CN102776178B - MicroRNA molecule marker associated with pig blood parameter properties, and application thereof - Google Patents
MicroRNA molecule marker associated with pig blood parameter properties, and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of the preparation of a livestock molecule marker, and concretely relates to a microRNA molecule marker applied as a pig marker auxiliary selection and associated with blood parameter (immune) properties. The molecule marker is obtained through pig microRNA miR-155 precursor and flanking sequence cloning, and the nucleotide sequence of the molecule marker is represented by SEQ ID NO:2 in a sequence table. Hha I-RFLP (restriction fragment length polymorphism) is caused by a T297-C297 base mutation at the 297th base of the sequence represented by the SEQ ID NO:2 in the sequence table. The invention also discloses a preparation method of the molecule marker, and an application of the molecule marker in polymorphism detection, and provides a new molecule marker for the pig marker auxiliary selection.
Description
Technical field
The invention belongs to the domestic animal gene engineering technology field, be specifically related to a kind of clone and application of the microRNA molecule marker relevant to blood parameters (immunity) proterties as the pig marker assisted selection.
Background technology
MicroRNAs (miRNAs) be a class size approximately 22 bases by with its said target mrna, being combined the little RNA of gene silencing (Bartel et al., 2004 after inducible transcription; Kim et al., 2005).In Mammals, the course of processing of miRNA can be divided into multistep.At first, most of miRNA is transcribed into long originally transcript (pri-miRNA) (Lee et al. by rna plymerase ii (RNA polymerase II), 2004), subsequently, pri-miRNA is cut into the precursor miRNA that size is 70～80nt (pre-miRNA) (Lee et al., 2003 by the little processing complex body that is comprised of III type RNA nuclease Drosha and double-strand RNA binding protein DGCR8; Gregory et al., 2004; Denli et al., 2004).Then by core output albumen Exportin-5 and synergy factor R an-GTP, together the hair clip precursor miRNA is transported out core (Yi et al., 2003; Lund et al., 2004; Bohnsack et al., 2004).In tenuigenin, precursor miRNA is formed double-stranded miRNA complex body by another III type RNA nuclease Dicer cutting.Untwist subsequently, wherein one becomes ripe miRNA and enters the nucleoglucoprotein complex body, forms RNA and disturbs silencing complex (RISC) (Preall et al., 2005).In RISC, miRNA regulates the expression of target gene by dual mode, if itself and target gene 3 ' UTR complete complementary make its degraded; If incomplete complementary combination, hinder its translation.
The biosynthesizing of miRNA is subject to tight regulation and control in body, and being present in sudden change on pri-miRNA, pre-miRNA and ripe miRNA sequence can affect the generation of final ripe miRNA to some extent.In Mammals, miRNA, as the endogenous repressor, checks the translation of encoding gene by the 3 ' UTR district in conjunction with said target mrna.Can affect the regulating and controlling effect of miRNA due to the SNP that exists on the target site sequence, the spontaneous SNP of target site is the key object (Saunders, et al.2007) of miRNA functional study.
Increasing research is in recent years found, the gene of the synthetic mankind miRNA of coding (comprises miRNA primary transcript pri-miRNA, miRNA precursor pre-miRNA and ripe miRNA) and the target gene binding sequence of miRNA in also have SNP (Duan, et al.2007).Find during the research has-miR-125a genes such as Duan, also there is SNP in this gene locus, thereby the miR-125a that contains miR-125a-G and two kinds of equipotentials of miR-125a-U, wherein combination and the montage of miR-125a-U energy remarkably influenced DGCR8 and pri-miRNA-125a, ripe miR-125a is generated to be reduced, make miR-125a weaken (Duan, et al.2007) to the translation restraining effect of target gene Lin-28.Sethupathy etc. study discovery, the SNP (rs5186) that is positioned at AGTR1 gene has-miR-155 binding site downstream can affect the combination with has-miR-155, has-miR-155 is that the binding ability of the AGTR1 mRNA of A equipotential strengthens to this site, downward effect to AGTRI genetic expression is more obvious, and the AGTR1 mrna expression that this site is the C equipotential is not subjected to has-miR-155 to affect (Sethupathy, et al., 2007).
