CN101892225A - HPSE (Heparanase) gene as molecular markers relevant to pig immune characters and application thereof - Google Patents
HPSE (Heparanase) gene as molecular markers relevant to pig immune characters and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of the preparation of livestock molecular markers and particularly relates to preparation and application of pig molecular markers. The preparation method comprises the steps: cloning from a human source HPSE (Heparanase) gene to obtain a gene fragment applied as a molecular marker relevant to pig immune characters, wherein the nucleotide sequence of the gene fragment is disclosed as SEQ ID No:2, and A80-G80 base substitution exists in the 80bp position of the SEQ ID No:2 to lead to the polymorphism of Alu I-RFLP (Restricted Fragment Length Polymorphisms). The invention also discloses a method for preparing molecular markers relevant to pig immune characters and application of the prepared molecular markers to the detection of the polymorphism of the pig immune characters. The invention provides a new marker for the assisted selection of pig markers.
Description
Technical field
The invention belongs to the molecular marker assisted selection technical field of pig, be specifically related to a kind of clone and application of the molecule marker relevant as the pig marker assisted selection with immune character.Molecule marker of the present invention is relevant with the HPSE gene.
Background technology
Since nearly 10 years, GENERALIZATION OF MODERN BREEDING TECHNIQUE makes the production performance (growth, trunk and meat proterties) of pig obtain significant improvement, and the genetic improvement of pig health and resist the disease ability is paid attention to inadequately.Disease is an important step during livestock industry is produced always.Raise the management level, improve preventive measures such as hygienic condition, injectable drug and vaccine, in preventing disease takes place, play a part important really.But they all can not fundamentally solve this difficult problem of preventing disease.The natural disease resistance that improves body has become emphasis, the especially breeding for disease resistance of present research.
In breeding for disease resistance, the searching of disease-resistant gene is crucial.The main disease-resistant candidate gene that has identified both at home and abroad has following at present:
(1) enterotoxin type intestinal bacteria (Enterotoxigenic Escherichia coli is to cause that piglet rushes down one of main pathogens of dysentery ETEC), and it can cause multiple colibacillosiss such as yellow scour of piglet, dysentery characterized by white mucous stool, bowel oedema disease.At present the focus of research mainly concentrates on (Shi Qishun, chitling toxin intestinal bacteria resistant gene and breeding for disease resistance on the acceptor gene of ETEC such as K88 (F4), K99 (F5), 987P, 1741 (F6), F17 (FY, Att25), F18, F42, F165.The 11st national Animal Genetics academic meeting paper collection, Chinese agriculture press, 2001,229-333).If studies show that and lack intestinal bacteria K88 acceptor or F18 acceptor in the animal body, will produce resistance to the diarrhoea that corresponding serotype intestinal bacteria cause, the K88 mutant of having identified at pig ETEC bacterial strain at present has three kinds: K88ab, K88ac and K88ad (Mooi etc., 1985.Molecular biology of fimbriae of enterotoxigenic Escherichia coli.Curr.Top.Microbiol.Immunol.118:119-138.).The gene of current encoder intestinal bacteria K88 acceptor (K88abR and K88acR) has been positioned at the position (Edfors-Lilja etc. of close Transferrins,iron complexes gene on No. 13 karyomit(e)s of pig, The porcine intestinal receptor for Escherichia coliK88ab.K88ac:regional localization on chromosome 13and influenve of IgG response to the K88antigen.Anim Genet, 1997,26:237-242), α-(1,2)-(α (1 for trehalose transferring enzyme 1,2) fucosyltransferase, FUT1) gene is the candidate gene of F18 acceptor (ECF18R), be positioned at (Meijerink etc. on No. 6 karyomit(e)s of pig, Two α (1,2) fucosyltransferase genes on porcine chromosome 6q11 are closely linked to the blood groupinhibitor (s) and Escherichia coli F18 receptor (ECF18R) loci.Mamm Genome, 1997,8:736-741).
