CN101255427A - Porcine SIM1 gene and genetic mark as porcine production deseription - Google Patents

Abstract

The invention discloses a pig SIM1 gene and using the same as genetic marker of pig production traits. The pig SIM1 gene has sequence of SEQ ID No.1 or SEQ ID No.2. The gene marker can be used in SIM1 gene polymorphism analysis of different pig varieties, and research relationship between gene and production traits, fat traits, meat quality traits.

Description

Pig SIM1 gene and as the genetic marker of pig production character

Technical field

The invention belongs to the biology field of pig, the clone and order-checking and single nucleotide polymorphism (the single nucleotide polymorphism that relate to pig SIM1 (single minded1) gene, SNP) detection method, especially, relate to a boar SIM1 gene and as the genetic marker of pig production character.

Background technology

Along with the raising of living standards of the people, meat quality has caused the concern that the producers and consumers is bigger, thereby the improvement of meat proterties is also paid attention to by the breeding work person gradually.Fatty character is an important indicator of meat proterties, and back-fat thickness commonly used is represented.Reduce back-fat thickness and can increase lean ratio, can adapt to consumers demand, increase economic benefit.By the research gene relevant with fatty character, utilize genotype to select to reduce back-fat thickness, be the effective way of meat character improvement.

SIM1 (Single-minded 1) gene is the homologue of fruit bat SIM gene, and it is playing the part of important role (Crews et al.1988) in the center line cell system of central nervous system.The homologue that is included in SIM gene in a lot of species such as people, mouse and zebra fish has all successfully separated acquisition (Ema et al.1996; Chrast et al.1997; Wen et al.2002), it is long-armed that this gene of people is located in No. 6 karyomit(e)s, and this gene of mouse is located in No. 20 karyomit(e)s long-armed (http://www.informatics.jax.org/searches/homology_form.shtml).SIM albumen is bHLH-PAS (basic helix-loop-helix+period, aryI hydrocarbon receptor, Single-minded) one of transcription factor family member, studies show that this family gene coding produces alkaline helix-loop-helix transcripton, is the functional gene (Holder et al.2000) of decision neurocyte differentiation.In Mammals, the SIM1 gene is mainly expressed in central nervous system is unified the liver organization of growing, and it is most important to look the formation of other nuclear (PVN) for hypothalamus.The neuroendocrine cell that nuclear was controlled by hypothalamus was looked quite crucial to the effect of some physiological change of body, for example energy balance and blood pressure etc.The mouse afunction experiment that SIM1 knocks out gene shows that this gene looks the differentiation extremely important (Michaud et al.1998) of other nucleus neuron for hypothalamus, lack SIM1, the growth that nearly all hypothalamus is looked other nucleus neuron all will be obstructed and the SIM1 transgenation can cause food consumption of people and mouse and the increase of body fat (Michaudetal.1998, Monica C et al.2006).With respect to above research, carry out the research of pig SIM1 gene, disclose the growth of SIM1 gene pairs pig, the influence of fatty character and meat proterties is significant.

Summary of the invention

The objective of the invention is at the deficiencies in the prior art, provide a boar SIM1 gene and as the genetic marker of pig production character.After obtaining pig SIM1 gene (DNA), obtain single nucleotide polymorphism by sequence comparing analysis; Set up suitable mononucleotide polymorphic classifying method in view of the above, provide the molecule marker of usefulness for the marker assisted selection of growth traits, fatty character and the meat proterties of pig.

The present invention is achieved through the following technical solutions:

One boar SIM1 gene, it has the sequence of SEQ ID No.1 or SEQ ID No.2.

A kind of genetic marker of pig production character, it is a pig SIM1 gene.

A kind of molecule marking method of genetic marker of pig production character may further comprise the steps:

(1) from pig blood, extracts genomic dna, according to people and mouse SIM1 homologous sequence design primer SEQ IDNo.3 and SEQ ID No.4;

(2) carry out pcr amplification and obtain pig SIM1 gene order SEQ ID No.1 and SEQ ID No.2;

(3) pig SIM1 gene order design primer SEQ ID No.5 that obtains based on described step (2) and SEQ IDNo.6 are used for the PCR-SSCP somatotype and detect a base mutation 834G-834A.

The invention has the beneficial effects as follows: genetic marker of the present invention can be used for the SIM1 Polymorphism Analysis of different pig varieties, and the relation of research gene and pig growth traits, fatty character and meat proterties.

Description of drawings

Fig. 1 is that Jinhua pig and Large White SIM1 gene order compare synoptic diagram;

Fig. 2 is the SNP synoptic diagram of the different banding pattern correspondences of pig SIM1 gene PCR-SSCP;

Fig. 3 is pig SIM1 homology amplified production partial sequence and people mouse SIM1 exon sequence comparison result synoptic diagram.

