CN101157923A - DNA fragment related to pig polydactyly - Google Patents

Abstract

The invention provides a DNA fragment relating to a swine polydactyly. The DNA fragment is one of the following polynucleotide: (1) the polynucleotide with the nucleotide sequence of SEQ NO.1 and SEQ NO.2; (2) the polypeptide polynucleotide with the encoding amino acid sequence of SEQ NO.2 and SEQ NO.4. The DNA fragment of the invention has a close relation with the development of swine limbs. The missense mutation of the former four codons of an Exon 16 directly results the polydactuly of swine toes. Therefore, the invention provides a method for detecting the development phenotype of the swine. The method adopts the following premiers to increase reverse transcription products of the general RNA of the swine: 5'-CGC CGT GGC TTG TAA CAT TCT CTG C-3', 5'-AAG CAG TGG TAT CAA CGC AGA GT-3'. The product of the normal swine is 197bp, the product of the pilydactyly swine is 636bp.

Description

A kind of and the relevant dna fragmentation of the many toes of pig

Technical field

The present invention relates to sudden change and field of genetic engineering, be specifically related to a kind of animal limb anormogenesis genes involved.

Background technology

Refer to that (toe) is in the animal foot forming process because a kind of cacomelia that signal error causes happen occasionally, and phenotype is similar between different plant species on people, mouse, chicken, pig more.At present, on many fingers (toe) of people, mouse, chicken, carried out a large amount of research, and having obtained certain progress, the types of presentation, the mode of inheritance that mainly show as many fingers (toe) are clear and definite, have located chromosome segment (people: 7q36,2q31 and the 7p13 of many fingers (toe) gene; Mouse: disconnected on No. 5 karyomit(e)s with people 7q36 homologous district; Chicken: 2p45), cloned such as Shh (sonic hedgehog), Lmbr1 (lamb region 1)/C7orf2 (chromosome 7 open reading frame 2), Lmbr2, Hox, Gli, EN2 (ENGRAILED2), FGF, FgF acceptor, RAR (retinoic acid receptors), WNT (wg and int), BMP a large amount of candidate genes such as (bone morphogenetic protein), and found the various mutations form or the montage type of gene.

In numerous candidate genes, the Lmbr1 gene is the indispensable gene that the limbs structure forms.On people and mouse, the difference of Lmbr1 gene vigor can cause opposite limbs abnormal phenotype: Lmbr1 function gain mutation can cause the formation of additional limb structure, produces extra limb or supernumerary toe; Function is lost the type sudden change can cause that losing of limb structure far away or toe number reduce.Chicken Lmbr1 gene 1244 bit base C mutation T can cause that also chicken produces five toes (chicken is generally four toes); And heavy, the complete clean thorax of thorax is heavy only, brisket weighs, das Beinfleisch is heavy, abdomen fat heavily waits in the carcass proterties in carcass body weight, half, and CT type chicken is obviously greater than CC type chicken.Therefore, select CT type individuality, can improve the speed of growth, increase carcass output at the breeding mass selection; In addition, chicken Lmbr1 gene also can be used as the mark that detects its development character, is that C or T can detect its genotype by detecting chicken Lmbr1 gene 1244 bit bases, can be used as molecular marker assisted selection, and great application prospect is arranged in a breed of chicken.CN02153389.X

In actual production, we find that many toes overwhelming majority of pig are the preceding many toes (PPD) of axle, but the research of many toes of control pig gene still belongs to blank.So far still there is not more complete pig Lmbr1 gene order, more do not find the difference of normal pig and many toes pig on the Lmbr1 gene order, therefore, can not carry out the genotype in mutational site and the correlation analysis between pig carcass proterties, and then inquire into the feasibility of Lmbr1 gene as pig carcass trait molecular marker assisted Selection.

Summary of the invention

The object of the present invention is to provide a kind of and the relevant dna fragmentation of the many toes of pig.

Another object of the present invention is to provide the application of this dna fragmentation in detecting pig development character method.

The present invention realizes that the technical scheme of above-mentioned purpose is:

A kind of and the relevant dna fragmentation of the many toes of pig, this dna fragmentation is one of following polynucleotide:

(1) nucleotides sequence is classified the polynucleotide of SEQ NO.1 and SEQ NO.3 as;

(2) encoding amino acid sequence is the polynucleotide of the polypeptide of SEQ NO.2 and SEQ NO.4.