MiRNA plays the part of very important role in immunomodulatory, microRNA than the damage associated molecular pattern, miR-155 is miRNA (the Hideho Okada relevant to pathogen-associated molecular pattern, 2010), and miR-155 is as a proto-oncogene (Tam et al., 1997), high expression level in kinds of tumors.The miR-155 function is extensive, and it participates in many bioprocesss (Isabella Faraoni, etal., 2009) such as hematopoiesis, inflammation and immunity.BIC/miR-155 play a significant role in keeping immunity system normal function and stable state (Rodriguez, et al., 2007).People and mouse mir-155 are positioned respectively separately all to derive from a non-coding long-chain RNA, BIC (B-cellIntegration Cluster) on genomic No. 21, No. 16 karyomit(e)s.BIC is accredited as the gene on a common retrovirus integration site that is present in the bone-marrow-derived lymphocyte cancer of by avian leukosis viruses, being induced at first, the promoter activity activated transcription that it can be inserted into (Tam et al., 1997).The discovery miR-155 such as Eis in 2005 are sheared and produce through processing by bic RNA, bic comprises three exons altogether, and the precursor of miR-155 is present in (mouse miR-155 precursor originates in the 88nt of the 3rd exon) in the 3rd exon (Eis et al., 2005).
In recent years, the further investigation of miRNA has been showed a brand-new immunoregulation mechanism to us.At present to the research and comparison that participates in immunoregulatory miRNA thorough miR-155 just arranged.Rodriguez etc. (2007) discovery, in the mouse of miR-155 gene knockout, the function of T cell, B cell and DC cell is all destroyed, and it is necessary that this explanation miR-155 keeps normal immunologic function for body.O ' Connell etc. (2007) are after stimulating the mouse scavenger cell with Polyinosinic-polycytidylic acid (Poly I:C) and IFN-β, utilize the differential expression of the relevant miRNA of genechip detection, find that miR-155 is that unique these two kinds of factors that are subjected to stimulate the rear miRNA that continues up-regulated expression.Suppress kinases JNK by the pharmacology mode and can stop the generation of Poly I:C or the beta induced miR-155 of IFN-, show that JNK path regulation and control are induced to produce miR-155 (O ' Connell etal., 2007).Tili etc. (2007) discovery, LPS can induce the up-regulated of scavenger cell miR-155, the down-regulated expression of miR-125b, miR-155 and miR-125b all participate in immune response.
Up to now, there is not yet the report of comprehensive research pig miR-155 gene function, and polymorphism and the proterties association analysis of research mutational site in colony is very strong means of research gene function., so the applicant has carried out polymorphic research and association analysis to the gene order of miR-155, to finding its related with the pig blood parameter, and then find the function of miR-155 at immunology.
Summary of the invention
The object of the invention is to overcome the prior art defect, precursor and the flanking sequence of clone and pig Immune interrelation microRNA-155, find the mutational site of this fragment, screen a kind of microRNA molecule marker relevant to pig blood parameter proterties, utilize the application of this molecule marker as the marker assisted selection of pig.
The present invention is achieved through the following technical solutions:
Applicant clone obtains microRNA-155 precursor and the flanking sequence relevant to pig blood parameter proterties, and its nucleotide sequence is as described in sequence table SEQ ID NO:2.
The microRNA-155 precursor of pcr amplification and flanking sequence total length are 472bp, and its nucleotide sequence is as described in sequence table SEQ ID NO:2 and (showing the base mutation position in Fig. 2 bracket) shown in Figure 2.
There is an allelic base mutation (T297-C297) in 297bp place at sequence table SEQ ID NO:2, and this sudden change causes Hha I-RFLP polymorphism (Restriction Fragment Length Polymorphism).
Amplification miR-155 precursor and flanking sequence, check order and to detect the nucleotide sequence of T297-C297 place base mutation primer pair used as follows:
Forward primer: 5 ' CCAAAGCAACTGCAGGATAG 3 ';
Reverse primer: 5 ' CTTCTTTGTCATCCTCCC 3 '.