Embodiment

The clone and the order-checking of pig SIM1 gene of the present invention, its step comprises: selection place of china kind blood lineage's Jinhua pig and adventive Large White are as the typical test material, from the blood of pig, extract genomic dna, according to people and mouse SIM1 gene conserved regions design two its sequences of outer primer, see sequence table respectively: SEQ ID No.3 and sequence table: SEQ ID No.4, carry out pcr amplification, PCR product purification and directly order-checking, sequence comparing analysis obtains pig SIM1 gene coding region partial dna sequence.Wherein Jinhua pig and Large White SIM1 gene DNA sequence are seen sequence table SEQ ID No.1 and sequence table SEQ ID No.2 respectively.

Relatively Jinhua pig and Large White SIM1 gene DNA sequence are seen the dna sequence dna of sequence table SEQ ID No.1 and sequence table SEQ ID No.2, have found the mononucleotide polymorphic SNP site (as shown in Figure 1) of 834 G → A of Nucleotide of SIM1 gene PCR amplified production.

The method of setting up PCR-SSCP detects the polymorphism of 834 G-A of pig SIM1 gene, see sequence table SEQ ID No.1 or sequence table SEQ ID No.2 according to known pig SIM1 sequence, the design primer, its sequence is respectively shown in sequence table SEQ ID No.5 and sequence table SEQ ID No.6, carry out pcr amplification, choose the discrepant representative individuality of electrophoresis banding pattern and carry out PCR and directly check order, by sequencing result this SNP site (as shown in Figure 2) as can be known.And the correlationship between the definite different genotype individuality and the production traits.

One, the foundation of the acquisition of gene fragment and pleiomorphism detecting method

(1) acquisition of SIM1 gene order,

Select place of china kind Jinhua pig and external kind Large White as the typical test material, adopt the homology amplification method, according to the adjacent exon region design of the SIM1 primer of people and mouse.

Upstream primer is SEQ ID No.3:5 '-AGGAGCTTCAGGGGGAAACGCCT-3 '

Downstream primer is SEQ ID No.4:5 '-ATTGGGCCCGACGTCGCATGCTC-3 '

PCR is reflected at and contains 50ng genomic dna, 15mmol/L Tris-HCl, 50mmol/L KCl (pH8.0), 2mmol/L MgCl ₂, 200 μ mol/L dNTPs, primers F ₁And R ₁Carry out in the 25 μ l reaction systems of 10 μ mol/L, 1U TaqDNA polysaccharase, reaction conditions is 94 ℃ of pre-sex change 5min, carries out program then and is: 94 ℃ of sex change 40s, 55 ℃ of renaturation 40s, 72 ℃ are extended 40s, 30 circulations, last 72 ℃ are extended 7min, 4 ℃ of preservations.After being purified, amplification PCR products (896bp) directly carries out sequencing.Dna sequence dna is arranged and is compared with DNAMAN 5.2.2 software, and Jinhua pig and Large White SIM1 gene DNA sequence are seen sequence table SEQ ID No.1 and sequence table SEQ ID No.2.With BLAST software relatively, the sequence of PCR product conclusive evidence PCR product is the SIM1 gene, with corresponding people and the exon sequence of mouse very high homology (Fig. 3) is arranged on dna level.Through compare of analysis Jinhua pig and Large White dna sequence dna, found that at 834 in the Nucleotide of SIM1 gene PCR amplified production (G → A), i.e. SNP (Fig. 1): pig is A to 1 sequence change in the kind Jinhua of pig, and Large White is G.

(2) the PCR-SSCP method detects the polymorphic of SIM1 gene G 834 → 834A

See sequence table SEQ ID No.1 or sequence table SEQ ID No.2 according to known pig SIM1 sequence, the design primer:

Upstream primer is SEQ ID No.5:F2:5 '-ATTGATTTGGGCCGGTTCAG-3 '

Downstream primer is SEQ ID No.6:R2:5 '-ATTGGGCCCGACGTCGCATGCTC-3 '

Pcr amplification system: 50ng genomic dna, 15mmol/L Tris-HCl, 50mmol/L KCl (pH8.0), 2mmol/L MgCl ₂, 200 μ mol/L dNTPs, primers F ₁And R ₁Carry out in the 25 μ l reaction systems of 10 μ mol/L, 1 U Taq archaeal dna polymerase, reaction conditions is 94 ℃ of pre-sex change 5min, carries out program then and is: 94 ℃ of sex change 40s, 55 ℃ of renaturation 40s, 72 ℃ are extended 40s, 30 circulations, last 72 ℃ are extended 7min, 4 ℃ of preservations.

