CN1253466C - Chicken polydactyly functional gene and uses thereof - Google Patents

Abstract

The present invention discloses a chicken polydactyly functional gene and the application thereof, which relates to a chicken polydactyly functional gene and the addition thereof in the field of biological engineering; the present invention particularly relates to a chicken polydactyly functional gene and the application thereof in single nucleotide polymorphism detection. The present invention aims to provide a chicken polydactyly functional gene which is one of the following nucleotide sequences: SEQ ID No. 1 in the sequence table, the polyribonucleotide of the SEQ ID No. 2 protein sequence in the coding sequence table, and functional protein DNA sequences which have more than 90% homology with DNA sequences limited by the SEQ ID No. 1 in the sequence table and have the same encoding. The present invention secondly aims to provide a convenient and reliable method for detecting chicken growth characters; for realizing the purpose, the present invention adopts the following technical scheme: in the method for detecting chicken growth characters, PCR amplification is carried out to the total DNA of chickens through the following primers: 5'-GATTTGATCTGCTTGGAGAC-3' in the positive direction and 5'-GTCATGATAGCAAAGAGCAAG-3' in the reverse direction; single nucleotide polymorphism (SNPs) detection is carried out for detecting whether the NO. 1244 basic group of the amplified gene is c or t. The present invention has important significance for culturing new species of chickens.

Description

Many apodization functions of chicken gene and application thereof

Technical field

The present invention relates to a kind of functional gene and the application thereof of chicken in the bioengineering field, particularly relate to many apodization functions gene and the application in single nucleotide polymorphism detects thereof of chicken.

Background technology

Many toes are vertebrate common unusual limb phenotypes, and different plant species has similarity.At present on people and mouse, carried out research to a certain degree for unusual limb phenotype.

Congenital limb is hereditary defect comparatively common among the crowd unusually, refers to that wherein (toe) is modal congenital hand (foot) deformity more.This class deformity both can independently take place, and can be used as a syndromic part again and occurred.At first, according to the position of finger (toe) of getting involved, independently, many fingers (toe) of non-syndrome type can be divided into axle before (the oar side, preaxialpolydactyly-PPD) and axle back (ulnar side, posterior polydactyly) two big classes.Many fingers (toe) family of being studied at present, major part is an autosomal dominant inheritance, also has minority to transmit in the mode of autosomal recessive inheritance.

The mankind refer to that the genes involved of (toe) family is positioned karyomit(e) No. 7 more more in recent years, and No. 7 dyeing embodies the location focus that has become with the limb development genes involved.At present existing several groups are with many fingers (toe) of their research, also the assignment of genes gene mapping of finger (toe) disease is in No. 7 karyomit(e)s.No. 7 karyomit(e)s of people long-armed with No. 5 karyomit(e)s of mouse are homologous regions, the En2 of mouse etc. and limb are grown relevant gene and all are positioned this.

Many toes candidate gene Lmbr1 (C7orf2) has 17 exons, comprises the genomic dna of about 200kb.The Northern analysis revealed, C7orf2 has the highest expression at human heart and pancreas, and transcription product has all shown the band of 1.9kb and 4.8kb in all test organizations.By adopting the scanning of 5 ' RACE and cDNA library, obtained the sequence of 4849 Nucleotide, C7orf2 transcription sequence open reading frame (ORF) is encoded one by 490 initial protein that amino-acid residue is formed of methionine residues, before this ORF 5 '-UTR of 176 Nucleotide, this 5 '-UTR comprises a terminator codon, is long relatively 3203 Nucleotide 3 '-UTR behind the ORF.By the heredity location, Lmbr1 and Lmbr2 are positioned in the critical area of mouse and the unusual limb mutant of people.People's homologous gene (C7orf2) of Lmbr1 is positioned in the middle part of critical area of the 450kb of human PPD phenotype, and the 3 ' part of C7orf2 is included in the pac clone RP5-982E9 (HEUS etc., 1999) of nearest order-checking.

Studies show that the Lmbr1 gene is the important gene that influences limb development.Discoveries such as Richard, Lmbr1 gene are that the normal limb structure far away of formation mouse is necessary, lose (Richard etc., 2001) that the orthomutation of disappearance Lmbr1 first exon causes mouse limb structure far away.No hand is not had pin, and (Lmbr1 (Corf2) gene also is that human limb structure far away forms necessary for Acheiropodia, ACHP) studies show that of patient.ACHP is an autosomal recessive disease, the molecule loss of ACHP has obtained evaluation recently, it is the 4th exon that the fragment loss of a 4-6kb has been eliminated the Lmbr1 gene, the losing of this exon causes before Lmbr1 reads the frame maturation clips, and produced a null mutation (Ianakiev, 2001) of this gene.ACHP syndromes patient shows as acrodysostosis far away, even agenesis of tibia, and its phenotype is more serious than mouse, and it has been positioned to the zonule of human chromosome 7q36, has covered definite zones of containing the TP-TPS sudden change such as Heus.The sudden change of people, mouse Lmbrl gene different loci all causes losing of limb structure far away, shows that Lmbr1/Lmbr1 has special effect to the g and D of normal limbs.And propose a model: the difference of Lmbr1 vigor causes mutual limb phenotype.Lmbr1 function acquisition type sudden change (GOF) causes the formation of additional limb structure; Function is lost type sudden change (LOF) and is caused losing of limb structure far away

At present, in human PPD family and Hx sudden change mouse, all do not detect the specific mutant of Lmbr1 gene.But Munis etc. (2001) adopt Northern blot to analyze total RNA that the mice embryonic limb forms the zone and find: in the E11.0 stage, the transcriptional level of Lmbr1 is identical in wild-type and Hx/Hx appendage bud; In the E11.5 phase, the expression level of Lmbr1 in forelimb descends but still is present in the hind leg of mutant mice; Arrived the E12.0 phase, the Lmbr1 expression level acutely descends, and limb all detects less than its expression signal in the front and back of Hx/Hx, but the Hx/+ mouse is consistent with wild-type at the expression level of E12.0 phase Lmbr1.Lmbr1 gene expression dose unusual just in time appears at Hx mutant mice visible unusual time (excessive increase of Hx mouse forelimb tissue has been considered to cause the formation of forelimb supernumerary finger) of limb the earliest.The transcriptional level of Lmbr1 returns to the wild-type level in the mouse limb etap in later stage (E12.2 and E13.5), and the expression amount of adult mice tissue (comprising thymus gland, lung, liver, kidney, the heart and brain) Lmbr1 is not subjected to the interference of Hx mutant yet.

