CN1654681A - Molecule marking method for foreshowing and identifying chicken abdomen fat content by A-FABP gene - Google Patents
Molecule marking method for foreshowing and identifying chicken abdomen fat content by A-FABP gene Download PDFInfo
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Abstract
The present invention provides the molecular labeling method of adopting A-FABP gene in predicting and identifying fat content in chicken belly. The method includes designing one pair of primers based on chicken A-FABP gene sequence; performing PCR amplification of chicken genome DNA with the primers and digesting amplified PCR product with restriction endonuclease Taq1; electrophoretically separating the digested PCR product with 2 % agarose gel to detect the C/T variant site in A-FABP gene exon 1; and naming C base, T base and the site of A-FABP gene exon 1 during heterozygosis. Test shows that the AA genotype individual has belly fat weight 8.67-13.02 g and belly fat rate 0.34-0.66 % lower than that of the BB genotype individual. The present invention has simple operation, low cost, high precision and fast detection.
Description
(1), affiliated field
The invention belongs to animal molecular genetics field, particularly relate to a kind of method with the Markers for Detection chicken abdomen fat content.
(2), background technology
Fast large-scale fryer body fat (especially abdomen fat) is accumulated and has too much been become distinct issues.It is many unfavorable that broiler chicken internal deposition excess fat has: (1) obviously reduces feed efficiency, because the fatty tissue of sedimentation unit weight consumes the triple energy than the lean meat of sedimentation unit weight more; (2) reduced the ratio of trunk lean meat, thereby reduced the output that cuts meat fatty tissue; (3) processor and human consumer abandon a big chunk (abdomen fat pad, muscular stomach are fatty, crop is spoken in a low voice fat and mesentery fat etc.) of these fat of broiler chicken internal deposition on every side, this has not only increased processor and human consumer's burden, and increased lipid content in refuse and the treating water, thereby contaminate environment.In view of this, broiler chicken internal deposition excess fat causes conspicuous financial loss will for the producer, processor and human consumer.Meat kind chicken overfertilization will have a strong impact on laying rate, rate of fertilization and hatching rate, and the generation of meeting induced lipolysis liver syndromes, thereby has strengthened the death rate of laying period.Therefore, control fat is too much accumulated in that chicken is intravital, further improves the feed efficiency of fryer and carcase quality and is China and be badly in need of the significant problem researched and solved.
Fatty acid binding protein (fatty acid binding protein FABP) belongs to the member of contaminated with lipid superfamily protein, is present in the cell of animal body, and be that the molecule amount is the albumen of 14-15KD.FABP has very strong binding ability to longer chain fatty acid, and its major function is the enzymolysis of signal conduction, genetic transcription and the protection free fatty acids of the longer chain fatty acid in the transport cells, the growth of regulating cell and differentiation, cell.Have at least 9 kinds of FABP to be separated in Mammals, they are respectively liver (L-) FABP, small intestine (I-) FABP, heart (H-) FABP, fat (A-) FABP, epidermis (E-) FABP, ileum (IL-) FABP, brain (B-) FABP, myelin (M-) FABP and testis (T-) FABP.
Have at least 5 kinds of FABP to be separated bird, they are respectively liver (L-) FABP, small intestine (I-) FABP, fat (A-) FABP, muscle (M-) FABP and retina (R-) FABP.These albumen are made up of 126-134 amino acid, and are named by first separated tissue.These proteic genes of encoding have identical structure, all are to be made of 4 exons and 3 introns, and the same length of exon or close, but the length variation of intron is very big.