One of dna fragmentation of the present invention (SEQ NO.1) is located away from normal pig (the supporting B of being of CRP is a pig) heart tissue, sequence total length 1797bp, the part coding region (1-1178bp) and the complete 3 untranslated region (1179-1797bp) that have comprised many toes of pig genes involved, encoding amino acid sequence is the polypeptide of SEQ NO.2, the Lmbr1 similarity of its coding region and people (NM-002458.3), mouse (NM-020295.3), chicken (AY251537.2) is high, and the nucleotide sequence similarity is respectively 90%, 87%, 80%; The encoded polypeptide sequence similarity is respectively 96%, 96%, 91%.

Two (SEQ NO.3) of dna fragmentation of the present invention separate from many toes pig (the supporting B of being of CRP is a pig) heart tissue; it is a fragment in many toes of pig genes involved; compare with normal gene SEQ NO.1; its sudden change zone is 755～768bp, is 922～934bp in the corresponding position of SEQ NO.1.In this zone, with respect to normal pig, many toes pig has the sudden change of 13 places: G755C, A756T, A757G, T758C, C759A, A760G, C761T, T762 G, A763C, G764T, A765G, T767G, T768A are (before the sudden change: GAATCACTAGATTT, sudden change back: CTGCAGTGCTGTGA).Wherein, preceding 11 places sport missense mutation, and 2 places, back are for being not intended to sudden change; Missense mutation causes corresponding proteins matter sequence to have four amino acid to change: G sports A, I and sports that A, T sport V, R sports L, is not intended to sudden change and causes reading the frame premature termination; With respect to normal pig SEQ NO.1, promptly lack part exons 16 and complete exons 17 amounts to 246 Nucleotide.

CDNA sequence after the dna fragmentation generation said mutation of the present invention is SEQ NO.3, and total length is 1069bp, has comprised part coding region (1-768bp) and complete 3 untranslated region (769-1069bp), and the encoded polypeptide sequence is SEQNO.4.

A large amount of preparations of dna fragmentation of the present invention can be by making up cloning vector, and be transformed into and carry out massive duplication in protokaryon or the eucaryon host, the method of carrier construction and conversion is method commonly used, used carrier and host cell can be carrier and the host cells of using always, those skilled in the art can select as required, just are not described further here.

Dna fragmentation of the present invention and pig limb development are closely related, exons 16 preceding 4 codon generation missense mutation of its dna fragmentation directly cause the preceding many toes of pig axle, the present invention utilizes the difference of this DAN molecule between normal pig and many toes pig, a kind of method that detects the pig development character is provided, specifically:

(1) extract for the total RNA that detects sample, utilize following primer to carry out RT-PCR:

GSP2：5′-GAC?TTG?CCG?CTC?ATT?GAC?AAC?GAC-3′

NUP：5′-AAG?CAG?TGG?TAT?CAA?CGC?AGA?GT-3′；

(2) utilize following primer that step (1) RT-PCR product is carried out PCR.

NGSP2：5′-CGC?CGT?GGC?TTG?TAA?CAT?TCT?CTG?C-3′

NUP：5′-AAG?CAG?TGG?TAT?CAA?CGC?AGA?GT-3′；

(3) detect the PCR product at 1% sepharose then.The product of normal pig is 1197bp, and the product of many toes pig is 636bp.

The present invention has obtained the sequence SEQ NO.1 of more complete pig Lmbr1 gene cDNA, and found that there are two kinds of montage types in this gene: compare with normal montage type, there is disappearance the exons 16 and 17 sudden change montage type of totally 246 Nucleotide in many toes pig Lmbr1 gene; Simultaneously, the present invention also finds the missense mutation site of many toes pig, and a kind of method that fast, accurately detects the pig development character is provided in view of the above, for dna fragmentation of the present invention is laid a good foundation as the feasibility of pig carcass trait molecular marker assisted Selection.

Embodiment

The acquisition of example 1 dna fragmentation of the present invention

1 sample collecting

After pig is slaughtered, gather tissues such as the heart, liver, spleen, lung, kidney, forelimb muscle immediately, being cut into small pieces is contained in the frozen pipe, is put in immediately to preserve in the liquid nitrogen, preserves in-80 ℃ of Ultralow Temperature Freezers behind time laboratory.

Searching and the design of primers of 2 pig homology EST

With the encoding sequence of many toes candidate gene C7orf2/Lmbr1 gene of people/mouse as object search, carry out BLAST in the NCBI website, obtain the homology EST (CV878120 of one section pig, 724bp), the 6bp-636bp part and people C7orf2 the gene order ((accession number: NM_022458 of this sequence, 2781bp altogether, wherein 155bp-1627bp partly is CDS)) the 497bp-1148bp portion homologous be 88%.Based on this section est sequence, use Primer5.0 software, the primer of design RT-PCR and RACE reaction.