High-resolution positioning experiment is effectively supported former imagination, promptly similar effect gene mouse and the sudden change of people's limb.The Lmbr1 gene is arranged in the zone of mouse and the many toe sudden changes of people, and has shown the intensive changes of expression level in the limb of Hx mouse.According to the location and the Lmbr1 expression of gene detected result of many toes proterties, can think that the Hx mutant of mouse should be the regulation and control allelotrope of Lmbr1 gene.

Chicken is four toes normally, and five toes are breedinesses of Dorkings, houndans, sikies, sultans.The four toe phenomenons of chicken are results that high vertebrates the 5th toe of the typical the five fingers is lost.Many results of study show, the supernumerary toe of chicken five toe kinds is not the recovery of the 5th toe lost, but has grown a new toe at the other end of pin.

The many toes of chicken (Po) sudden change is positioned to the 4th linkage group of traditional collection of illustrative plates.Richard etc. (2000) position segregating population with the compartment analysis method, many toes site are positioned the short arm of a chromosome No. 2, near microsatellite marker MCW007.But find that its exact position is subjected to the influence of the phenomenons such as molecular characterization of penetrance, the unknown.Accurately many toes site, location is still difficult.

2P zone chicken carries many toes gene, and this section karyomit(e) has proved a regional homology on No. 5 karyomit(e)s with human 7q36 zone and mouse.The SHH gene physically connects many toes of 2p critical area of mouse HX/Hm and human 7q36 and chicken.The MCW0071 mark is positioned at EN2 (engrailed), and EN2 is positioned on No. 5 karyomit(e)s of mouse, and a sudden change-Hx relevant with supernumerary toe (hemimelic extra-toes) has been positioned to the 5th karyomit(e) of mouse, from EN2 gene 1cm place.Hx is still unknown at the homologous gene of chicken, and it may can be used as the candidate gene (Richard etc., 2000) of the many toe sudden changes of chicken.

Summary of the invention

The many apodization functions gene and the encoded protein matter thereof that the purpose of this invention is to provide chicken.

Many apodization functions gene of chicken, it is one of following nucleotide sequences:

1) the SEQ ID № in the sequence table: 1;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;

3) with sequence table in SEQ ID №: 1 dna sequence dna that limits has 90% above homology, and the identical function protein DNA sequence of encoding.

Sequence 1 in the sequence table is made up of 1457 Nucleotide, the protein of sequence 2 in the code sequence tabulation.

The protein of many apodization functions of chicken genes encoding is SEQ ID № in the sequence table: 2 amino acid residue sequence or with SEQ ID №: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 2 is identical active by SEQ ID №: 2 deutero-protein.

The many apodization functions expression carrier and the clone that contain chicken all belong to the present invention's scope required for protection.

Second purpose of the present invention provides the method for a kind of convenience, reliable detection chicken development character.

Be to realize this purpose, the present invention by the following technical solutions:

A kind of method that detects the chicken development character is with following primer total DNA of chicken to be carried out pcr amplification,

Forward: 5 '-GATTTGATCTGCTTGGAGAC-3 '

Oppositely: 5 '-GTCATGATAGCAAAGAGCAAG-3 '

Carry out single nucleotide polymorphism (SNPs) then and detect, the 1244th base that detects the gene that amplifies is c or t.

Described single nucleotide polymorphism detects preferably carries out with single-strand conformation polymorphism (SSCP) method.

When the 1244th bit base was c, the genotype of its homozygous chicken was BB, and chicken toe type is four toes; When the 1244th bit base was t, the genotype of its homozygous chicken was AA, and chicken toe type shows as five toes, and the genotype of its heterozygous chicken is AB, and wherein 80% above AB type individuality is five toes, and 20% above AB type individuality is four toes.The AB genotype weighs at carcass, complete clean thorax is heavy, partly thorax is heavy only, brisket is heavy, the leg chicken is heavy, neck weighs and abdomen fat weighs in several the indexs all greater than the BB type.And AA type and BB type, AB type difference are not remarkable.

The present invention utilizes the PCR-SSCP method to detect SNPs dexterously, has found to influence the functional gene and the specific mutant site of many toes of chicken proterties.Opened the international standard kind, the secret of the five toe proterties of one of the good local variety silkiefowl black bone of China " perfect " feature, significant to the limb development mechanism of further research chicken.Chicken is as the fabulous animal model of mankind's growth simultaneously, and human limb development is also significant for studying.Discover, the different genotype and the growth traits of many toes of chicken character gene are utmost point significant correlation, show that this gene may also be one of gene that influences growth traits, or with the growth traits gene to a certain degree chain arranged at least, can be used as the marker assisted selection of growth traits.The AB genotype is significantly higher than the BB type, and the new variety of the chicken that might cultivate high-speed rapid growth are handed in prompting with pure pentadactyl chicken and pure four toe chicken giblets.

Embodiment

Used material and experimental technique among the present invention:

1, bacterial strain:

Strain name	Phenotype and genotype
		DH5α	supE44 ΔLac169(Φ80 LacZ ΔM15)hsdR17 RecA endA1 gyrA98 thi-1 recA1
JM109	recA1 supE44 endA1 hsdr17 gryA96 relA1 thiΔ(lac-proAB)F′〔traD36 proAB + laI q LacZ ΔM15〕

2, cloning vector:

pGEM 3zf(+/-)vectors

pGEM 7zf(+/-)vectors

PGEM-T Vectors is all from Promega company

3, tissues of experimental animals sample and blood sample

The F2 that produces with the hybridization of star's fryer, silkiefowl black bone and star's fryer and silkiefowl black bone is on behalf of the experiment material, and wherein to do male parent be orthorhombic system to fryer, and it is reciprocal cross system that Gallus Domesticus is cooked male parent, and each two family of reciprocal cross are totally 312 chickens.During hatching and 12 weeks added up the toe type during ages respectively; Wing venous blood collection during 12 ages in week, the oxalate anti-freezing, after the imitative extracting of phenol, TE dissolves-20 ℃ of preservations.

4, enzyme and reagent

Reagent such as various enzymes, RNA test kit, RACE test kit are respectively available from Huamei Bio-Engrg Co.,, Biolab company and Promega company; The PCR primer is synthetic by Shanghai bio-engineering corporation and Xi'an Mei Lian company.

5, DNA analysis software

Homology analysis software: DANMAN

PCR primer-design software: OLIG04.0,

Data analysis software: SAS (6.12 version).

6, the preparation of damping fluid and common agents

TE damping fluid: 10mM TrisCl, 1mM EDTA, pH8.0, autoclaving.The autoclaving condition is 1.034 * 105Pa, 20min.

STE damping fluid: 0.1M NaCl, 10mM TrisCl, 1mMEDTA pH8.0, autoclaving.