The preparation method of microRNA molecule marker of the present invention is:
Manned under in miRBase (http://microrna.sanger.ac.uk/sequences/)/mouse miR-155 precursor sequence, carry out the BLASTn comparison with NCBI (http://www.ncbi.nlm.nih.gov/) pig genome database, angle the partial dna sequence (comprising precursor sequence and flanking sequence) of getting homology pig miR-155 gene, design PCR primer, extract the pig genomic dna, carry out pcr amplification, PCR product purification and order-checking, obtain the nucleotide sequence as shown in sequence table SEQ ID NO:2.
The method of the PCR-RFLP that application is conventional detects the 297th bit base sudden change shown in sequence table SEQ ID NO:2, and tentatively carry out the application of the association analysis between the Traits of its genotype and pig, for the molecular marker assisted selection of pig provides a new molecule marker.
More detailed technical scheme is referring to " embodiment ".
Description of drawings
Sequence table SEQ ID NO:1 splices with the method for information biology the pig microRNA-155 gene fragment that obtains.The sequence total length is 960bp.
Sequence table SEQ ID NO:2 is the nucleotide sequence of the present invention's microRNA molecule marker relevant to pig blood parameter proterties of cloning.
Fig. 1: techniqueflow chart of the present invention.
Fig. 2: be the nucleotide sequence of clone's the microRNA molecule marker relevant to part blood parameters proterties in the present invention, sequence length is 472bp, is allelic sudden change in bracket.
Fig. 3: the comparison chart of the miR-155 gene order (472bp) of Du Luoke, the purebred pig of plum mountain pig in the present invention.
Fig. 4: the comparison chart of the miR-155 gene order (471bp) of painted face in Beijing opera, the purebred pig of landrace in the present invention.
Fig. 5: the comparison chart of landrace, the purebred pig miR-155 of Du Luoke gene order (467bp) in the present invention.
Fig. 6: many sequencing sequence comparisons of Du Luoke, the purebred pig of plum mountain pig in the present invention.
Fig. 7: three kinds of genotype (CC, TC, TT) electrophoretogram of the Hha I-RFLP of pig microRNA-155 gene in the present invention.The M swimming lane is DNA molecular amount standard (DL2000)
Embodiment
Embodiment 1:
(1) amplification of pig microRNA-155 precursor and part flanking DNA sequence
manned under in miRBase (http://microrna.sanger.ac.uk/sequences/)/mouse miRNA-155 precursor sequence (gene accession number: MI0000681/MI0000177), carry out the BLASTn comparison at NCBI (http://www.ncbi.nlm.nih.gov/) pig genome database, angle and get homology pig miRNA-155 gene order, selection angles the homology got greater than 98% more than 20 bar sequences, import in DNASTAR software seqman program, (its total length is 960bp to splice a higher sequence of confidence level, see shown in SEQ ID NO:1) for follow-up analysis.According to splicing gained sequences Design PCR primer (as follows), extract the pig genomic dna, carry out pcr amplification, PCR product purification and order-checking, obtain the nucleotide sequence as shown in sequence table SEQ ID NO:2.By the order-checking of the amplification to miRNA-155 precursor and flank section sub-sequence (472bp) comparison, found 1 allelic sudden change (being T297-C297) in the 297bp place, restriction analysis is carried out in this sudden change, find that this sudden change just can be by the identification of restriction endonuclease HhaI.
Amplification miRNA-155 precursor and flanking sequence, check order and to detect the nucleotide sequence of T297-C297 place base mutation primer pair used as follows:
Forward primer: 5 ' GGGTGGGAATCGTTCAAAGG 3 ',
Reverse primer: 5 ' CATCCATCTTCCAGGAGCCA 3 '.
(2) purifying of PCR product, Cloning and sequencing
Add DNA profiling 1 μ L in the reaction system of pcr amplification condition: 10uL, distilled water 7 μ L, 10 * PCR buffer, 1 μ L, each 0.3 μ L before and after dNTP0.3 μ L, 10mM primer, Taq enzyme 1U.The PCR reaction conditions is: after 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ of extension 30s, 33 circulations, last 72 ℃ are extended 5min.The PCR product detects through 1.5% agarose gel electrophoresis.