ESTF：5′-GCC?TTG?CTC?ATT?CTC?GGG?AT-3′；

ESTR：5′-TCC?CTT?TGG?CAT?CGC?TGT?T-3′；

GSP1：5′-TAT?AAG?CCG?ACC?GGG?GTG?CAC?AAG?A-3′；

NGSP1：5′-CCC?ACA?CCA?TCC?CGA?GAA?TGA?GCA?A-3′；

GSP2：5′-GAC?TTG?CCG?CTC?ATT?GAC?AAC?GAC-3′；

NGSP2：5′-CGC?CGT?GGC?TTG?TAA?CAT?TCT?CTG?C-3′；

NUP：5′-AAG?CAG?TGG?TAT?CAA?CGC?AGA?GT-3′；

3 total RNA extract and detect

3.1RNA the preparation work before extracting

(1) mortar, vial etc. are cleaned, placed after 37 ℃ of oven dry again with 180 ℃ of bakings of high temperature oven 8h;

(2) preparation of RNase-free water: use the vial of RNase-free, after adding DEPC spends the night to final concentration 0.1% (v/v) placement in ultrapure water, autoclave sterilization;

(3) rifle head, centrifuge tube, PCR pipe etc. is placed on soaked overnight in 0.1% the DEPC water, autoclave sterilization;

(4) 75% ethanol: with the preparation of DEPC treating water.

3.2RNA extraction step

(1) tissue grinds: the sample that very low temperature is preserved takes out weighing 50-100mg heart tissue, is transferred to rapidly in the mortar with the liquid nitrogen precooling, grind with pestle, during constantly add liquid nitrogen, Powdered until being ground to;

(2) ground tissue is transferred in the 1.5ml centrifuge tube that is added with 1ml RNAiso Reagent in advance with little spoon, uses the forced oscillation mixing, leave standstill 5min;

(3) 4 ℃ of centrifugal 5min of 12000g;

(4) carefully draw supernatant liquor, be transferred in the new centrifuge tube;

(5) to the chloroform that wherein adds 1/5 volume RNAiso Reagent, use forced oscillation, room temperature leaves standstill 5min;

(6) 4 ℃ of centrifugal 10min of 12000g;

(7) draw supernatant liquor and be transferred in the new centrifuge tube, to wherein adding isopyknic Virahol, the mixing that turns upside down, 15-30 ℃ leaves standstill 10min;

(8) 4 ℃ of centrifugal 10min of 12000g;

(9) careful supernatant discarded, the ethanol of adding 1ml 75%, the washing of turning upside down gently, 4 ℃ of centrifugal 5min of 12000g;

(10) carefully discard ethanol, drying at room temperature precipitation 2-5min adds 40ul RNase-free water dissolution precipitation;

3.3RNA quality examination

(1) electrophoresis detection: get 5ul RNA+1ul 6 * Loading Buffer, 150v electrophoresis 15min, observation band situation, the preservation of taking pictures;

(2) OD pH-value determination pH: open ultraviolet spectrophotometer power supply and program, preheating 30min cleans micro-cuvette with the DEPC treating water, zeroing, take out the cuvette of outside, the testing sample dilution is put in (2ul RNA+98ul DEPC treating water) in the cuvette for 50 times, measure OD ₂₆₀, OD ₂₈₀, D ₂₆₀/ OD ₂₈₀, the record analysis result.

4 reverse transcriptions

In the PCR pipe, add following reagent: For preparation of 5'-RACE-Ready cDNA:RNA sample 3ul, 5'-CDSprimerlul, BD SMART II A oligolul; For preparation of 3 '-RACE-Ready cDNA:RNA sample 3ul, 3'-CDS primerlul, ddH ₂O 1ul

↓

Mix also of short duration centrifugal

↓

Hatch 2min for 70 ℃

↓

Place 2min on ice

↓

The of short duration centrifugal reagent that makes arrives the pipe end

↓

In each pipe, add 5 * First-Strand Buffer 2ul, DTT (20mM) 1ul,

dNTP?Mix(10mM)1ul、BD?PowerScript?Reverse?Transcriptase?1ul

↓

Blow and beat mixing gently with the rifle head, of short duration centrifugal

↓

Hatch 90min (carrying out in the PCR instrument) for 42 ℃

↓

Add 100ul Ticine-EDTA Buffer cut back

↓

2 ℃ of water-bath 7min

↓

-20 ℃ of preservations are standby

53 ', 5 '-RACE amplification

Reaction system: in the PCR pipe of a sterilization, add following reagent, add PCR-Grade Water at first, add 50 * BD Advantage, 2 Polymerase Mix at last.