50X TAE damping fluid: Tris alkali 242g, glacial acetic acid 57.1ml, 0.5MEDTA (pH8.0) 100ml adds water to 1L.

5 * tbe buffer liquid: Tris alkali 54g, boric acid 27.5g, 0.5M EDTA (pH8.0) 20ml adds water to 1L.

Plasmid DNA is extracted solution I: 50mM glucose, and 2.5mM TrisCl (pH8.0), 10mM EDTA (pH8.0), 10 pounds of 15min of autoclaving are stored in 4 ℃.

Plasmid DNA is extracted solution II: 0.2M NaOH, and 1%SDS, now with the current.

Plasmid DNA is extracted solution III: 5M potassium acetate solution 60ml, glacial acetic acid 11.5ml, aqua sterilisa 28.5ml.

IPTG solution: IPTG 1g is dissolved in the 50ml distilled water, and filtration sterilization, packing are stored in-20 ℃.

1M TrisCl: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:EDTA 186.1g is dissolved in the 800ml distilled water, with NaOH adjust pH to 8.0, is settled to 1000ml, autoclaving.

3M NaAc (pH7.0): NaAc3H2O 408.1g is dissolved in the 800ml distilled water, transfers pH to 7.0 with glacial acetic acid, is settled to 1000ml, autoclaving.

3M NaAc (pH5.2): NaAc3H2O 408.1g is dissolved in the 800ml distilled water, transfers pH to 5.2 with glacial acetic acid, is settled to 1000ml, autoclaving.

1M CaCl2:CaCl26H2O 27g adds water to 100ml, and filtration sterilization is stored in-20 ℃.

10%SDS:SDS 100g is dissolved in the 900ml distilled water, is heated to 68 ℃ of hydrotropies, with HCl adjust pH to 7.2, is settled to 1000ml.

RNaseA solution: 100mg/ml is dissolved in 10mM TrisCl (pH7.5), and 100 ℃ are boiled 10min among the 15mM NaCl, after the room temperature cooling, is stored in-20 ℃.

Blood DNA extracting solution: 10mM TrisCl (pH8.0), 0.1M EDTA (pH8.0), 20 μ g/ml Pancreatic RNases, 0.5%SDS.

The tissue DNA extracting solution:

	Stock concentration	Working concentration
			Rnase	10mg/ml	20ug/ml
Proteinase K	10mg/ml	100-200ug/ml
			Tissue DNA extraction solution	1M Tris Cl(pH8.0)	50mM
0.5M EDTA(pH8.0)	100mM
		2M NaCl		100mM
10％ SDS	1％

7, the preparation of competent cell (CaCl2 method)

Picking DH5 α or JM109 intestinal bacteria original seed, streak culture on the LB agar plate

↓ picking is cultured fresh single bacterium colony just, is inoculated in the 100ml LB substratum, and 37 ℃ are cultured to OD600 and reach

0.4-0.5。Bacterium is transferred in the sterilization 100ml centrifuge tube of a precooling ice bath 10-15min

↓

4000rpm, 4 ℃ of centrifugal 10min are to reclaim bacterial precipitation

↓

The 0.1M CaCl that adds the precooling of 20ml ice ₂Suspension bacterial precipitation, ice bath 10-15min then

↓

4000rpm, 4 ℃ of centrifugal 10min reclaim bacterial precipitation

↓

Add 4ml 0.1M CaCl ₂The resuspended bacterial precipitation of solution, cell at this moment can be directly used in transformation experiment.

↓

Glycerol adding to final concentration is 15%-20%, and mixing is distributed into 200 μ l, one aliquot, and is frozen in-70 ℃

8, the small-scale of plasmid DNA is extracted---alkaline lysis

(1). inoculate a single bacterium colony and contain in the LB liquid nutrient medium of 70ug/ml Amp, 220rpm, 37 ℃ of shaking culture spend the night (8-10hr) in 3ml.

(2) .10,000rpm, 4 ℃ of centrifugal 5min (do not have 4 ℃ of condition normal temperature also can) collect thalline.

(3). an amount of resuspended bacterial precipitation of STE, the 3ml culture is deposited in the 1.5ml centrifuge tube, and 4 ℃ of centrifugal 5min collect thalline.

(4). use up STE liquid, add the 200ul solution I, vibrate abundant suspension cell in solution I.

(5). add the solution II 200ul of new preparation, will manage and put upside down mixing rapidly 10 times, room temperature leaves standstill 5min.

(6). add solution III 200ul, will manage and be inverted vibration 1min, ice bath 5min.

(7) .1, the centrifugal 8min of 2000rpm carefully gets supernatant in centrifuge tube.

(8). use with the saturated phenol of the isopyknic Tris of supernatant, phenol/chloroform, chloroform in turn extracting once keep water.

(9). add the 3M NaAc of the pH5.2 of 1/10 volume, add the dehydrated alcohol of 2 volumes again, precipitation 15min in-70 ℃ of refrigerators.

(10). the centrifugal supernatant of abandoning adds 70% washing with alcohol precipitation.

(11). the centrifugal supernatant of abandoning, vacuum is drained, and removes trace ethanol.

(12). add 50ul TE add simultaneously 1ulRNase put into 37 ℃ 2 hours.

(13). electrophoretic examinations content.

9, a large amount of preparations of plasmid DNA

Inoculate the single bacterium colony of intestinal bacteria in 100ml LB liquid nutrient medium, 37 ℃ of overnight incubation

↓

4000rpm, 4 ℃ of centrifugal 10min collect thalline, abandon supernatant

↓

Bacterial precipitation is resuspended, centrifugal again as stated above with the STE solution of 20ml ice precooling, remove supernatant, be inverted, blot debris, the plasmid that adds the ice precooling extracts solution I 2ml, and bacterial precipitation is resuspended

The lysozyme soln of the new preparation of ↓ adding 200 μ l (10mg/ml is dissolved in 10mM TrisCl, pH8.0), and mixing

↓

The plasmid that adds the new preparation of 4ml extracts solution II, covers tight centrifuge tube lid, slowly put upside down for several times,

Fully the mixing content is placed 5-10min in room temperature

↓

Add the ice-cold plasmid of 2ml and extract solution III, centrifuge tube is put upside down for several times,

Ice bath 15min can see that the adularescent flocks produces

↓

12,000rpm, 4 ℃ of centrifugal 20min

↓ supernatant is transferred in another centrifuge tube, add 0.6 times of volume Virahol, fully mixing is placed 10min in room temperature

↓

10000rpm, the centrifugal 15min of room temperature reclaims the plasmid precipitation

↓

Remove supernatant, wash precipitation once, drain slightly, be dissolved among the 1ml TE with 70% ethanol

↓

Add 5 μ l RNaseA (5mg/ml), 37 ℃ of digestion 1hr

↓

Be transferred to little centrifuge tube, use isopyknic phenol, phenol: chloroform, chloroform, each extracting is once

↓ add the 10M NH4AC of 1/5 volume, add the dehydrated alcohol of 2 times of volumes again, mixing, room temperature is placed 30min

↓

12,000rpm, the centrifugal 10min of room temperature

↓

Abandon supernatant, wash precipitation twice with 70% ethanol, vacuum is drained, and is dissolved among an amount of TE, is stored in-20 ℃.