(2) NRAMP1 (natural resistance associatedmacrophage protein 1, the huge albumen of biting that natural resistance is relevant) gene finds that at first pathogenic micro-organism in the mouse pair cell infects the resistance that had or susceptibility and is subjected to dominant gene Bcg, Ity or Ish control on No. 1 karyomit(e), is NRAMP1 with such unnamed gene thus.The phosphoglucoprotein that the NRAMP1 coding has complete film, contain 10-12 commentaries on classics diaphragm area of inferring, feature (Cellier etc., Resistance to intracellular infections:comparative genomic analysis of NRAMP1.Trends Genet.1996Jun with ionic channel and transport function; 12 (6): 201-4; Vidal etc., Naturalresistance to intracellular infections:Nramp1 encodes a membrane phosphoglycoprotein absentin macrophages from susceptible (Nramp1 D169) mouse strains.J Immunol.1996Oct 15; 157 (8): 3559-68.1996).NRAMP1 albumen can be resisted infecting of multiple born of the same parents' endoparasitism such as mycobacterium, Salmonellas pathogenic bacteria and bring into play important immunologic function, to the big (Wu Hongmei etc. of livestock and poultry body disease-resistant power influence, NRAMP1 gene studies progress and the application in breeding for disease resistance thereof. Chinese animal and veterinary, 2005,32 (4): 26～28.).
In addition, MHC be proved to be with antibody to multiple antigenic replying, the phagocytosis of bacterium, proterties such as susceptibility to spiral Trichinella spiralis and malignant melanoma have relevant (Peelman etc., A detail physical map of the porcine major histocompatibilitycomplex (MHC) class III region:comparison with human and mouse MHC class III regions.Mamm Genome, 1996,7:363-367), other gene relevant with disease resistance that research is arranged is MX1 (Myxovirus (influenza) resistance 1, myxovirus (influenza) resistance factor 1), (Interleukins 1 for IL1, interleukin 1), PPARG genes such as (Peroxisome proliferatoractivated receptor gamma, peroxisome proliferation activated receptor γ).
Said gene all is the disease-resistant candidate gene that identifies according to epidemiology and economic selecting scale select target disease basically.The pig high-pathogenicity blue ear disease that was called as " hyperpyrexia disease " in 2006 breaks out in Chinese big area, and epidemic situation involves more than 10 province (city, district).This epidemic situation impacts about 2,120,000 pigs and causes at least 40 ten thousand pig death.Blue otopathy has become the No.1 transmissible disease that the restriction pig industry develops in a healthy way.Blue otopathy is caused by high-pathogenicity blue ear disease virus, this virus is by the receptor-mediated host cell that enters, the acceptor that has identified at present has four, be respectively: Suleparoid (Heparan sulfate, HS) (Delputte etc., Involvement of the matrix protein in attachmentof porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcinealveolar macrophages.J Virol.2002,76 (9): 4312-20.), sialoadhesin (sialoadhesin, SN) (Delputte etc., Analysis of porcine reproductive and respiratory syndrome virus attachment andinternalization:distinctive roles for heparan sulphate and sialoadhesin.J Gen Virol.2005,86 (Pt 5): 1441-5), CD151 (Shanmukhappa etc., Role of CD151, A tetraspanin, in porcine reproductiveand respiratory syndrome virus infection.Virol.2007,4:62.) and CD163 (Calvert waits CD163expression confers susceptibility to porcine reproductive and respiratory syndrome viruses.JVirol.2007.81 (14): 7371-7379).Wherein HS can will be free on the virus absorption of surface of cell membrane and be fixed on the cytolemma, plays the effect of viral adsorption.This process has determined viral infective dose, plays crucial effects in virus enters the process of cell.HS in body be with a kind of protein-polysaccharide (Heparan sulfate proteoglycan, HSPG) form exists and works, HSPG is made up of core protein and HS side chain.And heparitinase (heparanase, HPSE) be in the mammalian cell of finding at present unique can the incising cell epimatrix in sulfate-proteoglycan side chain--Suleparoid (Heparan sulfate, HS) endogenous Glycosylase, be antitumor, the desirable target spot of anti-inflammatory (Mammalian heparanase:gene cloning such as Vlodavsky, expression and function intumor progression and metastasis.Nat med.1999,5:193).This enzyme is by gene HP SE coding, and the people HPSE assignment of genes gene mapping is in HAS4q21.3, and its cDNA comprises the open reading frame of a 1629bp, the polypeptide chain of forming by 543 amino-acid residues of encoding, and its relative molecular mass is 61200.Many kinds of important physical pathological effects of HSPG are all relevant with the enzymolysis of its polysaccharide side chain Suleparoid (HS).Heparanase not only can histoclastic integrity to the cutting of HS, and can discharge and activate various and HS bonded bioactive molecule, thereby influences various physiology and pathologic process.