PCR-Grade?Water 34.5ul

10×BD?Advantage?2?PCR?Buffer 5.0ul

dNTP?Mix(10mM) 1.0ul

50×BD?Advantage?2?Polymerase?Mix?1.0ul

3’(5’)-RACE?Ready?cDNA 2.5ul

UPM(10×) 5.0ul

GSP 1.0ul

Total 50.0ul

Reaction conditions:

Get 5ulPCR product agarose gel electrophoresis and detect,, react according to following condition if there is not band that remaining PCR product is put back in the PCR instrument:

Electrophoresis detection (condition is the same) if still do not have the purpose band, is then carried out nest-type PRC.

First round PCR product is got 5ul, with the template of 245ul Tricine-EDTA Buffer dilution as nest-type PRC.System is as follows:

PCR-Grade?Water 36ul

10×BD?Advantage?2?PCR?Buffer 5.0ul

dNTP?Mix(10mM) 1.0ul

50×BD?Advantage?2?Polymerase?Mix?1.0ul

Template 5.0ul

NUP 1.0ul

NGSP 1.0ul

Total 50.0ul

Condition:

Get the 5ul electrophoresis detection.

EST part pcr amplification in the middle of 6

PCR system: 10 * Buffer 2.5ul, dNTP 2.0ul, Mg ²⁺2.0ul, each 0.5ul of ESTF, ESTR, Taq enzyme 0.125ul, cDNA2.0ul adds the sterilization tri-distilled water to 25ul.The PCR condition: 95 ℃ of pre-sex change 5min, 35 circulations (72 ℃ are extended 1min for 94 ℃ of sex change 30sec, 55 ℃ of annealing 40sec), 72 ℃ are extended 10min.Parallelly do two pipes, the cDNA with No. 734 and No. 741 pigs increases as masterplate respectively, and other condition is identical.Getting the 5ulPCR product detects with 1.5% agarose electrophoresis.

7 glue reclaim purifying

(1) electrophoresis: make glue with the macropore comb.EB dyeing, 10 * Loading Buffer+PCR product, full as far as possible hole; 100V voltage electrophoresis 30min;

(2) cut glue: cut off the agar sugar that contains DNA under the ultraviolet lamp, make it as far as possible little, and action is rapid, put into the 1.5ml centrifuge tube of weighing in advance, weighs, and calculates the weight of agarose;

(3) dissolving: the ratio that adds 300ulS1 liquid according to every 100g agarose adds S1 liquid, puts 10min in 50 ℃ of water-baths, puts upside down mixing once in per 2 minutes, and the agar sugar is dissolved fully;

(4) during purpose segment＜500bp, add the long-pending Virahol of 1/3S1 liquid, mixing, after 50 ℃ of temperature are bathed 1min, mixing; During purpose segment＞500bp, can omit this step;

(5) the Agarose liquid after will dissolving moves into adsorption column, and centrifugal 30sec outwells the liquid in the collection tube, and adsorption column is put into same collection tube;

(6) add 500ulW1 liquid in adsorption column, centrifugal 15sec outwells the liquid in the collection tube, and adsorption column is put into same collection tube;

(7) add 500ulW1 liquid in adsorption column, leave standstill 1min, centrifugal 15sec outwells the liquid in the collection tube, and adsorption column is put into same collection tube;

(8) centrifugal 1min;

(9) adsorption column is put into a clean 1.5ml centrifuge tube, after adsorption film central authorities add 30ulT1 liquid, leave standstill 1min, centrifugal 1min will reclaim product and be stored in-20 ℃.

The preparation of 8 competent cells

(1) take out one from-80 ℃ and manage DH5 α intestinal bacteria original seed, after dissolving on ice, at LB agar lining out, incubated overnight (12-16h) in 37 ℃ of constant incubators;

(2) picking list bacterium colony is inoculated in the 100ml LB liquid nutrient medium, and 37 ℃ are shaken about bacterium 12h, to OD ₆₀₀Reach 0.4-0.5;

(3) bacterium liquid is transferred in the 50ml centrifuge tube of two precoolings, ice bath 10-15min, 4 ℃ of centrifugal 10min of 4000rpm reclaim bacterial precipitation;

(4) every pipe adds the CaCl of the 0.1M of 10ml precooling ₂, resuspended bacterial precipitation; Ice bath 10-15min;

(5) 4 ℃ of centrifugal 10min of 4000rpm reclaim bacterial precipitation;

(6) repeat (4), (5) step;

(7) every pipe adds 2ml CaCl ₂Resuspended bacterial precipitation, cell at this moment can be directly used in transformation experiment.Add glycerine to concentration 20%, be distributed into 200ul/ part ,-80 ℃ of preservations.