PCR-SSCP analyzes: reaction of degeneration system 12 μ l, 5 μ l PCR products wherein, the loadingbuffer of 7 μ l (98% formyl ammonia, 0.025% tetrabromophenol sulfonphthalein, 0.025% dimethylbenzene green grass or young crops, 10mmol/L EDTA (pH=8.0) and 2% glycerine), 98 ℃ of sex change 10 minutes are then immediately more than the ice bath 5min.12%PAGE glue (29: 1) electrophoresis 12 hours, electrophoresis finish back silver and dye apparent band, utilize gel imaging system to observe and record.Choose the discrepant representative individuality of electrophoresis banding pattern and carry out PCR and directly check order, by sequencing result this SNP site (Fig. 2) as can be known.

Two, the polymorphic distribution of molecule marker in different swinerys

Employing PCR-SSCP analytical technology is carried out the SNP somatotype in 4 external swinerys (Large White, long white, Pietrain, duroc) and 2 Chinese swinerys (Jinhua pig, the black pig in Jiaxing).Detected result is as shown in table 1.External swinery Large White, long white, the dominant A gene frequency of duroc are respectively 0.80,0.58 and 0.55 in the several pig kinds that detected, and the black pig A gene frequency of Chinese swinery Jinhua pig, Jiaxing all reaches 1.00, A allelotrope and B gene frequency are approaching in the swinery Pietrain abroad, are respectively 0.50.Following table 1 is different varieties pig SIM1 genotype and gene frequency synoptic diagram.

Table 1

Three, the association analysis of the molecule marker and the production traits

Whether relevant in order to determine SIM1 gene SNP polymorphism with the pig phenotypic difference, select 337 Jinhua pig * Pietrain pigs F ₂In generation, is as test materials, adopt embodiment 2 described PCR-SSCP methods to carry out polymorphism scanning, and adopt the SAS glm of statistical software program to carry out the correlationship of single mark variance analysis pig SIM1 gene different genotype and pig production character, adopt the reg program to calculate additive effect of gene and dominant effect simultaneously, and carry out test of significance, the model that adopts is:

Y _ijkl＝μ+G _i+F _j+S _k+Y ₁+b _ijklX _ijkl+e _ijkl

Y _IjklBe the proterties phenotypic number, μ is a mean number, G _iFor the genotype effect (comprises additive effect of gene and dominant effect; Additive effect is represented GG, AG and AA genotype with-1,0 and 1, and dominant effect is with 1, and-1 and 1 represents GG, AG and AG genotype respectively) F _j, S _k, Y _lBe fixed effect, represent family, sex and annual effect respectively, b _IjklFor slaughter weight or butcher the regression coefficient of age in days, carcass trait is concomitant variable with the slaughter weight, and the meat proterties is a concomitant variable to butcher age in days, e _IjklBe the residual error effect.

337 Jinhua pig * Pietrain pigs F that detected ₂In individuality, SIM1 gene GG genotype observation number is 43 (genotype frequency is 0.13), and AG genotype observation number is 173 (genotype frequency is 0.51), and AA genotype observation number is 121 (genotype frequency is 0.34).Statistics between the different genotype and the production traits is summarized in table 2.Table 2 is the statistical study hoist pennants of SIM1 genotype and growth, trunk and meat proterties.

Table 2

Annotate: above numerical value is least square mean value standard error; Contain same letter and represent that difference is not remarkable, different letter representation differences are extremely remarkable; The additive effect negative value represents that A allelotrope reduces the proterties phenotypic number; ^*Expression p＜0.05, ^*Expression p＜0.01.

As can be seen from Table 2, the SIM1 genotype is not simultaneously, the birth weight of growth traits, weaning weight, 90 ages in days are heavy, 120 ages in days are heavy, 150 ages in days are heavy, average daily gain, nose heave, 6 ~ 7 ribbed back fat thickness of carcass trait, the last rib thickness of backfat, waist are recommended the junction thickness of backfat, the triadic mean thickness of backfat, heavy, the eye muscle area of leaf fat, and the intramuscular moisture content of meat proterties and intramuscular fat content are all pure in remarkable or utmost point significant difference.And mainly based on the additivity mode of action, in effect direction and inconsistent, A allelotrope has reduced last rib and waist when having improved pig growth later stage body weight recommends the junction thickness of backfat and has improved eye muscle area, intramuscular moisture content and lipid content simultaneously.This shows that this mark is quite suitable for improving pig production character.