In Mammals, the function of various dissimilar FABP there has been research more deeply.Wherein the mouse of A-FABP gene knockout shows normal physiological status, can normotrophic fatty tissue, on the stability of lipid metabolism, change very little (HotamisLigiL etc., 1996).This mainly is because the up-regulated expression of E-FABP gene has produced compensation effect (Coe etc., 1999 to it; Shaughnessy etc., 2000), and the expression of E-FABP gene lower level in fatty tissue under normal circumstances (Krieg etc., 1993).Compare with wild-type mice, lipid acid flow velocity or lipoprotein function in the mouse fatty tissue of A-FABP gene knockout do not change, but basic lipolysis has reduced about 40% (Coe etc., 1999).In the pathogeny of type ii diabetes, A-FABP may bring into play the effect of its regulation and control hyperinsulinism and insulin resistant by regulating lipolysis and insulin secretion.The A-FABP gene experiment that knocks out obesity mice shows that the disappearance of A-FABP not only can be improved peripheral insulin resistant, can also safeguard the normal function of pancreas beta cell, helps normally the carrying out of lipid metabolism (UysaL etc., 2000).Recently, the expression of A-FABP in scavenger cell also is studied (Makowski etc., 2001).A-FABP demonstrates its important effect in the scavenger cell biological respinse, and influential to the generation of arteriosclerosis.The mouse of apo E and A-FABP while defective can prevent the generation (Makowski etc., 2001) of arteriosclerosis.Oxidized low-density lipoprotein can be induced mRNA and the proteic expression (Fu etc., 2000) of A-FABP in people's the scavenger cell.These data show that A-FABP has different effects in adipocyte and scavenger cell, and this prevention for arteriosclerosis provides a new treatment target spot.Gerbens etc. (1998) have detected a microsatellite sequence at first intron of A-FABP gene, and the test of six pig kinds is found to have polymorphism.In Du Luoke colony, the heritable variation of A-FABP is relevant with IMF content.
FABP in liver, muscle, small intestine, retina and the fatty tissue of bird, also be separated (SeweLL etc., 1989a, 1989b; Sams etc., 1990,1991; Gotz etc., 1996; SeLLner etc., 1993).Chicken lard type FABP (A-FABP) also is separated, its molecular weight is 14KD, iso-electric point is 5.1, analyze from amino acid composition aspect, this albumen and Mammals H-FABP and A-FABP be the height homology structurally, and part studies show that in conjunction with characteristics, chicken A-FABP has extremely strong binding ability to linolic acid, next is oleic acid, palmityl CoA and palmitinic acid, and can not be in conjunction with cholesterol (Sams etc., 1990).The research of bird FABP gene pleiomorphism is at the early-stage.Wang Qigui etc. (2001) studies show that the polymorphism of EX-FABP gene relevant extremely significantly with abdomen fat content (p＜0.01).
Wang Qigui etc. (2002) have cloned the chicken A-FABP gene, studies show that this gene constitutes (Genbank Accession No.AF432507) by 4 exons and 3 introns, and only express in fatty tissue.
(3), summary of the invention
Main purpose of the present invention provides a kind of Protocols in Molecular Biology, promptly adopts the PCR-RFLP method to detect the chicken genotype, thus the molecule marking method of indication and evaluation chicken abdomen fat content.
The object of the present invention is achieved like this:
1, design a pair of primer AFF and AFR according to the chicken A-FABP gene order:
AFF：5’-AGA?CTG?CTA?CCT?GGC?CTG?ACA-3’
AFR：5’-CAT?CCT?ACT?GGA?ATA?CGG-3’
2, utilize this primer that the genomic dna of chicken is carried out pcr amplification, pcr amplified fragment length is 702bp;
3, utilize restriction enzyme Taq I digest amplification PCR products;
4, the sepharose of use 2% carries out electrophoretic separation to postdigestive PCR product, detects the C/T variant sites among the A-FABP gene extron I;
5, when variant sites is the C base among the chicken A-FABP gene extron I, can produce the restriction enzyme site (T/CGA) of restriction enzyme Taq I, can produce 2 fragments (629bp and 73bp) behind the Taq I digestion PCR product, with its called after BB genotype; When this site is the T base (TTGA), then there is not Taq I restriction enzyme site, has only 1 fragment (702bp) behind the Taq I digestion PCR product, its called after AA genotype; When this site is the C/T heterozygosis, can produce 3 fragments (702bp, 629bp and 73bp) behind the Taq I digestion PCR product, with its called after AB genotype.
The present invention can also comprise:
1, wherein the method for analyzing gene polymorphism is the genotype of classifying by the sudden change that detects base.
2, the position that wherein is used for the analyzing gene polymorphism is an exon.