9 ligations

Calculate the mol ratio of external source segment and carrier and (generally insert the segment mole number: carrier molecule mole number=3～8: 1).Ligation system (10ul): PMD18-T Vector 0.5ul, Solution I 5.0ul, foreign DNA is an amount of, adds aqua sterilisa to 10ul.Reaction conditions: 16 ℃ of incubation 12h, carry out in the PCR instrument.Get 5ul and connect liquid conversion, all the other 20 ℃ of preservations.

10 transform

(1) get a competent cell, dissolving on ice adds 5ul and connects product, ice bath 30min;

(2) 40 ℃ of heat shock 90sec, ice bath 2min immediately;

(3) add 500ul LB liquid nutrient medium, 37 ℃ bring back to life 50min, shop system X-Gal, Amp, IPTG flat board;

(4) 37 ℃ of constant incubators are cultivated 12-19h;

(5) flat board is placed 4 ℃ place a few hours, make locus coeruleus more obvious;

(6) picking hickie is inoculated in the test tube that the 3mlLB liquid nutrient medium is housed, and 37 ℃ are shaken bacterium 12h.

The evaluation of 11 positive colonies and order-checking

The picking white colony is cultivated 12h in the 3mlLB liquid nutrient medium is housed

↓

The a small amount of bacterium liquid of picking is applied in the centrifuge tube that contains the certain volume aqua sterilisa

↓

Add other composition according to the PCR system

↓

The PCR rear electrophoresis detects

↓

The positive colony order-checking

The Molecular Detection of example 2 pig development characters

(1) extraction of the total RNA of pig blood;

Gather 5 milliliters of the jugular vein blood of pig individuality to be detected, anticoagulant heparin.After separating white corpuscle, utilize the total RNA extraction reagent (D312) of Dalian precious biotechnology company limited, according to the total RNA of explanation.

(2) RT-PCR amplification;

With SMARTTM RACE cDNA Amplification test kit, reverse transcription reaction: total RNA 1-3ul, 3 '-CDSprimer 1ul, add deionized water to 5ul, 70 ℃ of 2min, put 2min on ice, add 5 * First-Strand Buffer 2ul, DTT (20mM) 1ul, dNTP (10mM) 1ul, BD PowerScript Reverse Transcriptase 1ul, 42 ℃ of 90min, add 100ul Tricine-EDTA Buffer dilution, 72 ℃ of water-bath 7min obtain 3 '-RACE-Ready cDNA.

Utilize UPM (test kit provides) and GSP2 (5 '-GAC TTG CCG CTC ATT GAC AAC GAC-3 ') to carry out first round PCR.PCR system and reaction conditions are seen the test kit specification sheets.First round PCR product is got 5ul, with 245ul Tricine-EDTA Buffer dilution, utilize NGSP2 (5 '-CGC CGT GGC TTG TAA CAT TCT CTGC-3 ') and NUP (5 '-AAG CAG TGG TAT CAA CGC AGA GT-3 ') to carry out second and take turns PCR.PCR system and reaction conditions are seen example 1.

(3) electrophoresis detection is judged determining of clip size and pig growth and development type

The PCR product is carried out electrophoresis at 1% sepharose.Judge the type of pig growth and development according to the size that detects the PCR product.The product of normal pig is 1197bp, and the product of many toes pig is 636bp.