Sequence table

＜110〉Zhejiang University

＜120〉pig SIM1 gene and as the genetic marker of pig production character

<160>2

<170>PatentIn?version?3.1

<210>1

<211>896

<212>DNA

＜213〉Jinhua pig

<400>1

aggagcttca?gggggaaacg?cctggtatct?ttatagtcct?gtcgggtttc?gccacctctg 60

acttgagcgt?cgatttttgt?gatgctcgtc?aggggggcgg?agcctatgga?aaaacgccag 120

caacgcggcc?tttttacggt?tcctggcctt?ttgctggcct?tttgctcaca?tgttctttcc 180

tgcgttatcc?cctgattctg?tggataaccg?tattaccgcc?tttgagtgag?ctgataccgc 240

tcgccgcagc?cgaacgaccg?agcgcagcga?gtcagtgagc?gaggaagcgg?aagagcgccc 300

aatacgcaaa?ccgcctctcc?ccgcgcgttg?gccgattcat?taatgcagct?ggcacgacag 360

gtttcccgac?tggaaagcgg?gcagtgagcg?caacgcaatt?aatgtgagtt?agctcactca 420

ttaggcaccc?caggctttac?actttatgct?tccggctcgt?atgttgtgtg?gaattgtgag 480

cggataacaa?tttcacacag?gaaacagcta?tgaccatgat?tacgccaagc?tatttaggtg 540

acactataga?atactcaagc?tatgcatcca?acgcgttggg?agctctccca?tatggtcgac 600

ctgcaggcgg?ccgcgaattc?actagtgatt?attgatttgg?gccggttcag?aagcaggttc 660

atttaccgtc?agctgccttc?tgtctctcca?ccagttctgg?taaaggggca?ggtgaccacc 720

aagtactacc?ggttcctggc?caagcacggc?ggctgggtct?gggtacagag?ctacgcgacc 780

atcgtgcaca?acagccgctc?ctccaggcca?cactgcatcg?tcagcgtcaa?ctaagtcctc 840

aaatcgaatt?cccgcggccg?ccatggcggc?cgggagcatg?cgacgtcggg?cccaat 896

<210>2

<211>896

<212>DNA

＜213〉Large White

<400>2

aggagcttca?gggggaaacg?cctggtatct?ttatagtcct?gtcgggtttc?gccacctctg 60

acttgagcgt?cgatttttgt?gatgctcgtc?aggggggcgg?agcctatgga?aaaacgccag 120

caacgcggcc?tttttacggt?tcctggcctt?ttgctggcct?tttgctcaca?tgttctttcc 180

tgcgttatcc?cctgattctg?tggataaccg?tattaccgcc?tttgagtgag?ctgataccgc 240

tcgccgcagc?cgaacgaccg?agcgcagcga?gtcagtgagc?gaggaagcgg?aagagcgccc 300

aatacgcaaa?ccgcctctcc?ccgcgcgttg?gccgattcat?taatgcagct?ggcacgacag 360

gtttcccgac?tggaaagcgg?gcagtgagcg?caacgcaatt?aatgtgagtt?agctcactca 420

ttaggcaccc?caggctttac?actttatgct?tccggctcgt?atgttgtgtg?gaattgtgag 480

cggataacaa?tttcacacag?gaaacagcta?tgaccatgat?tacgccaagc?tatttaggtg 540

acactataga?atactcaagc?tatgcatcca?acgcgttggg?agctctccca?tatggtcgac 600

ctgcaggcgg?ccgcgaattc?actagtgatt?attgatttgg?gccggttcag?aagcaggttc 660

atttaccgtc?agctgccttc?tgtctctcca?ccagttctgg?taaaggggca?ggtgaccacc 720

aagtactacc?ggttcctggc?caagcacggc?ggctgggtct?gggtacagag?ctacgcgacc 780

atcgtgcaca?acagccgctc?ctccaggcca?cactgcatcg?tcagcgtcaa?ctaggtcctc 840

aaatcgaatt?cccgcggccg?ccatggcggc?cgggagcatg?cgacgtcggg?cccaat 896

Claims (3)

1, a boar SIM1 gene is characterized in that, it has the sequence of SEQ ID No.1 or SEQ ID No.2.

2, a kind of genetic marker of pig production character is characterized in that, it is the described pig SIM1 of claim 1 gene.

3, the molecule marking method of the genetic marker of the described pig production character of a kind of claim 2 is characterized in that, may further comprise the steps:

(1) from pig blood, extracts genomic dna, according to people and mouse SIM1 homologous sequence design primer SEQ IDNo.3 and SEQ ID No.4.

(2) carry out pcr amplification and obtain pig SIM1 gene order SEQ ID No.1 and SEQ ID No.2.

(3) pig SIM1 gene order design primer SEQ ID No.5 that obtains based on described step (2) and SEQ IDNo.6 are used for the PCR-SSCP somatotype and detect a base mutation 834G-834A.

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