10, the Rapid identification of recombinant plasmid size---Cracking method

Picking transforms the back at the dull and stereotyped white colony of cultivating of x-gal, contains Amp's at another piece

The LB lining out is cultivated 12-16hr, chooses a locus coeruleus simultaneously and does contrast

↓

With a small amount of thalline of toothpick picking, be applied in the centrifuge tube that contains 20 μ l aqua sterilisas

↓ vibration behind the thalline that fully suspends, adds 2 * Cracking buffer of equal-volume 20 μ l, thermal agitation 2min

↓ 12, behind the centrifugal 5min of 000rpm, get sample electrophoresis on the supernatant 10-20 μ l, compare with the lysate of locus coeruleus, detect

Whether plasmid has is inserted and approximate size.

11, the extraction of chicken blood DNA

Get 20 μ l fresh bloods, add the ACD anti-freezing, add 600 μ l fowl lysates, adding proteolytic enzyme k is 100-200 μ g/ml to final concentration, and 55 ℃ of digestion of mixing 6-10hr no longer includes the heavy-gravity agglomerate in solution

↓

Solution is cooled to room temperature, adds equal-volume phenol, put upside down centrifuge tube repeatedly,

Mix formation emulsion, 12,000rpm, the centrifugal 10min of room temperature until two-phase

↓

Get supernatant, use equal-volume phenol again: chloroform, each extracting of chloroform 1 time

↓

Get reset and add 1/10 volume NaAc (3M, pH5.2) and 2 times of volume dehydrated alcohol deposit D NA

↓

DNA chosen be put in the 1.5ml centrifuge tube, wash twice with 70% ethanol

↓

(attention can not be too dried) after the DNA drying is dissolved in an amount of TE or the sterilization distilled water.

12, the segmental preparation of purpose (freeze-thaw method)

(1). get 10 μ g plasmid DNA, 200 μ l systems, 37 ℃ of enzymes of 10u restriction endonuclease are cut 3hr.

(2). add 25.8 μ l 1M TrisCl (pH7.5), the 10u restriction endonuclease adds ddH ₂37 ℃ of enzymes of O to 300 μ l are cut 2hr.

(3). the enzyme that takes a morsel is cut product inspection enzyme and is cut effect.

(4) .0.8% agarose, electrophoretic separation purpose fragment on the glue.

(5). cutting-out contains purpose fragment gel, and chopping places the 1.5ml centrifuge tube.

(6). add equal-volume phenol mixing.

(7). place 3-5min in the liquid nitrogen, room temperature is thawed, and repeats secondary.

(8) .8000g, centrifugal 8min.

(9). get that to reset and add equivalance phenol imitative, chloroform is respectively taken out one time.

(10). add 2 times of volume dehydrated alcohols, 1/10 times of volume 3M NaAc (pH5.2), put 20min in-20 ℃.

(11) .1, the centrifugal 10min of 2000rpm.

(12) .70% ethanol is washed one time, drains to be dissolved in 20 μ l TE (pH8.0).

(13). survey the OD value, electrophoretic examinations.

13, the PCR product is linked on the T carrier

Linked system:

10 * T4 dna ligase damping fluid, 1 μ l

T4DNA ligase enzyme (3u/ μ l) 1 μ l

T carrier (30ng/ μ l) 1 μ l

PCR product 60ng

Add dd H ₂O to 10 μ l

16 ℃ of water-baths are spent the night

14, transform

(1). from-70 ℃ of competent cells that take out preservation, ice bath helps and melts.

(2) .200 μ l competent cell adds 5 μ l and connects product, ice bath 30min.

(3) .42 ℃ of water-bath heat shock is 90 seconds, ice bath 2min.

(4). add 800 μ lLB, 37 ℃ of water-baths bring back to life 50min.

(5). spread 200 μ l in containing X-gal, IPTG, the LB flat board of Amp.

(6) cultivate 9-16hr for .37 ℃.

15, screening recon

(1). the picking hickie lines and scribbles X-gal, IPTG, the flat board of Amp.

(2). connect the line bacterium in 2ml LB, incubated overnight.

(3). alkaline denaturation extracts recombinant plasmid.

(4). restriction endonuclease digests 0.5 μ g DNA.

(5). electrophoretic examinations.

16, preparation template

(1). alkaline denaturation prepares template.

(2). transfer concentration to 0.1-0.2 μ g/ μ l.

17, Dye Primer does the part order-checking to positive colony

(1). alkaline lysis is prepared template, is diluted to 0.1-0.2 μ g/ μ l

(2) .PCR sequencing reaction system:

Base	A C G T
		Template (μ l)	1 1 2 2
Premix(μl)	4 4 8 8
		Cumulative volume (μ l)	5 5 10 10

(3) .PE9600 PCR reaction

A.95 ℃, 30 seconds; 55 ℃, 30 seconds; 70 ℃, 1min; 15 circulations.

B.95 ℃, 30 seconds; 70 ℃, 1min, 15 circulations.

(4). precipitation PCR product

A. reactant is joined and contain 100 μ l, 95% ethanol, in the 1.5ml centrifuge tube.

B. place 15min on ice.

C.12,000rpm, centrifugal 15min.

D.250 the ethanol of μ l 70% is washed one time.

E. drain ,-20 ℃ of preservations are prepared order-checking and are gone up sample.

18, utilize glass milk elution method to reclaim the PCR product

(1). downcut required dna fragmentation from sepharose, be placed in the 1.5ml Eppendorff pipe and smash to pieces, action is soft.

(2). add the sol solutions of 3 times of volumes, place 5min (or 50 ℃ of insulation 3min) under the room temperature, jog Eppendorff pipe dissolves glue several times fully therebetween.

(3). add 10 μ l glass milk, put upside down mixing, ice bath is placed 10min down.And the 2-3min mixing once at interval.

(4) .12, centrifugal 30 seconds of 000rpm inhales and abandons supernatant.

(5). add 250 μ l rinsing liquids, inhale rinsing liquid with sample injector and gently glass milk is broken up evenly the centrifugal supernatant of abandoning.