We can draw the HPSE gene and participate in multiple physiological and pathological process and the effect wanted emphatically of performance by above data.Seek the variant sites in the gene, by and proterties between association analysis find that the relation between gene and proterties is an important means of research gene function, also be the basis of carrying out marker assisted selection.The clone of pig HPSE gene, detection and SNP examination, detection and and the proterties association analysis of differential expression have been carried out for this reason.
Summary of the invention
The complete CDS sequence that the objective of the invention is to clone pig HPSE gene, the detection method of the mutational site of searching HPSE gene and the polymorphism of gene is set up the molecular marker method that is applicable to pig immune trait.
The present invention is achieved through the following technical solutions:
The clone obtains a kind of molecule marker relevant with immune character as the pig marker assisted selection from pig HPSE gene fragment, its complete coding region sequence such as sequence table SEQ ID NO:1 and shown in Figure 2, its partial dna sequence is as described in sequence table SEQ ID NO:2 and the accompanying drawing 3.There is an A/G base mutation at the 782nd bit base place in sequence shown in the SEQ ID NO:1, the base mutation that an A80-G80 is promptly arranged at the 80th bit base place of sequence shown in the sequence table SEQ ID NO:2, this sudden change is positioned at the 5th exon, causes Alu I-RFLP polymorphism.
The applicant has designed the CDS primer sequence of a pair of clone pig HPSE gene, and the CDS sequence of this primer amplification is as described in the SEQ ID NO:1.Simultaneously the applicant designed a pair of comprise the 5th exon be used to increase the primer of pig HPSE genome sequence, the sequence of this primer amplification is as described in the SEQID NO:2.
Prepare above-mentioned molecular marker method, according to following steps:
Extract DNA from the pig blood genome, personnel selection HPSE gene mRNA is the information probe, does the homologous sequence screening, obtains the expressed sequence tag (EST) of homology more than 80%; Splice pig EST-contig then, with the comparison of people's homology, according to expressed sequence tag-contig design primer; Organizing sample to extract total RNA with adult Large White is template, makes RT-PCR amplification, PCR product purification and cloning and sequencing, carries out sequential analysis, obtains as the described cDNA sequence of sequence table SEQ ID NO:1 (comprising whole CDS); Extract genomic dna, the design primer, pcr amplification, PCR product purification and order-checking obtain the nucleotide sequence shown in sequence table SEQ ID NO:2.
The method of the PCR-RFLP that application is conventional detects the 80th bit base sudden change of sequence table SEQ ID NO:2, and tentatively carry out the application of the association analysis between its genotype and the pig immune trait, for the molecular marker assisted selection of pig provides a new molecule marker.
Description of drawings
Sequence table SEQ ID NO:1 is the CDS sequence of the pig immune trait genes involved HPSE that clones of the present invention;
Sequence table SEQ ID NO:2 is that the pig immune trait genes involved HPSE that the present invention clones is used for the PCR-RFLP partial dna sequence;
Fig. 1: the schema that is HPSE gene preparation of the present invention;
Fig. 2: be the CDS fragment that pig HPSE gene is used to clone among the present invention.Used primer sequence marks with underscore;
Fig. 3: be that pig HPSE gene is used for the dna fragmentation that PCR-RFLP detects among the present invention.Used primer sequence marks with underscore;
Fig. 4: three kinds of genotype (the AA AG GG) electrophoresis result that is pig HPSE gene A lu I-RFLP among the present invention.M:DNA molecular weight standard among the figure (DL2000 ladder).