SEQUENCE?LISTING

＜110〉Chongqing Academy of Animal Sciences

＜120〉a kind of and the relevant dna fragmentation of the many toes of pig

<160>4

<170>PatentIn?version?3.3

<210>1

<211>1797

<212>DNA

＜213〉the supporting B of being of CRP is a pig

<400>1

acgcggggat?ccatggtttg?tggaaccttg?cctctctctt?ttccaatctt?tgtttatttg 60

tattgatgcc?ctttgccttt?ttctttctgg?aatcagaagg?ttttgccggc?ctgaaaaagg 120

gaatccgagc?ccgaatttta?gaaaccctgg?tcatgcttgt?tctcctggcc?ttgctcattc 180

tcgggatggt?gtgggtggcc?tcggcgctca?ttgacaacga?cgcggcgagc?atggaatctc 240

tatatgatct?ctgggaattc?tacctaccgt?atttatactc?ctgtatatca?ttgatgggat 300

gtttgttact?tctcttgtgc?accccggtcg?gcctctcccg?catgtttacg?gtcatggggc 360

agctgttggt?gaagccagcg?atcctggaag?acctggatga?acagatttac?attatcaccc 420

tagaggaaga?agctctccag?agacgactaa?atggactgtc?ttcatcagtg?gaatccaatg 480

taatggagtt?ggaacaagaa?cttgaaaatg?taaagactct?taagacaaaa?ttagagaggc 540

gcaaaaaggc?ttcagcatgg?gaaagaaatt?tggtgtatcc?tgctgttatg?gttctccttc 600

ttattgaaac?atccatttcc?gtcctcttgg?tggcttgtaa?cattctctgc?ctgttggtgg 660

atgaaacagc?gatgccaaag?ggaacaaggg?gacctggaat?aggaagtgct?tctctttctg 720

cttttggttt?tgtgggagct?gcacttgaaa?tcattttgat?tttctatctt?atggtgtcct 780

ctgttgtcgg?tttctatagc?ctccgatttt?ttggagactt?tattcccaag?aaggatgaca 840

caactatgac?aaagatcatt?ggcaactgtg?tatcgatctt?ggtcctgagc?tctgcgctgc 900

ctgtgatgtc?aagaacactg?ggaatcacta?gatttgatct?acttggagac?tttggaaggt 960

ttaattggct?gggaaatttc?tatattgtgt?tgtcctacaa?tttgcttttt?gctgttgtga 1020

caaccttgtg?tctggtcaga?aaattcacct?ctgcagtgcg?agaagaactt?ttcaaggccc 1080

tagggcttca?taaactccac?ttatcaaata?cttcgaggga?ctcagaaaca?gcaaagcctt 1140

cggccaacgg?gcatcagaaa?gcactgactt?ggagctgaaa?tggttgcagc?tgtatttttt 1200

cacctcctta?aagaggcgaa?gggcaccgtt?gtgatctgtg?ttacaggaag?agactgccta 1260

gactttgagt?ccaggggcgg?gtagagaatt?ctcctgggcc?ccgagccttg?gtcacacatg 1320

ccccgggaat?accgtctgct?gcccagtggc?gtaagctatg?gtgggaagga?ctgtgtactc 1380

atagttcgct?gagagttgct?gtgtatctga?ttcactgtta?cctctcatgg?catagttgcc 1440

agtgctgaag?acacttgtaa?cttagatttt?gccttatagg?ctgtatgtac?cctcagtttt 1500

tttaaacaat?ttttttgttt?gtttccaaaa?atcccttaat?acccctgcgc?tcctgcgtcc 1560

attatggagc?tataaatgct?ccggtaggga?ctccttttgt?caccaccctc?ttcaggcttc 1620

ggtcacttct?gtacatgcca?atcgaaaacc?tcagacttcg?gaaaagtacc?atgcagcacc 1680

cagacccaca?gcagcagcat?tcatgatgta?ggaaccggtc?tgcagtgtat?catgatctga 1740

cgggcgttca?gttattaaat?tgtaaataat?tcttgaaaaa?aaaaaaaaaa?aaaaaaa 1797

<210>2

<211>391

<212>PRT

＜213〉the supporting B of being of CRP is a pig

<400>2

Ala?Gly?Ile?His?Gly?Leu?Trp?Asn?Leu?Ala?Ser?Leu?Phe?Ser?Asn?Leu

1 5 10 15

Cys?Leu?Phe?Val?Leu?Met?Pro?Phe?Ala?Phe?Phe?Phe?Leu?Glu?Ser?Glu

20 25 30

Gly?Phe?Ala?Gly?Leu?Lys?Lys?Gly?Ile?Arg?Ala?Arg?Ile?Leu?Glu?Thr

35 40 45

Leu?Val?Met?Leu?Val?Leu?Leu?Ala?Leu?Leu?Ile?Leu?Gly?Met?Val?Trp

50 55 60