(6). repeated for the 5th step once.After having drawn rinsing liquid, in centrifugal again 10 seconds, last a little rinsing liquid is blotted only with the Tip head.Then, be positioned over the dry 20min of 37 ℃ of incubators.

(7). add an amount of sterile distilled water or TE (10-30 μ l), mixing.60 ℃ of water-bath 5min.

(8) .12, the centrifugal 1min of 000rpm, it is standby to reclaim supernatant, repeats 7-8 and goes on foot 1-2 time.

19, the extraction of total RNA

Method one: guanidinium isothiocyanate---hot phynol method

1) outfit that will test usefulness spends the night with 0.1% DEPC water logging bubble, autoclaving then, glassware more than 180 ℃ of baking 8h, 80 ℃ of oven dry of plastics.

2) 0.1g is organized sample rapid grinding powder in mortar, put into the homogenate pipe homogenate that 1ml GITC reagent is housed.Homogenate takes out and is placed in the 1.5ml centrifuge tube, inhales and beats several times to shear chromatin dna, 60 ℃ of temperature bath 10min.

3) add 60 ℃ of phenol of equal-volume, mixing, extracting.

4) cooling in the ice, the centrifugal 10min of 10000rpm.Get supernatant, repeat extracting once with hot phenol.

5) after the equal-volume chloroform extracting at room temperature 1-2 time, on reset and add 1/10 volume 3M sodium acetate and 3 times of volume dehydrated alcohols, spend the night-20 ℃ of precipitations.

6) the centrifugal 15min of 12000rpm, precipitation is dissolved among the 0.5ml TE, adds Proteinase K to 200 μ g/ml, 56 ℃ of digestion 1h.The hot phenol of equal-volume: the chloroform extracting, cool off centrifugally, repeat once.

7) reset and add 2.5 times of volume dehydrated alcohols on and precipitated liquid.Centrifugal, 70% ethanol is washed precipitation, after the drying, is dissolved in an amount of DEPC water.

Method two: extract total RNA with the TRIZOL test kit

1) homogenate

Add the 50-100mg tissue in the TRIZOL of 1ml reagent, sample volume should not surpass 10% of TRIZOL.With refiner high-speed homogenization 2 minutes.

2) (optional)

Want protein isolate, fat, polysaccharide or ECM such as muscle, fatty tissue, 2-8 ℃ of 12000g is centrifugal 10 minutes after homogenate.With the even new Eppendof pipe that is transferred to of supernatant.

3) phase-splitting

15-30 ℃ of incubation 5 minutes, nucleoprotein complex body fully dissociates.Add the 0.2ml chloroform, firmly shook 15 seconds, placed 2-3 minute for 15-30 ℃.Centrifugal 15 minutes less than 12000g 2-8 ℃.

4) precipitation of RNA

Shifting water newly manages in one.Add the 0.5ml Virahol, mixing, room temperature was placed 10 minutes.4-8 ℃ of centrifugal 10 minutes of 12000g (RNA is invisible usually before centrifugal, forms glue sample precipitation at the tube wall or the pipe end).

5) the sedimentary washing of RNA

Abandon supernatant, the washing with alcohol RNA that every 1ml TRIZOL adds 1ml 75% at least once.2-8 ℃ less than centrifugal 5 minutes of 7500g.

6) weight of RNA is molten

Outwell ethanol, air drying or vacuum-drying 5-10 minute.Add suitable DEPC treating water, inhale and beat several times, be put in 55-60 ℃ of dissolving 10 minutes.-70 ℃ of preservations.

20, RNA sex change electrophoresis

Method one: in a sterilization Eppendorf tube, mix following liquid, with preparation RNA sample.

RNA 9μl

10×TAE 4μl

Formaldehyde 7 μ l

Methane amide 20 μ l

Cumulative volume 40 μ l

Behind the mixing, 65 ℃ of incubation 15min take out and put on ice.

Add the RNA sample-loading buffer, the agarose gel electrophoresis with 1%, electrophoresis buffer are 1 * TAE.

Method two

1) contain the preparation of the sex change glue of formaldehyde: take by weighing the 1g agarose in 73ml distilled water, dissolve after cooling slightly, add 20ml 5 * MOPS buffer, 17ml formaldehyde falls glue behind the mixing.

2) preparation of RNA sample: the following liquid of mixing in an Eppendorf tube:

RNA (being no more than 30 μ g) 5.5 μ l

5 * formaldehyde gel electrophoretic buffer, 2.0 μ l

Formaldehyde 3.5 μ l

Methane amide 10.0 μ l

Cumulative volume 20.0 μ l

65 ℃ of incubation 15min cool off in the ice, and are centrifugal slightly.

Add 2 μ l formaldehyde sample loading buffers.Prerunning is 5 minutes before the application of sample.Electrophoresis buffer is 1 * formaldehyde gel electrophoretic buffer, and voltage is 5V/cm.

21, the removal of DNA in the cell total rna

1) mixes following reagent with the DNA among the total RNA of peptic cell, 37 ℃ of incubations 30 minutes

52μl 1μg/μlRNA

10 μ l, 1 μ/μ l people placenta RNA enzyme inhibitors

10 μ l, 1 μ/μ l does not contain the DNA enzyme I of RNA enzyme

8 μ l 10 * reaction Buffer

Cumulative volume 80 μ l

2) add equal-volume phenol/chloroform, acutely mixed postposition 10 minutes on ice.4 ℃ of 12000g are centrifugal 5 minutes.

3) move supernatant and newly manage in one, add 5 μ l 3mol/L sodium acetates, 200 μ l, 100% ethanol, mixing is placed 30 minutes precipitated rnas for-70 ℃.

4) 4 ℃ centrifugal 10 minutes, abandon supernatant.Ethanol (preparation of DEPC water) washing precipitation with 500 μ l 70%.

5) with the DEPC water dissolution RNA of 20 μ l, get 1 μ l RNA sample in 1ml water, with the light absorption value of spectrophotometric determination A260, the concentration of accurately quantitative RNA.

6) RNA that 3 μ g are clean carries out electrophoresis on 6% sex change sepharose, detects the integrity of RNA.All the other RNA are stored in-80 ℃.