Embodiment
The clone of embodiment 1, HPSE gene
(1) design of primers
(the GenBank number of including: NM_006665.3) be the information probe of personnel selection HPSE gene cDNA, utilize the BLAST instrument among the NCBI in GenBank pig est database, to do the homologous sequence screening, obtain a series of homologys and be the ESTs (fragment length is greater than 100bp) more than 80%, the number of including of these ESTs is inquired about corresponding sequence with ENTREZ (http://www.ncbi.nlm.nih.gov/Web/Search/index.html) in NCBI, use the ASSEMBLY program construction pig EST-contig among the GeneTool then.According to EST splicing a pair of primer M-F of sequences Design and M-R, sequence is as follows
HPSE：M-F：5′-AGCCAGGTGAGCCCGAGATG-3′，
M-R：5′-GCATCTGCTCGTGTTCCTAC-3′。
(2), the clone of PCR product and order-checking
PCR product behind the purifying spent the night 4 ℃ of water-baths with pMD-18T carrier (available from Takara) be connected; Get 100～120 μ l competent cells under the sterile state in 1.5ml Ependorff pipe,, place 30min on ice the connection product adding mixing of 5 μ l, 42 ℃ of heat shock 90s, back ice bath 3～4min adds the LB liquid nutrient medium of 400 μ l antibiotic-frees, 37 ℃ of shaking culture 45min.Get on the agar plate that 100 μ l coat isopropylthio-(IPTG) X-gal, 37 ℃ keep flat to be inverted behind the 1h and cultivate.Single bacterium colony on the picking flat board is inoculated among the 2-3ml LB, 37 ℃ of 300r/min overnight incubation.Collect thalline with the centrifugal several seconds of 1.5ml EP pipe 12000r/min and be prepared a spot of plasmid.Recombinant plasmid after the checking adopts the terminal cessation method of two deoxidations to check order on automatic dna sequencer, and sequencing is finished by Beijing AudioCodes Bioisystech Co., Ltd.Splice the CDS sequence (seeing that SEQ ID NO:1 is described) that to obtain a length be 1735bp with the ASSEMBLY program in the GeneTool1.0 software.
The dna sequence dna homology search is identified:
By the American National biotechnology (NCBI of information center, National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov) BLAST of website (Basic Local Alignment Search Tool) software, the known physiological function gene of announcing in the dna sequence dna that order-checking back is obtained and the GenBank database carries out sequence homology relatively, with evaluation with obtain the function information of this dna sequence dna.Result for retrieval show check order row (the GenBank number of including: partial sequence homology NM_006665.3) reaches 83% with people HPSE gene DNA.
The foundation of embodiment 2, PCR-RFLP diagnostic method
(1), primer sequence
HPSE P-SF 5 '-GGATGAAGGCTGGTATTT-3 ' (corresponding to SEQ ID NO:3)
P-SR 5 '-TGGGATAAGGCAATACAG-3 ' (corresponding to SEQ ID NO:4)
(2) pcr amplification condition
PCR reaction cumulative volume 20 μ l, wherein the about 100ng of pig genomic dna contains 1 times damping fluid (available from Promega company), 1.5mmol/LMgCl
2, the dNTP final concentration is 150 μ mol/L, the primer final concentration is 0.4 μ mol/L, 2U Taq archaeal dna polymerase (Promega).The pcr amplification program is: 94 ℃ of 3min, and the 94 ℃ of 30s that circulate 35 times, 54 ℃ of 30s, 72 ℃ of 25s then, last 72 ℃ are extended 5min.The PCR reaction product detects with 2% agarose gel electrophoresis.Obtain 374bp specific amplified fragment, this fragment is positioned at 4-5 intron (as Fig. 2).There is an Alu I restriction enzyme site in found that in this 374bp fragment of order-checking, and (AG ↓ CT), wherein the 80bp place is the polymorphism point of contact, is arranged in exon 5.