3, the position that wherein is used for the analyzing gene polymorphism is the exon district of the primer amplification represented by AFF and AFR.
4, the site that wherein is used for analyzing polymorphism is selected from the base mutation of the C-T of the 74th of the following sequence of chicken A-FABP gene.
1????AGACTGCTAC?CTGGCCTGAC?AAAATGTGCG?ACCAGTTTGT?GGGCACCTGG?AAGCTCCTTT
61???CTAGTGAAAA?CTTCGAGGAC?TATATGAAAG?AGCTGGGTGA?GAAATGTTAC?CTGAAATTAT
121??TGTGTCTGGG?GAAGGGAGGG?AATGCTGAAC?TTGAGCTCTA?TTCCTTACTT?GCTTTTTCAG
181??TCTATGCTGT?TTACCAAGGT?CTTTTTAGTC?CAACTACTTT?TATTAAATAG?TTCTAAACTG
241??GAAAACTTGG?CTGTTTGGAT?TAGCGTTGTC?ACATTCCTAT?AACATAGGAA?AGCTTGACTG
301??GGAGGAATAG?TTTCAGGTAG?GCCAAGTCTA?TGCATGCCTA?ACTGCTAGTG?CTATAAATGA
361??AAGTAAACAC?TTTGCTGCAG?TATTTTATGT?GAATTTGCTT?TTAAACTTTT?GGAAGTACAA
421??AGTGATGTAA?TGCTAGAATG?CAGGAAGCTT?CTCTGTGTCT?ATGAGCAACA?CACCTAAATT
481??TGCTCTTGGA?CTAGGACACT?GATTAATTGT?CATGGCTATT?ATAGTAAGTG?AATGAAACTC
541??TGCGATTGCC?TCCAGTGAAT?ATTAACAAAG?AAAGTTGCAC?AGATTCATGA?CAGGTACTGC
601??AGTATACCTG?CAAAAGTATT?CAAGGAATAA?CACTCTAAAG?GTGTTGACTT?CTCACTTAGA
661??ATTAAATCTG?GGCTTTAAAA?TTTTCCGTAT?TCCAGTAGGA?TG
5, wherein chicken abdomen fat content is the heavy and abdomen fat rate of abdomen fat of chicken.
6, the restriction enzyme that wherein is used to digest the PCR product is Taq I.
7, the sepharose concentration that wherein is used for agarose gel electrophoresis is 2%.
Experimental results show that the abdomen fat with AA genotype individuality weighs and abdomen fat rate significantly is lower than the heavy and abdomen fat rate (P＜0.05) of the abdomen fat with BB genotype chicken.In 2 colonies that we studied, has the heavily low 8.67-13.02 gram of abdomen fat of the abdomen fat anharmonic ratio BB genotype individuality of AA genotype individuality; Have the abdomen fat rate low 0.34-0.66% of the abdomen fat rate of AA genotype individuality than BB genotype individuality.
The present invention adopts the method for PCR-RFLP to detect C/T variant sites among the chicken A-FABP gene extron I dexterously.
The present invention is simple to operate, expense is low, tolerance range is high, can carry out rapid detection.When using marker gene type of the present invention that the abdominal fat weight of chicken is selected, abdomen fat will heavily be reduced about 10 grams.China is annual to want 4,000,000,000 commercial meat birds of large-scale production, if every chicken can reduce by 10 gram abdomen fat, is equivalent to save 2.4 hundred million kilograms of feeds at least, can bring very big economic benefit.In a word, this mark of discovering can be used for the marker assisted selection of chicken fatty character, and important production meaning is not only arranged, and has opened new window for the research of fowl fat metabolism on molecular level.
(4), description of drawings
Accompanying drawing is that A-FABP gene Taq I restriction enzyme site is analyzed collection of illustrative plates.
(5), specific embodiments
For example the present invention is done in more detail below and describes:
One, experiment material
1. laboratory animal and property determination
What Northeast Agricultural University breeding base was set up is the F that the parent is hybridized generation with high fat strain of fryer and white eared pheasant
2Resource colony.5 familys are chosen in this research, amount to 455 chickens.What China Agricultural University set up is the F that the parent is hybridized generation with star's fryer and silkiefowl black bone
2Resource colony, wherein to do male parent be quadrature to fryer, it is reciprocal cross that Gallus Domesticus is cooked male parent.Each 4 family of reciprocal cross are chosen in this research, amount to 558 chickens.