22, reverse transcription

1) gets the RNA that 1 μ g does not contain DNA, be diluted to 0.1 μ g/ μ l with DEPC water, and place ice bath.Set up the reverse transcription reaction system of RNA by following method:

The reaction system μ l of 20 μ l

Water 9.4

5×RTbuffer 4.0

dNTP(250μM) 1.6

Total RNA (no DNA) 2.0 (0.1 μ g/ μ l, interim dilution)

T(18) 2.0

Total system 19.0

2) 65 ℃ of temperature are bathed and were made the sex change of mRNA secondary structure in 5 minutes; 37 ℃ of temperature are bathed and were made primer annealing in 10 minutes.Every pipe adds 1 μ l 100U/ μ l MMLV ThermoScript II.Rapid mixing, 37 ℃ were continued incubation 50 minutes.5 minutes deactivation ThermoScript II of 75 ℃ of incubations, centrifugal collection drop.Reaction tubes is placed on ice, be used for pcr amplification immediately, or be stored in-20 ℃ and be used for later experiment.

Embodiment 1, RACE experiment

One, synthesizing single-stranded cDNA

1, in the centrifuge tube of 0.5ml, adds

10×PCR buffer 2.5ul

GSP1 1ul(25pmol/ul)

Total heart RNA 4ul (0.8ug/ul removes DNA)

DEPC water 10.5ul

Add up to 15.5ul

2, mixture is taken out after 10 minutes 70 ℃ of hatchings placed cooled on ice 1 minute, add on ice

10×PCR buffer 2.5ul

2.5mM Mgc12 2.5ul

10mM dNTP mix 1ul

0.1M DTT 2.5ul

1M DTT 2.5ul

3, softly mix, 42 ℃ of hatchings added luisuperscript after 1 minute ^TMII RT softly mixes, and is centrifugal, hatches 50 minutes for 42 ℃

4,15 minutes termination reactions of 70 ℃ of hatchings.

5, place 37 ℃ centrifugal 10-20 second, adds the RNase mix of 1ul, thorough and equably mixing, and 37 ℃ of hatchings were placed on ice in 30 minutes, and product is stored in-20 ℃.

Two, cDNA purifying

1, under the room temperature balance in conjunction with damping fluid; 65 ℃ of following balance 100ul aqua sterilisas;

2, add 120ul in conjunction with liquid (6M NaI) in straight chain reactions;

3, mixed solution is forwarded to GlassMax Spin Cartridge, centrifugal 20 seconds of 13000g;

4, outwell waste liquid in the pipe, again pillar is put back to (reservation waste liquid) in the pipe;

5, the 1 * WASH buffer that adds 0.4ml precooling (4 ℃) is in post, and centrifugal 20 seconds of 13000g abandons waste liquid, again 3 times again;

6, as above wash twice with 70% ethanol (4 ℃) of 400ul precooling;

7, the centrifugal 1min of 13000g again after the last washing with alcohol;

8, pillar is forwarded to one and newly manage, add 40ul aqua sterilisa (being preheating to 65 ℃), 20 seconds centrifugal eluted dnas of 13000g.

Three, cDNA TDT tailing

1, adds following composition, and mix

DEPC water 6.5ul

5×tail buffer 5.0ul

2mM dCTP 2.5ul

Purifying cDNA 10ul

Add up to 24ul

2, hatched 2-3 minute for 94 ℃, cooled on ice 1 minute adds lulTDT on ice, hatches 10 minutes for 37 ℃.

3,65 ℃ made the TDT inactivation in 10 minutes, placed on ice.

Four, tailing cDNA PCR

PCR liquid is preheating to 94 ℃, adds following component on ice:

Aqua sterilisa 15.25ul

10×PCR buffer 2.5ul

25mMMgcl2 1.5ul

10mMdNTP 0.5ul

GSP2(10uM) 2ul

AAP(25uM) 1ul

Dc-tailing cDNA 2.5ul

Add up to: 24.5ul

Add 0.5ul Taq enzyme mixing, place immediately on the PCR instrument that is preheating to 94 ℃,

The PCR reaction conditions:

94 ℃ of 2min, 94 ℃ of 1min then, 55 ℃ of 30S, 72 ℃ of 2min, 35 circulations, last 72 ℃ of 10min, 4 ℃ of long-time preservations.

Five, nest-type PRC takes out tailing PCR product 5ul, adds the aqua sterilisa dilution of 495ul, adds on ice:

Aqua sterilisa 15.5ul

10×PCRbuffer 2.5ul

dNTP 2ul

GSP3(25uM) 1ul

UAP(25uM) 1ul

Dilution PCR product 2.5ul

Add up to 25ul

Add 0.5ul Taq enzyme, put into immediately and be preheating to 94 ℃ PCR

The PCR reaction conditions:

94 ℃ of 2min, 94 ℃ of 1min then, 55 ℃ of 30S, 72 ℃ of 2min, 35 circulations, last 72 ℃ of 10min, 4 ℃ of long-time preservations.

The condition of embodiment 2, single-strand conformation polymorphism analysis (SSCP)

25 μ l PCR reaction systems: 2.5 μ l, 10 * Taq dna polymerase buffer liquid (500mmol/L KCl, 100mmol/L TrisCl, 15mmol/L MgCl ₂, 0.01% gelatin), 2 μ l dNTPs (2.5mmol/L for each), primer final concentration 0.5 μ mol/L, 1U Taq archaeal dna polymerase, template DNA 50-100ng.Final volume 25 μ l.

PCR reaction conditions: 94 ℃ of 5min, 94 ℃ of 30S then, 62 ℃ of 30S, 72 ℃ of 40S, 35 circulations, last 72 ℃ of 7min, 4 ℃ of long-time preservations.

Agarose gel electrophoresis with 2% detects the pcr amplification result, carries out sscp analysis afterwards.As follows with 20% non-denaturing polyacrylamide gel (30ml volume, 0.1cm adhesive tape) making, electrophoresis and dyeing for the example detailed process:

(1) cleans sheet glass that glue uses and clean, after the oven dry, with the slit between 0.8% agarose closed glass plate and adhesive tape with distilled water flushing.

(2) in the 100ml beaker, add 30% acrylamide (29: 1) 20ml, 50% glycerine of 3ml, 3ml 10 * TBE, 10% ammonium persulphate 200ul, TEMED12ul adds pure water 4ml, mixes back encapsulating rapidly.

(3) stop encapsulating to from sheet glass upper edge 0.1cm the time when pouring, insert comb, polymerized at room temperature half an hour, unnecessary acrylamide is preserved.At any time observe the gel polymerisation situation, and add acrylamide.

(4) gel polymerisation good after, add 1 * TBE to electrophoresis chamber, use the irrigation with syringe well.

(5) prerunning is 10 minutes, prepares point sample simultaneously.

(6) get the 1ulPCR product and place the PCR pipe, add 5ul sex change Buffer, centrifugal mixing, sex change is 10 minutes under the 98 degree conditions.

(7) rapid ice bath is 10 minutes, uses the microsyringe point sample.

(8) opening power, 120 volts, electrophoresis 24 hours.