(3), PCR-RFLP testing conditions
PCR product endonuclease reaction volume is 10 μ l, 1 * buffer, 1 μ l wherein, and PCR product 3～5 μ l, restriction enzyme A lu I are 0.3 μ l (10U), use H
2O supplies 10 μ l, and with centrifugal behind the sample mixing, 37 ℃ of water-bath 4h detect enzyme with 2% agarose gel electrophoresis and cut the result, and the record genotype is taken pictures under ultraviolet lamp.Two the homozygous sequencing results in this site are shown that when the 80bp position was G, then this Alu I restriction enzyme site did not exist, and Alu I enzyme is cut the back detected result and is had only 1 fragment, and length is 374bp (being decided to be allelotrope G); But when having the replacement of G80 → A80, its result causes the generation of the Alu I in 80bp place restriction enzyme site, obtains 2 fragments, and length is respectively 294bp and 80bp (being decided to be allelotrope A), three kinds of frequency of genotypes AA, and AG, GG are as described in Figure 5.
(4), the application of molecule marker of the present invention in the association analysis of pig immune trait mark property
Test has detected the polymorphic of 189 Large Whites altogether, determine its genotype, and carry out genotype and immune character (white blood cell count(WBC) (WBC), red blood cell count(RBC) (RBC), oxyphorase (HGB), pcv (HCT), mean corpuscular volume (MCV), mean corpusular hemoglobin (MCH), immunoglobulin G (IgG)) association analysis.Set up least square model as described below:
y
ijk＝μ+G
i+C
j+P
k+B
j+ε
ijk
Wherein, y
IjkBe the character observation value, μ is a population mean, G
iBe genotype effect, C
jBe variety effect, P
kBe male parent effect, B
jBe a batch effect, ε
IjkBe random error, suppose ε
IjkSeparate, and obedience N (0, σ
2) distribute.Genotype detection result shows AA genotype in 189 individualities, has 13, and the AG genotype has 84 individualities, and the GG genotype has 92 individualities.Genotype with the result of proterties association analysis is: HPSE gene and red blood cell count(RBC) and white blood cell count(WBC) are significantly relevant.
As shown in Table 1, the HPSE gene SNP site has remarkably influenced (P＜0.05) to red blood cell count(RBC) (RBC), white blood cell count(WBC) (WBC) and pcv (HCT), wherein the red blood cell count(RBC) of AG genotype individuality, white blood cell count(WBC) and pcv all are significantly higher than the respective value of GG gene individuality, and the AA genotype is individual placed in the middle.Therefore the red blood cell count(RBC) and the white blood cell count(WBC) of GG genotype individuality are lower as can be seen.
Table 1: the polymorphism of pig HPSE gene A lu I-RFLP restriction enzyme site is to the influence of physiochemical indice and partial immunity proterties
Annotate:
*Expression P＜0.05,
*Expression P＜0.01.Character value is mean number ± standard deviation in the table.
Claims (5)
1. HPSE gene relevant of using as molecule marker with pig immune trait, its partial nucleotide sequence is shown in sequence table SEQ ID NO:2, there is the base of an A80-G80 to replace at the 80bp place of sequence table SEQ ID NO:2, causes Alu I-RFLP polymorphism.
2. gene according to claim 1, the right dna sequence dna of the used primer of HPSE gene the 5th exon that wherein increases is as follows:
Forward primer: 5 '-GGATGAAGGCTGGTATTT-3 ',
Reverse primer: 5 '-TGGGATAAGGCAATACAG-3 '.
