased laggard row agarose gel electrophoresis, then uses specific restriction endonuclease to carry out enzyme to the object fragment amplified and cut; Afterwards again agarose gel electrophoresis is carried out to digestion products, detect the clip size of digestion products, analyze the nucleotide polymorphisms site determining chicken FABP1 gene, when the 83rd of FABP1 gene order Second Exon is C or T, and genotype is when being TT type, can judge that chicken breed to be detected is as the kind chicken with excellent slaughter trait;
It is characterized in that:
The nucleotides sequence of described primer pair FABP1 E8F/E8R is classified as:
FABP1 E2F：CTTGAGAGGCTCTTCCACAATAG；
FABP1 E2R：GTTGGACTGGATAGCCTTTAAGTTC；
The response procedures of described pcr amplification is: 95 DEG C of denaturation 4-5min; 94 DEG C of sex change 30-35s, 55-61 DEG C of annealing 30-35s, 72 DEG C extend 1min, 28-35 circulation, and last 72 DEG C extend 10min, and are placed in 4 DEG C of preservations;
Described specific restriction endonuclease is MboI, and it is the following sequence amplified in goal gene that its enzyme cuts action site:
5 '
↓gATC ... 3 ' or 3 ' ... CTAG
↑5 '.
4. application according to claim 3, is characterized in that: for FABP1 E2F/E2R primer pair, pcr amplification program is: 95 DEG C of denaturation 4min; 94 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C extend 1min, 35 circulations, and last 72 DEG C extend 10min, and are placed in 4 DEG C of preservations.
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