(9) electrophoresis is shut electrophoresis apparatus after finishing, and emits electrophoresis liquid, carefully takes off gel, places 70% ethanol, and the water-bath oscillator slowly shakes up fixes 15 minutes.(ethanol uses up and can reclaim)

(10) deionization (or distilled water) washing glue is 2 times, and each 2 minutes, flush away ethanol.

(11) with 200ml staining fluid dyeing 40 minutes.

(12) deionization washing glue is 3 times, and each 2 minutes, the staining fluid that flush away is unnecessary.

(13), about 10-30 minute, when the intensity of being with as DNA is suitable, outwell colour developing liquid with the colour developing of 200ml colour developing liquid.

(14) distilled water is washed unnecessary colour developing liquid off, and preservative film is sealed, scanography or preservation.

Embodiment 3, single-strand conformation polymorphism method (SSCP) detection functionality mutational site

With primer of the present invention the whole blood DNA of experimental chicken is carried out pcr amplification, carry out sscp analysis then, when the 1244th bit base of many apodization functions of chicken gene (Lmbr1) gene was C, the genotype of its homozygous chicken was BB, and chicken toe type is four toes; When the 1244th bit base of Lmbr1 gene was T, the genotype of its homozygous chicken was AA, and chicken toe type shows as five toes; The genotype of its heterozygous chicken is AB, and wherein 80% above AB type individuality is five toes, and 20% above AB type individuality is four toes.Table 1 is to utilize this primer that the F2 filial generation of silk plumage Gallus Domesticus and star's fryer is carried out SSCP somatotype result.

Table 1 lmbr1 gene is in the card side comptibility test (unit: only) of resource family F2 for separation case

Genotype	The toe type	51 familys	74 familys	85 familys	87 familys	Add up to	Theoretical value
								AA	Five toes	24	12	9	12	59	72.5
Four toes	1	0	0	1
					AB	Five toes	41		42	25	32	166	145
Four toes	10	5	3	8
						BB	Five toes	1	1	1	0			65	72.5
Four toes	21	14	13	14
					Add up to (290)		98	74	51	67	x 2＝3.2130 prob＝0.2066

Card side's independence test of table 2 lmbr1 genotype and toe type (unit: only)

Genotype	Left and right sides pin four toes	Left and right sides pin five toes	Other *	Add up to	Chi square test
						AA	2	49	8	59	x 2＝153.8524 ** prob＜0.0001
AB	26	117	23	166
					BB	62	3	0	65

Annotate: in this table, " other " comprises that other many dactylitis type result that non-left and right sides toe is four toes or five toes shows:

1) F0 is AA for individual pentadactyl in these four familys, and four toe types all are BB.

2) F1 is for being pentadactyl, and genotype all is AB.

3) F2 generation then the AA genotype be five toes, the BB genotype is four toes, wherein in 63 AA types 2 four toes is arranged, and 3 five toes is arranged, through x in 67 BB types ²Check difference is all not remarkable.

4) the AB genotype 85% is five toes then, and 15% is four toes, shows that many toes proterties is the Incomplete dominance proterties.

5) table 1 result shows, each genotype at the segregation ratio in F2 generation through x ²Check is not remarkable with the difference of theoretical value, shows to meet the Mendelian inheritance law of segregation, i.e. AA: AB: BB=1: 2: 1.

6) card side's independence test (table 2) difference of genotype and toe type is extremely remarkable, shows that the goodness of fit of genotype and toe type is good.

It is the functional gene of many toes proterties that these results have convincingly demonstrated this gene.

The correlation analysis of embodiment 4, genotype and carcass proterties

ANOVA showed significant (as shown in table 3), chicken Lmbr1 different genotype has remarkable effect to growth traits.Heavy at carcass, heavy, the complete clean thorax of half clean thorax is heavy, brisket is heavy, das Beinfleisch is heavy, in the abdomen fat principal characteristic shape, be the genotypic least square average of AB (LSM) greater than the BB type, and the difference between AA type and BB type is not remarkable.This heterozygous genes type that shows many toes character gene Lmbr1 is favourable to the growth of chicken, and therefore, the ratio that improves the Lmbr1 gene hybridizing type in seed selection is expected to improve the speed of growth, increases carcass output.

Production traits least square mean (LSM) comparative result of table 3 different genotype

Genotype	Carcass heavy (g)	Half clean thorax (g)	Complete clean thorax (g)	Brisket heavy (g)	Das Beinfleisch (g)	Neck heavy (g)	Abdomen fat heavy (g)
								AA	1557.9 ab	1234.9 ab	1140.8 ab	98.5 ab	136.9 ab	139.1 ab	56.6 ab
AB	1607.3 a	1280.7 a	1167.3 a	100.4 a	140.4 a	145.4 a	58.5 a
								BB	1484.7 b	1174.5 b	1075.1 b	90.1 b	128.5 b	134.5 b	45.5 b

Conspicuous level: 0.05＜p＜0.01

Sequence table

<160>2

<210>1

<211>1457

<212>DNA

＜213〉jungle fowl be born in the year of chicken kind of (a Gallus gallus)

<220>

<222>(1244)