Val?Ala?Ser?Ala?Leu?Ile?Asp?Asn?Asp?Ala?Ala?Ser?Met?Glu?Ser?Leu

65 70 75 80

Tyr?Asp?Leu?Trp?Glu?Phe?Tyr?Leu?Pro?Tyr?Leu?Tyr?Ser?Cys?Ile?Ser

85 90 95

Leu?Met?Gly?Cys?Leu?Leu?Leu?Leu?Leu?Cys?Thr?Pro?Val?Gly?Leu?Ser

100 105 110

Arg?Met?Phe?Thr?Val?Met?Gly?Gln?Leu?Leu?Val?Lys?Pro?Ala?Ile?Leu

115 120 125

Glu?Asp?Leu?Asp?Glu?Gln?Ile?Tyr?Ile?Ile?Thr?Leu?Glu?Glu?Glu?Ala

130 135 140

Leu?Gln?Arg?Arg?Leu?Asn?Gly?Leu?Ser?Ser?Ser?Val?Glu?Ser?Asn?Val

145 150 155 160

Met?Glu?Leu?Glu?Gln?Glu?Leu?Glu?Asn?Val?Lys?Thr?Leu?Lys?Thr?Lys

165 170 175

Leu?Glu?Arg?Arg?Lys?Lys?Ala?Ser?Ala?Trp?Glu?Arg?Asn?Leu?Val?Tyr

180 185 190

Pro?Ala?Val?Met?Val?Leu?Leu?Leu?Ile?Glu?Thr?Ser?Ile?Ser?Val?Leu

195 200 205

Leu?Val?Ala?Cys?Asn?Ile?Leu?Cys?Leu?Leu?Val?Asp?Glu?Thr?Ala?Met

210 215 220

Pro?Lys?Gly?Thr?Arg?Gly?Pro?Gly?Ile?Gly?Ser?Ala?Ser?Leu?Ser?Ala

225 230 235 240

Phe?Gly?Phe?Val?Gly?Ala?Ala?Leu?Glu?Ile?Ile?Leu?Ile?Phe?Tyr?Leu

245 250 255

Met?Val?Ser?Ser?Val?Val?Gly?Phe?Tyr?Ser?Leu?Arg?Phe?Phe?Gly?Asp

260 265 270

Phe?Ile?Pro?Lys?Lys?Asp?Asp?Thr?Thr?Met?Thr?Lys?Ile?Ile?Gly?Asn

275 280 285

Cys?Val?Ser?Ile?Leu?Val?Leu?Ser?Ser?Ala?Leu?Pro?Val?Met?Ser?Arg

290 295 300

Thr?Leu?Gly?Ile?Thr?Arg?Phe?Asp?Leu?Leu?Gly?Asp?Phe?Gly?Arg?Phe

305 310 315 320

Asn?Trp?Leu?Gly?Asn?Phe?Tyr?Ile?Val?Leu?Ser?Tyr?Asn?Leu?Leu?Phe

325 330 335

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

340 345 350

Arg?Glu?Glu?Leu?Phe?Lys?Ala?Leu?Gly?Leu?His?Lys?Leu?His?Leu?Ser

355 360 365

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

370 375 380

Gln?Lys?Ala?Leu?Thr?Trp?Ser

385 390

<210>3

<211>1069

<212>DNA

＜213〉the supporting B of being of CRP is many toes of pig mutant strain

<400>3

gccttgctca?ttctcgggat?ggtgtgggtg?gcctcggcgc?tcattgacaa?cgacgcggcg 60

agcatggaat?ctctatatga?tctctgggaa?ttctacctac?cgtatttata?ctcctgtata 120

tcattgatgg?gatgtttgtt?acttctcttg?tgcaccccgg?tcggcctctc?ccgcatgttt 180

acggtcatgg?ggcagctgtt?ggtgaagcca?gcgatcctgg?aagacctgga?tgaacagatt 240

tacattatca?ccctagagga?agaagctctc?cagagacgac?taaatggact?gtcttcatca 300

gtggaatcca?atgtaatgga?gttggaacaa?gaacttgaaa?atgtaaagac?tcttaagaca 360

aaattagaga?ggcgcaaaaa?ggcttcagca?tgggaaagaa?atttggtgta?tcctgctgtt 420

atggttctcc?ttcttattga?aacatccatt?tccgtcctct?tggtggcttg?taacattctc 480

tgcctgttgg?tggatgaaac?agcgatgcca?aagggaacaa?ggggacctgg?aataggaagt 540

gcttctcttt?ctgcttttgg?ttttgtggga?gctgcacttg?aaatcatttt?gattttctat 600

cttatggtgt?cctctgttgt?cggtttctat?agcctccgat?tttttggaga?ctttattccc 660

aagaaggatg?acacaactat?gacaaagatc?attggcaact?gtgtatcgat?cttggtcctg 720

agctctgcgc?tgcctgtgat?gtcaagaaca?ctggctgcag?tgctgtgaga?ctagaaacta 780

cgatttaaaa?aaaccctcaa?aataccaatg?tggacggcag?ctaaacaata?cgttgctaaa 840

caaccagtgg?gtctctttga?agaaatgaag?aaatcaaaga?agaaatttta?aaatactcta 900