Two F
2In 12 weeks wing venous blood collection during ages, the oxalate anti-freezing is butchered after the weighing live-weight, peels off abdomen fat and weighing abdomen fat heavy (g) then for colony, and divided by 12 age in week live-weight calculate abdomen fat rate.
2. medicine and enzyme
Tutofusin tris (Tris), Sigma ChemicaLs Co; The saturated phenol of Tris, Beijing ancient cooking vessel state biotech development center; Proteinase K (Proteinase K), MMERCK Co; DL 2000, dNTP (dATP; DTTP; DCTP; DGTP), the Taq enzyme, the precious biotech firm in Dalian; Agarose (Agarose), acrylamide, methylene diacrylamide, the white company in Yuanping City.
3. key instrument
PTC-200PCR instrument (PERKIN ELMER), Biometra grads PCR instrument, the multi-functional imaging system of UVP, ULtrospec 1000 ultraviolet spectrophotometers, BECKMAN refrigerated centrifuge, MiLLi-Q ultrapure water instrument, DYY-III2 voltage stabilizing electrophoresis apparatus and supporting electrophoresis chamber.
Two, experimental technique
1. the preparation of damping fluid and common agents
1M Tris CL:121.14g Tris alkali is dissolved in the 800mL distilled water, with hydrochloric acid adjust pH to 8.0, is settled to 1000mL, autoclaving.
TE damping fluid: 10mM Tris CL, 1mM EDTA, pH8.0, autoclaving.
20 * SET damping fluid: 3MNaCL, 1M Tris CL (pH 8.0), 20mM EDTA (pH 8.0), autoclaving.
5 * tbe buffer liquid: 54g Tris alkali, 27.5g boric acid, 20mL 0.5M EDTA (pH8.0) adds water to 1L.
50 * TAE damping fluid: 242g Tris alkali, the 57.1mL glacial acetic acid, 100mL 0.5MEDTA (pH8.0) adds water to 1L.
1M Tris CL:121.14g Tris alkali is dissolved in the 800mL distilled water, with hydrochloric acid adjust pH to 8.0, is settled to 1000mL, autoclaving.
0.5M EDTA:186.1g EDTA is dissolved in the 800mL distilled water, with NaOH adjust pH to 8.0, is settled to 1000mL, autoclaving.
3M NaAc (pH5.2): 408.1g NaAc 3H
2O is dissolved in the 800mL distilled water, transfers pH to 7.0 with glacial acetic acid, is settled to 1000mL.
200mL silver dye liquor: NH
3H
2O 2mL; 3.6%NaOH 4.2mL; 20%AgNO
33.6mL, add deionized water to 200mL.
The 200mL liquid that develops the color: 1% Trisodium Citrate 1mL; Formaldehyde 100uL; Add deionized water to 200mL.
Fowl blood lysate liquid: 10mM TrisCL (pH8.0), 0.1M EDTA (pH8.0), 0.5%SDS.
2. primer design is with synthetic
Following primer is synthetic by Shanghai Bo Ya biotech firm:
AFF：5’-AGA?CTG?CTA?CCT?GGC?CTG?ACA-3’
AFR：5’-CAT?CCT?ACT?GGA?ATA?CGG-3’
3. a small amount of of chicken genomic dna is extracted
Method one:
(1) get 20 μ L anticoagulated bloods, add 500 μ L fowl lysates, adding Proteinase K to final concentration is 100-200 μ g/mL, and 55 ℃ of digestion of mixing 12hr no longer includes the heavy-gravity agglomerate in solution.