＜223〉n=t or c

<400>1

acgaggtgtc gatccgcgag cagatcttcc acagccaagt gcgggagtac acgatctgtt 60

ttctcctatt tgctgttctc tacatagtgt cctacttcat aatcacaaga tacaaaagaa 120

aagcagatga gcaagaggat gaagatgcca tagttaacag aatatcgctg ttcttgagca 180

ccttcactct agcagtttca gctggtgcag tcctgcttct gcctttctca atgatcagca 240

atgagatcct gctttccttt ccacaaaact actatattca gtggttaaat ggctcactaa 300

ttcatggttt atggaatctt gcttctcttt tttccaattt atgtttgttt gtgttgatgc 360

cctttgcctt tttcttcttg gagtcggaag gatttgctgg cttaaaaaag gggatcagag 420

cacgcattct ggagaccctt gtaatgctca tacttcttgc actgcttatc cttggaatcg 480

tatgggtggc ttcagctctc atagacaatg atgctgccag tatggagtct ttgtacgacc 540

tctgggaatt ctacctccca tatttatatt cctgtatatc actgatggga tgcttgttac 600

ttctgttatg cacgccagtg ggactttcgc ggatgtttac agtaatgggt cagttgctgg 660

tgaagccaac aattcttgag gacctagatg agcagatgta tatcatcact ttagaggaag 720

aagcaattca gaggaagctt aatgggatat cttccacatt ggaaaaccag acagtggagt 780

tggaacagga gcttgaaaaa gtgaagtgca agaaaacaaa tctagaacgt cggaaaaaag 840

cttctgcttg ggagaggaat ttagtgtatc cagctgttat gatattgcta ctgattgaga 900

catccatctc agtcctatta gttgctttca acatccttta cctgttggtt gatgagactg 960

caatgccaaa aggatcaggg ggacctggaa taggaaatgc atccctatcc acctttggtt 1020

ttgtgggagc agcacttgaa atcattttga ttttctatct catggtatcc tctgtcgtcg 1080

gcttctacag ccttcgtttt tttgaaaatt tcattcccag gaaggatgat acaactatga 1140

caaagataat tggaaactgt gtctcaatct tggtgctgag ctcggctttg ccagtgatgt 1200

caaggacact gggaattact cgatttgatc tgcttggaga cttnggaagg tttaactggc 1260

tgggaaactt ctatattgta ttatcttaca acttgctctt tgctatcatg acaacgttgt 1320

gtctggtcag aaagttcact tctgctgtgc gagaagagct cctgaaggca ttaggattag 1380

ataaacttca tctgtcgaac aatccaagag actcagagac aaagccgtct gcaaatgggc 1440

atcagaaagc actgtga 1457

<210>2

<211>484

<212>PRT

＜213〉jungle fowl be born in the year of chicken kind of (a Gallus gallus)

<400>2

Glu Val Ser Ile Arg Glu Gln Ile Phe His Ser Gln Val Arg Glu

1 5 10 15

Tyr Thr Ile Cys Phe Leu Leu Phe Ala Val Leu Tyr Ile Val Ser

20 25 30

Tyr Phe Ile Ile Thr Arg Tyr Lys Arg Lys Ala Asp Glu Gln Glu

35 40 45

Asp Glu Asp Ala Ile Val Asn Arg Ile Ser Leu Phe Leu Ser Thr

50 55 60

Phe Thr Leu Ala Val Ser Ala Gly Ala Val Leu Leu Leu Pro Phe

65 70 75

Ser Met Ile Ser Asn Glu Ile Leu Leu Ser Phe Pro Gln Asn Tyr

80 85 90

Tyr Ile Gln Trp Leu Asn Gly Ser Leu Ile His Gly Leu Trp Asn

95 100 105

Leu Ala Ser Leu Phe Ser Asn Leu Cys Leu Phe Val Leu Met Pro

110 115 120

Phe Ala Phe Phe Phe Leu Glu Ser Glu Gly Phe Ala Gly Leu Lys

125 130 135

Lys Gly Ile Arg Ala Arg Ile Leu Glu Thr Leu Val Met Leu Ile

140 145 150

Leu Leu Ala Leu Leu Ile Leu Gly Ile Val Trp Val Ala Ser Ala

155 160 165

Leu Ile Asp Asn Asp Ala Ala Ser Met Glu Ser Leu Tyr Asp Leu

170 175 180

Trp Glu Phe Tyr Leu Pro Tyr Leu Tyr Ser Cys Ile Ser Leu Met

185 190 195

Gly Cys Leu Leu Leu Leu Leu Cys Thr Pro Val Gly Leu Ser Arg

200 205 210

Met Phe Thr Val Met Gly Gln Leu Leu Val Lys Pro Thr Ile Leu

215 220 225

Glu Asp Leu Asp Glu Gln Met Tyr Ile Ile Thr Leu Glu Glu Glu

230 235 240

Ala Ile Gln Arg Lys Leu Asn Gly Ile Ser Ser Thr Leu Glu Asn

245 250 255

Gln Thr Val Glu Leu Glu Gln Glu Leu Glu Lys Val Lys Cys Lys

260 265 270

Lys Thr Asn Leu Glu Arg Arg Lys Lys Ala Ser Ala Trp Glu Arg

275 280 285

Asn Leu Val Tyr Pro Ala Val Met Ile Leu Leu Leu Ile Glu Thr

290 295 300

Ser Ile Ser Val Leu Leu Val Ala Phe Asn Ile Leu Tyr Leu Leu

305 310 315

Val Asp Glu Thr Ala Met Pro Lys Gly Ser Gly Gly Pro Gly Ile

320 325 330

Gly Asn Ala Ser Leu Ser Thr Phe Gly Phe Val Gly Ala Ala Leu

335 340 345

Glu Ile Ile Leu Ile Phe Tyr Leu Met Val Ser Ser Val Val Gly

350 355 360

Phe Tyr Ser Leu Arg Phe Phe Glu Asn Phe Ile Pro Arg Lys Asp

365 370 375

Asp Thr Thr Met Thr Lys Ile Ile Gly Asn Cys Val Ser Ile Leu

380 385 390

Val Leu Ser Ser Ala Leu Pro Val Met Ser Arg Thr Leu Gly Ile

395 400 405

Thr Arg Phe Asp Leu Leu Gly Asp Phe Gly Arg Phe Asn Trp Leu

410 415 420

Gly Asn Phe Tyr Ile Val Leu Ser Tyr Asn Leu Leu Phe Ala Ile

425 430 435

Met Thr Thr Leu Cys Leu Val Arg Lys Phe Thr Ser Ala Val Arg

440 445 450

Glu Glu Leu Leu Lys Ala Leu Gly Leu Asp Lys Leu His Leu Ser

455 460 465

Asn Asn Pro Arg Asp Ser Glu Thr Lys Pro Ser Ala Asn Gly His

470 475 480

Gln Lys Ala Leu

484

Claims (7)

1, many apodization functions of chicken gene, it is one of following nucleotide sequences:

1) the SEQ ID № in the sequence table: 1;

2) with sequence table in SEQ ID №: 1 dna sequence dna that limits has 90% above homology, and the identical function protein DNA sequence of encoding.

2, gene according to claim 1 is characterized in that: many apodization functions gene of described chicken is SEQ ID №: 1.

3, the protein of many apodization functions of chicken genes encoding, its amino acid residue sequence are SEQ ID № in the sequence table: 2.

4, contain the described expression carrier of claim 1.

5, the clone that contains the described expression vector of claim 4.

6, a kind of method that detects the chicken development character is with following primer total DNA of chicken to be carried out pcr amplification,

Forward: 5 ' GATTTGATCTGCTTGGAGAC-3 '

Oppositely: 5 ' GTCATGATAGCAAAGAGCAAG-3 '

Carry out single nucleotide polymorphism then and detect, the 1244th base that detects the gene that amplifies is c or t, and when the 1244th bit base was c, the genotype of its homozygous chicken was BB, and chicken toe type is four toes; When the 1244th bit base was t, the genotype of its homozygous chicken was AA, and chicken toe type shows as five toes.

7, the method for detection chicken development character according to claim 6 is characterized in that: described single nucleotide polymorphism detects carries out with the single-strand conformation polymorphism method.