gacaaatgaa?aatgaaaata?cagtgatcca?aaatatatgg?gattcagcaa?aagcaagtat 960

aagagggaca?tttatagtaa?tataagccta?tctcaggaaa?caagaaaaat?ctcaaatcaa 1020

gaatcttacc?atatacccaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaa 1069

<210>4

<211>255

<212>PRT

<213>1?GCCTTGCTCA?TTCTCGGGAT?GGTGTGGGTG?GCCTCGGCGC?TCATTGACAA?CGACGCGGCG

<400>4

Ala?Leu?Leu?Ile?Leu?Gly?Met?Val?Trp?Val?Ala?Ser?Ala?Leu?Ile?Asp

1 5 10 15

Asn?Asp?Ala?Ala?Ser?Met?Glu?Ser?Leu?Tyr?Asp?Leu?Trp?Glu?Phe?Tyr

20 25 30

Leu?Pro?Tyr?Leu?Tyr?Ser?Cys?Ile?Ser?Leu?Met?Gly?Cys?Leu?Leu?Leu

35 40 45

Leu?Leu?Cys?Thr?Pro?Val?Gly?Leu?Ser?Arg?Met?Phe?Thr?Val?Met?Gly

50 55 60

Gln?Leu?Leu?Val?Lys?Pro?Ala?Ile?Leu?Glu?Asp?Leu?Asp?Glu?Gln?Ile

65 70 75 80

Tyr?Ile?Ile?Thr?Leu?Glu?Glu?Glu?Ala?Leu?Gln?Arg?Arg?Leu?Asn?Gly

85 90 95

Leu?Ser?Ser?Ser?Val?Glu?Ser?Asn?Val?Met?Glu?Leu?Glu?Gln?Glu?Leu

100 105 110

Glu?Asn?Val?Lys?Thr?Leu?Lys?Thr?Lys?Leu?Glu?Arg?Arg?Lys?Lys?Ala

115 120 125

Ser?Ala?Trp?Glu?Arg?Asn?Leu?Val?Tyr?Pro?Ala?Val?Met?Val?Leu?Leu

130 135 140

Leu?Ile?Glu?Thr?Ser?Ile?Ser?Val?Leu?Leu?Val?Ala?Cys?Asn?Ile?Leu

145 150 155 160

Cys?Leu?Leu?Val?Asp?Glu?Thr?Ala?Met?Pro?Lys?Gly?Thr?Arg?Gly?Pro

165 170 175

Gly?Ile?Gly?Ser?Ala?Ser?Leu?Ser?Ala?Phe?Gly?Phe?Val?Gly?Ala?Ala

180 185 190

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

195 200 205

Phe?Tyr?Ser?Leu?Arg?Phe?Phe?Gly?Asp?Phe?Ile?Pro?Lys?Lys?Asp?Asp

210 215 220

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

225 230 235 240

Ser?Ser?Ala?Leu?Pro?Val?Met?Ser?Arg?Thr?Leu?Ala?Ala?Val?Leu

245 250 255

Claims (5)

1. One kind with the relevant dna fragmentation of the many toes of pig, this dna fragmentation is one of following polynucleotide:

   (1) nucleotides sequence is classified the polynucleotide of SEQ NO.1 and SEQ NO.3 as;

   (2) encoding amino acid sequence is the polynucleotide of the polypeptide of SEQ NO.2 and SEQ NO.4.
2. 2. the relevant dna fragmentation of the many toes of the described pig of claim 1, the aminoacid sequence of its encoded polypeptides is SEQ NO.2 or SEQ NO.4.
3. 3. the expression vector that contains the described dna fragmentation of claim 1.
4. 4. the clone that contains the described expression vector of claim 3.
5. 5. method that detects the pig development character, this method is:

   (1) extract for the total RNA that detects sample, utilize following primer to carry out RT-PCR:

   GSP2：5′-GAC?TTG?CCG?CTC?ATT?GAC?AAC?GAC-3′，

   NUP：5′-AAG?CAG?TGG?TAT?CAA?CGC?AGA?GT-3′；

   (2) utilize following primer that step (1) RT-PCR product is carried out PCR.

   NGSP2：5′-CGC?CGT?GGC?TTG?TAA?CAT?TCT?CTG?C-3′，

   NUP：5′-AAG?CAG?TGG?TAT?CAA?CGC?AGA?GT-3′；

   (3) detect the PCR product at 1% sepharose then.The product of normal pig is 1197bp, and the product of many toes pig is 636bp.