(2) solution is cooled to room temperature, adds 5M NaCL to final concentration 1.5M, mixing 10min.Add equal-volume phenol/chloroform, put upside down centrifuge tube mixing 10min repeatedly.□
(3) 12,000rpm, the centrifugal 10min of room temperature.Get supernatant, add equal-volume chloroform mixing 10min.□
(4) 12,000rpm, the centrifugal 10min of room temperature.Get 2 times of volume dehydrated alcohols of supernatant deposit D NA.□
(5) DNA is chosen be put in the 1.5mL centrifuge tube, wash 1 time with 70% ethanol.□
(6) 7,500rpm, the centrifugal 5min of room temperature abandons supernatant.
(7) (attention can not be too dried) after the DNA drying is dissolved among the 200 μ L TE.
Method two:
(1) adding of 20 μ L whole bloods is equipped with in the 1.5mL centrifuge tube of 700 μ L, 1 * SET, gently mixing.
(2) add Proteinase K (10mg/mL) to the SDS of final concentration 100-200 μ g/ μ L and 10% to 0.5%, 55 ℃ of digestion of final concentration 12h.
(3) after waiting to digest fully, add the saturated phenol of isopyknic Tris, put upside down back and forth, make its mixing
The centrifugal 10min of (4) 12,000rpm carefully moves into the upper strata water in another centrifuge tube with cutting off most advanced and sophisticated suction nozzle, discards organic phase.Repeat third and fourth step once.
(5) add isopyknic phenol, chloroform, primary isoamyl alcohol mixed solution (volume ratio is 24: 23: 1) to aqueous phase, mix 10min.12,000rpm., centrifugal 10min shifts out water to another centrifuge tube.
(6) add isopyknic chloroform, primary isoamyl alcohol mixed solution (23: 1) to aqueous phase, put upside down back and forth and mix 10min, 12,000rpm, centrifugal 10min shifts out water to another centrifuge tube.
(7) (3M pH5.2) and the dehydrated alcohol of 2 times of volumes, puts upside down deposit D NA back and forth to add 1/10 volume NaAc to aqueous phase.
(8) DNA is chosen be put in the 1.5mL centrifuge tube, wash 1 time with 70% ethanol.
The centrifugal 5min of (9) 7,500rpm.Carefully outwell ethanol in the pipe, will be upside down on the filter paper, allow ethanol flow to end, place air drying.
(10) TE of adding 200 μ L puts the dissolving DNA that spends the night in 50 ℃ of water-baths.Be stored in after the dissolving-20 ℃ standby.
4.PCR reaction
(1) be that template is carried out pcr amplification with chicken DNA, comprise following solution or reagent in the 10uL reaction system:
10×PCR?reaction?buffer?????????????1μL
DNTP Mixture (each 2.SmM) 0.8 μ L
Primer 1 (10 μ M) 0.2 μ L
Primer 2 (10 μ M) 0.2 μ L
EX-Taq(5U/μL)??????????????????????0.1μL
Deionized water 6.7 μ L;
Genomic dna (50ng/ μ L) 1.0 μ L
(2) carry out the PCR reaction with above-mentioned solution mixing and by following condition.
94 ℃ of sex change 5min; 94 ℃ of 30sec, 58 ℃ of 30sec, 72 ℃ of 50sec, 32 circulations; 72 ℃ are extended 7min.
(3) after reaction finishes, get PCR reaction solution (3 μ L) and carry out agarose gel electrophoresis, detect the PCR product.
5.PCR-RFLP reaction system and condition
It is as follows that enzyme is cut system:
Taq I restriction endonuclease 10U/ μ L 0.5 μ L
10×Buffer：??????????????????????2μL
PCR?Production????????????????????5μL
Add deionized water to 20 μ L
With above-mentioned reaction solution mixing, in 65 ℃ of waters, digested 3～5 hours.
6. agarose gel electrophoresis detects
After will digesting total overall reaction liquid is carried out 2% agarose gel electrophoresis, detect PCR product endonuclease bamhi polymorphism.
7. statistical model is set up
According to F
2The characteristics of resource colony, it is as follows to make up linear model:
Y＝μ+G+F+S+H+R+e
Y is the character observation value, μ is colony's average, a is the residue multigentic effect, G is the genotype fixed effect, F is the family fixed effect, and S is the sex fixed effect, and R is reciprocal cross (combination) fixed effect (the resource colony that northeast agricultural university sets up does not have this effect), H is hatching batch fixed effect, and e is a residual value shi.Use the JMP of statistical software 4 (SAS Institute Inc., Cary, the NC) degree of correlation between check genotype and proterties, and the least square average of estimation proterties.