The purifying of PCR product: to carry out follow-up clone, according to the amplification condition of the first step, amplification 100 μ lPCR products, can put 4 degree Refrigerator stores in the PCR product short period of time for the PCR product that obtains q.s.Take the 0.3g agarose and add the 20mlTAE electrophoretic buffer, boil in microwave oven, until the liquid bleach, pour into after cooling a little and be plugged canine tooth and reclaim the gel slab of comb.Treat the gel cooling curing, take off comb, put in electrophoresis chamber, add the TAE electrophoretic buffer of suitable volumes, to have gel, be not advisable.Add 20 μ l 6 * sample-loading buffers (6 * loading buffer) in 100 μ l pcr products, wherein added GelRed (substitute of EB, safer).Get simultaneously 10 μ l DL2000 DNA Ladder as marker.Voltage 120V, electric current 100mA, treat that the tetrabromophenol sulfonphthalein dyestuff runs from glue hole 3-4cm at a distance, carefully gets gel and puts into the gel imaging system imaging.Then in bale cutting instrument, the careful fritter gel that contains the purpose band that downcuts, put into a clean 1.5ml centrifuge tube.Reclaim test kit (DP209) specification sheets according to the common DNA gel of sky root, carry out gel purification.Roughly comprise following steps:
Column equilibration: (adsorption column is put into collection tube) adds 500 μ l balance liquid BL in adsorption column CA2, and centrifugal 1 minute of 12000rpm, outwell the waste liquid in collection tube, and adsorption column is relay and reclaims in collector.
Add 3 times of volume PN (gel heavily is 0.1g, and its volume can be considered 100 μ l) in the 1.5ml centrifuge tube that the gel strips that scales off is housed.50 ℃ of water-baths were placed 10 minutes, constantly leniently spun upside down centrifuge tube therebetween, to guarantee blob of viscose, fully dissolved.
Previous step gained solution is added (balance) in an adsorption column CA2, and room temperature was placed 2 minutes, the centrifugal 30-60 of 12000rpm second, outwell the waste liquid in collection tube, adsorption column CA2 is put into collection tube.
Add 600 μ l rinsing liquid PW in adsorption column CA2, the centrifugal 30-60 of 12000rpm second, outwell the waste liquid in collection tube, adsorption column CA2 is put into collection tube.
Add 600 μ l rinsing liquid PW in adsorption column CA2, the centrifugal 30-60 of 12000rpm second, outwell waste liquid.
Adsorption column CA2 is put back in collection tube, and centrifugal 2 minutes of 12000rpm eliminates rinsing liquid as far as possible.Adsorption column CA2 is placed in room temperature placed several minutes, dry up hill and dale, to prevent residual rinsing liquid, affect next step experiment.
Adsorption column CA2 is put in a clean centrifuge tube, and to the appropriate elutriant EB of the unsettled dropping in adsorption column mid-way, room temperature was placed 2 minutes.12000rpm collected DNA solution in centrifugal 2 minutes.(generally adding 35 μ l elutriants).
The purpose fragment that purifying reclaims is just in the liquid of wash-out gained; Portion of product is served extra large handsome biotech firm direct Sequencing.
(3) the DNA sequence dna homology search is identified
By the American National biotechnology (NCBI of information center, http://www.ncbi.nlm.nih.gov) BLAST of website (Basic LocalAlignment Search Tool) software, the known physiological function gene of announcing in the DNA sequence dna of acquisition after order-checking and GenBank database is carried out sequence homology relatively, to identify and to obtain the function information of this DNA sequence dna.
In the present embodiment, pcr amplification product shows and is special PCR product through 1.5% agarose gel electrophoresis detected result.The PCR product is reclaimed the order-checking of purifying rear clone, sequencing result shows that the DNA sequence dna total length that PCR obtains is 472bp, comprise the miRNA-155 mature sequence, precursor sequence and part flanking sequence (as shown in sequence table SEQ ID NO:2), sequencing result shows and has T297-C297 sudden change at the 297bp place of this DNA sequence dna, and sequencing result as shown in Figure 3.