3. one kind prepares the molecular marker method relevant with pig immune trait, and its method may further comprise the steps:
(1) personnel selection HPSE gene cDNA is the information probe, does the homologous sequence screening, obtains the expressed sequence tag (EST) of homology more than 80%, then EST is spliced; Extract pig lungs total tissue RNA and do the cDNA first chain reverse transcription; The design primer is right, the right forward primer of wherein said primer is 5 '-AGCCAGGTGAGCCCGAGATG-3 ', reverse primer is 5 '-GCATCTGCTCGTGTTCCTAC-3 ', cDNA fragment with RT-PCR method amplification pig HPSE gene, PCR product purification clone and order-checking obtain as the described sequence of sequence table SEQ ID NO:1 by sequential analysis;
(2) right according to the described sequences Design primer of SEQ ID NO:1, the right forward primer of described primer is 5 '-GGATGAAGGCTGGTATTT-3 ', reverse primer is 5 '-TGGGATAAGGCAATACAG-3 ' amplification pig genomic dna; The PCR product purification is cloned and check order, obtain as the described nucleotide sequence of sequence table SEQ ID NO:2 by sequential analysis.
4. the application of the described gene of claim 1 in the pig molecule mark assistant breeding.
5. the described primer of claim 2 is to the application in the pig molecule mark assistant breeding.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103255203A (en) * | 2012-02-21 | 2013-08-21 | 华中农业大学 | Application of molecular marker in pig birth weight trait correlation analysis |
| CN103320426A (en) * | 2012-03-19 | 2013-09-25 | 华中农业大学 | Angiogenin gene as molecular marker of swine immune trait |
| CN105002268A (en) * | 2015-06-21 | 2015-10-28 | 甘肃农业大学 | Molecular marker of HPSE gene as sheep immunity character and application thereof |
| CN109112218A (en) * | 2017-09-14 | 2019-01-01 | 华中农业大学 | Molecular labeling and application of the IL1R1 genetic fragment as pig immune trait |
| CN109609503A (en) * | 2017-10-04 | 2019-04-12 | 广西扬翔股份有限公司 | Molecular labeling and its application of the INVS gene as pig immune trait |
Citations (1)
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| CN1715419A (en) * | 2004-06-29 | 2006-01-04 | 华中农业大学 | Clone of pig immunity character relative gene PSMC5 and its use |
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| CN1715419A (en) * | 2004-06-29 | 2006-01-04 | 华中农业大学 | Clone of pig immunity character relative gene PSMC5 and its use |
Non-Patent Citations (3)
| Title |
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| 《Leukemia》 20070705 O Ostrovsky et al Association of heparanase gene (HPSE) single nucleotide polymorphisms with hematological malignancies 第21卷, 2 * |
| 《Reprod Fertil Dev》 20091231 Miles JR et al Molecular cloning and characterisation of heparanase mRNA in the porcine placenta throughout gestation 第21卷, 第6期 2 * |
| 《猪业科学》 20070930 易永宏等 猪分子标记辅助选择研究进展 , 第9期 2 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103255203A (en) * | 2012-02-21 | 2013-08-21 | 华中农业大学 | Application of molecular marker in pig birth weight trait correlation analysis |
| CN103320426A (en) * | 2012-03-19 | 2013-09-25 | 华中农业大学 | Angiogenin gene as molecular marker of swine immune trait |
| CN105002268A (en) * | 2015-06-21 | 2015-10-28 | 甘肃农业大学 | Molecular marker of HPSE gene as sheep immunity character and application thereof |
| CN109112218A (en) * | 2017-09-14 | 2019-01-01 | 华中农业大学 | Molecular labeling and application of the IL1R1 genetic fragment as pig immune trait |
| CN109112218B (en) * | 2017-09-14 | 2021-08-24 | 华中农业大学 | IL1R1 gene fragment as molecular marker of swine immune trait and application thereof |
| CN109609503A (en) * | 2017-10-04 | 2019-04-12 | 广西扬翔股份有限公司 | Molecular labeling and its application of the INVS gene as pig immune trait |
| CN109609503B (en) * | 2017-10-04 | 2022-01-04 | 广西扬翔股份有限公司 | Molecular marker of INVS gene as pig immune trait and application thereof |
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