The polymorphism of embodiment 1, chicken A-FABP gene and the F of northeast agricultural university
2Being correlated with of resource colony ventral fat character
The F that utilizes primer of the present invention (AFF and AFR) that Northeast Agricultural University is set up
2The genomic dna of 455 individualities of resource colony carries out pcr amplification, and utilizes Taq I digestion with restriction enzyme PCR product, carries out 2% agarose gel electrophoresis analysis then.At F
2Detect 3 kinds of genotype in the resource colony altogether, when variant sites is the C base among the chicken A-FABP gene extron I, the restriction enzyme site (T/CGA) of restriction enzyme Taq I can be produced, 2 fragments (629bp and 73bp) can be produced behind the Taq I digestion PCR product, its called after BB genotype; When this site is the T base (TTGA), then there is not Taq I restriction enzyme site, has only 1 fragment (702bp) behind the Taq I digestion PCR product, its called after AA genotype; When this site is the C/T heterozygosis, can produce 3 fragments (702bp, 629bp and 73bp) behind the Taq I digestion PCR product, with its called after AB genotype (accompanying drawing).
3 kinds of genotype that A-FABP gene C/T variant sites is produced and the F of northeast agricultural university
2The abdomen fat of 455 individualities of resource colony weighs and abdomen fat rate is carried out the least square analysis, and the result shows that genotype is to F
2The abdomen fat of resource colony weighs and abdomen fat rate has utmost point remarkably influenced (P＜0.01) (table 1).
The least square analytical results (P value) of the table 1. northeast F2 of agricultural university resource colony ventral fat character
| Abdomen fat is heavy | Abdomen fat rate | |
| Genotype sex hatching batch family | ????0.0048 ????0.1047 ????0.9241 ????0.0001 | ????0.0050 ????0.0001 ????0.1658 ????0.0001 |
To F between 3 kinds of genotype
2The least square average of resource colony ventral fat character is carried out multiple comparisons, and the result shows the abdomen fat weight of AA genotype individuality and the heavy and abdomen fat rate (P＜0.05) of abdomen fat that abdomen fat rate significantly is lower than AB and BB type individuality; Have abdomen fat heavy and abdomen fat rate respectively low 8.67 gram and 0.34% (tables 2) of the heavy and abdomen fat rate of the abdomen fat of AA genotype individuality than BB genotype individuality.
Table 2.A-FABP gene different genotype is to the F of northeast agricultural university
2The influence of resource colony chicken ventral fat character
| Genotype | Number of individuals | The heavy least square average (gram) of abdomen fat | Abdomen fat rate least square average |
| ????AA ????AB ????BB ????a ????d ????D | ????115 ????227 ????113 | ????73.90±2.57 b????84.08±1.58 a????82.57±2.62 a????4.335 ????5.845 ????1.348 | ??0.0375±0.0011 b??0.0419±0.0008 a??0.0409±0.0011 a??0.0017 ??0.0027 ??1.588 |
During the average comparison same row do not have same letter person's significant difference (
A-bP＜0.05)
D: degree of dominance D=d/a; A=(BB-AA)/2; D=AB-(AA+BB)/2
The polymorphism of embodiment 2, chicken A-FABP gene and the Chinese F of agricultural university
2Being correlated with of resource colony ventral fat character
The F that utilizes primer of the present invention (AFF and AFR) that China Agricultural University is set up
2The genomic dna of 588 individualities of resource colony carries out pcr amplification, and utilizes Taq I digestion with restriction enzyme PCR product, carries out 2% agarose gel electrophoresis analysis then.The result is at the Chinese F of agricultural university
2Also detect 3 kinds of genotype in the resource colony, and with the F of agricultural university northeastward
2Detected genotype is identical in the resource colony.When variant sites is the C base among the chicken A-FABP gene extron I, can produce the restriction enzyme site (T/CGA) of restriction enzyme Taq I, can produce 2 fragments (629bp and 73bp) behind the Taq I digestion PCR product, with its called after BB genotype; When this site is the T base (TTGA), then there is not Taq I restriction enzyme site, has only 1 fragment (702bp) behind the Taq I digestion PCR product, its called after AA genotype; When this site is the C/T heterozygosis, can produce 3 fragments (702bp, 629bp and 73bp) behind the Taq I digestion PCR product, with its called after AB genotype (accompanying drawing).
A-FABP gene Taq I enzyme is cut the 3 kinds of genotype and the Chinese F of agricultural university of polymorphic site
2The abdomen fat of 558 individualities of resource colony weighs and abdomen fat rate is carried out the least square analysis.The result shows that genotype is to F
2The abdomen fat of resource colony weighs and abdomen fat rate has remarkably influenced (P＜0.05) (table 3).
The least square analytical results (P value) of table 3. F2 of Chinese agricultural university resource colony ventral fat character
| Abdomen fat is heavy | Abdomen fat rate | |
| Genotype | ????0.0224 | ????0.0778 |
| Sex hatching batch family reciprocal cross (combination) | ????0.0001 ????0.0001 ????0.0065 ????0.6818 | ????0.0001 ????0.0001 ????0.0003 ????0.3434 |
To F between 3 kinds of genotype
2The least square average of resource colony ventral fat character is carried out multiple comparisons, and the result shows the abdomen fat weight of AA genotype individuality and the heavy and abdomen fat rate (P＜0.05) of abdomen fat that abdomen fat rate significantly is lower than AB and BB type individuality; Have abdomen fat heavy and abdomen fat rate respectively low 13.02 gram and 0.66% (tables 4) of the heavy and abdomen fat rate of the abdomen fat of BB genotype individuality than AA genotype individuality.
Table 4.A-FABP gene different genotype is to the Chinese F of agricultural university
2The influence of resource colony chicken ventral fat character
| Genotype | Number of individuals | The heavy least square average (gram) of abdomen fat | Abdomen fat rate least square average |
| ????AA ????AB ????BB ????a ????d ????D | ????35 ????200 ????323 | ????35.46±6.69 b????49.21±5.12 a????48.47±5.04 a????6.51 ????7.25 ????1.11 | ????0.0260±0.0038 b????0.0322±0.0030 a????0.0326±0.0028 a????0.0033 ????-0.0132 ????-0.88 |
During the average comparison same row do not have same letter person's significant difference (
A-bP＜0.05)
D: degree of dominance D=d/a; A=(BB-AA)/2; D=AB-(AA+BB)/2
Claims (8)
1, with the molecule marking method of indication of A-FABP gene and evaluation chicken abdomen fat content, it is characterized in that:
(1), designs a pair of primer according to the chicken A-FABP gene order
AFF：5’-AGA?CTG?CTA?CCT?GGC?CTG?ACA-3’
AFR：5’-CAT?CCT?ACT?GGA?ATA?CGG-3’
(2), utilize this primer that the genomic dna of chicken is carried out pcr amplification, pcr amplified fragment length is 702bp;
(3), utilize restriction enzyme Taq I digest amplification PCR products;
(4), use 2% sepharose that postdigestive PCR product is carried out electrophoretic separation, detect the C/T pleomorphism site among the A-FABP gene extron I;
(5), when pleomorphism site is the C base among the chicken A-FABP gene extron I, can produce the restriction enzyme site (T/CGA) of restriction enzyme Taq I, can produce 2 fragments (629bp and 73bp) behind the Taq I digestion PCR product, with its called after BB genotype; When this site is the T base (TTGA), then there is not Taq I restriction enzyme site, has only 1 fragment (702bp) behind the Taq I digestion PCR product, its called after AA genotype; When this site is the C/T heterozygosis, can produce 3 fragments (702bp, 629bp and 73bp) behind the Taq I digestion PCR product, with its called after AB genotype.
2, the molecule marking method of indication according to claim 1 and evaluation chicken abdomen fat content, it is characterized in that: wherein the method for analyzing gene polymorphism is the genotype of classifying by the sudden change that detects base.
3, the molecule marking method of indication according to claim 2 and evaluation chicken abdomen fat content, it is characterized in that: the position that wherein is used for the analyzing gene polymorphism is an exon.
4, indication according to claim 3 and identify the molecule marking method of chicken abdomen fat content is characterized in that: the position that wherein is used for the analyzing gene polymorphism is the exon district of the primer amplification represented by AFF and AFR.
5, indication according to claim 4 and identify the molecule marking method of chicken abdomen fat content is characterized in that: the site that wherein is used for analyzing polymorphism is selected from the base mutation of the C-T of the 74th of the following sequence of chicken A-FABP gene.
1??????AGACTGCTAC?CTGGCCTGAC?AAAATGTGCG?ACCAGTTTGT?GGGCACCTGG?AAGCTCCTTT
61?????CTAGTGAAAA?CTTCGAGGAC?TATATGAAAG?AGCTGGGTGA?GAAATGTTAC?CTGAAATTAT
121????TGTGTCTGGG?GAAGGGAGGG?AATGCTGAAC?TTGAGCTCTA?TTCCTTACTT?GCTTTTTCAG
181????TCTATGCTGT?TTACCAAGGT?CTTTTTAGTC?CAACTACTTT?TATTAAATAG?TTCTAAACTG
241????GAAAACTTGG?CTGTTTGGAT?TAGCGTTGTC?ACATTCCTAT?AACATAGGAA?AGCTTGACTG
301????GGAGGAATAG?TTTCAGGTAG?GCCAAGTCTA?TGCATGCCTA?ACTGCTAGTG?CTATAAATGA
361????AAGTAAACAC?TTTGCTGCAG?TATTTTATGT?GAATTTGCTT?TTAAACTTTT?GGAAGTACAA
421????AGTGATGTAA?TGCTAGAATG?CAGGAAGCTT?CTCTGTGTCT?ATGAGCAACA?CACCTAAATT
481????TGCTCTTGGA?CTAGGACACT?GATTAATTGT?CATGGCTATT?ATAGTAAGTG?AATGAAACTC
541????TGCGATTGCC?TCCAGTGAAT?ATTAACAAAG?AAAGTTGCAC?AGATTCATGA?CAGGTACTGC
601????AGTATACCTG?CAAAAGTATT?CAAGGAATAA?CACTCTAAAG?GTGTTGACTT?CTCACTTAGA
661????ATTAAATCTG?GGCTTTAAAA?TTTTCCGTAT?TCCAGTAGGA?TG
6, according to the molecule marking method of any one described indication of claim 1-5 and evaluation chicken abdomen fat content, it is characterized in that: wherein chicken abdomen fat content is the heavy and abdomen fat rate of abdomen fat of chicken.
7, according to any one described indication of claim 1-5 with identify the molecule marking method of chicken abdomen fat content, it is characterized in that: the restriction enzyme that wherein is used to digest the PCR product is Taq I.
8, according to the molecule marking method of any one described indication of claim 1-6 and evaluation chicken abdomen fat content, it is characterized in that: the sepharose concentration that wherein is used for agarose gel electrophoresis is 2%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101603086B (en) * | 2009-03-18 | 2011-12-07 | 东北农业大学 | Molecular marking method for indicating and identifying chicken abdominal fat weight by using ACC alpha gene |
| CN106591467A (en) * | 2017-01-04 | 2017-04-26 | 安徽农业大学 | Molecular marker assisted selection method for improving breast and leg muscle percentages of young Columba pigeon |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1274841C (en) * | 2001-09-25 | 2006-09-13 | 李宁 | Mononucleotide polymorphism analyzing method for detecting chicken ventral fat character |
| CN1453357A (en) * | 2003-04-07 | 2003-11-05 | 东北农业大学 | Fat-type fatty acid bindin gene of chicken and its cloning and identification method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101603086B (en) * | 2009-03-18 | 2011-12-07 | 东北农业大学 | Molecular marking method for indicating and identifying chicken abdominal fat weight by using ACC alpha gene |
| CN106591467A (en) * | 2017-01-04 | 2017-04-26 | 安徽农业大学 | Molecular marker assisted selection method for improving breast and leg muscle percentages of young Columba pigeon |
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