(2) the PCR-RFLP diagnostic method is set up
RFLP detects: with PCR product 6 μ L, 10 * Buffer, 1 μ L, restriction enzyme Hha I is 0.2 μ L (2U), adding distilled water mends to 10 μ L, with centrifugal after sample blending, 37 ℃ of incubators are placed 12h, detect enzyme with 2% agarose gel electrophoresis and cut result, record genotype, take pictures under ultraviolet lamp.
Obtained 472bp specific amplification fragment with above-mentioned primer pair amplification pig genomic dna, the sequencing results shows and exists T297-C297 to suddenly change at 297bp place, and causes Hha I polymorphism.This gene mutation site is controlled by two allelotrope, and wherein T is the allelotrope that does not form restriction enzyme site, and C is the allelotrope that forms restriction enzyme site.These two allelotrope can form three kinds of genotype wherein the TT type for homozygous (the only having DNA band of 472bp during electrophoresis detection) that enzyme cuts do not occur, homozygous (the occurring two DNA bands of 256bp and 216bp during electrophoresis detection) that the CC type is cut for enzyme occurs, TC is heterozygous (occurring three DNA bands of 472bp, 256bp and 216bp during electrophoresis detection).
Embodiment 2:
Experiment swinery Du Luoke * painted face in Beijing opera resource colony is that the Animal Genetics, Breeding and Reproduction Molecular Biology Lab at applicant place sets up, produced by 8 Duroc boars and 13 Erhualian sow hybridization, it is individual for the association analysis of the present embodiment SNP proterties that colony amounts to 392 F2.All 392 F2 individualities have been carried out detection and the record of routine blood test proterties, preserved perfect proterties data.To Du Luoke * painted face in Beijing opera F2 colony totally 392 pigs carry out the miR-155 genotype tests, determine every pig miR-155 genotype, arrange every pig label and genotypic corresponding relation.The applicant detects the genotype (wherein 6 pigs do not detect genotype) of 386 pigs altogether.Use mixed linear model (Mixed Model, the Mixed) program in SAS (form V8 version) software to carry out association analysis to the T/C of miR-155 existence is polymorphic with proterties.Model is as follows: Y=Genotype+Sire+Dam (Sire); Wherein Genotype represents the genotype effect, and Sire represents the male animal effect, and Dam represents the dam effect.
Pig miR-155 gene polymorphism sites genotype detection result is shown that the TT genotype has 113 in 386 individualities, gene frequency 0.29; The TC genotype has 185, and gene frequency is 0.48; The CC genotype has 88, and gene frequency is 0.23.The proterties of analyzing has main routine blood test index.Respectively the F2 individuality is detected at 20 ages in days, 33 ages in days, 35 ages in days, 80 ages in days, draw finally the SNP site of miR-155 gene with the hemoglobin concentration (HGB) of 33 ages in days, the basophilic granulocyte (BA) of 35 ages in days, mean corpuscular hemoglobin (MCH) significant correlation of 35 ages in days, with eosinophilic granulocyte (EO) utmost point significant correlation of 80 ages in days, with production traits teat number utmost point significant correlation.Relevant design parameter is as shown in following table.
The association analysis of table 1miR-155 gene polymorphism sites different genotype and 20 age in days part-blood conventional indexs detects
* represent significant difference, P＜0.05; * represents extremely significantly P＜0.01 of difference
As shown in Table 1, the SNP site of miR-155 gene has no significant effect the routine blood test index of 20 ages in days.
The association analysis of table 2miR-155 gene polymorphism sites different genotype and 33 age in days part-blood conventional indexs detects
* represent significant difference, P＜0.05; * represents extremely significantly P＜0.01 of difference
As shown in Table 2, the hemoglobin concentration (HGB) of the SNP site of miR-155 gene to 33 ages in days, have remarkably influenced (p＜0.05), and this site has no significant effect other routine blood test index.The hemoglobin concentration (HGB) of 33 ages in days there is is the least squares means of SNP loci gene type of miR-155 gene of remarkably influenced in Table 3:
The least squares means of table 3SNP site (miR-155) 33 age in days HGB Tile Width
* represent significant difference, P＜0.05; * represents extremely significantly P＜0.01 of difference
As shown in Table 3, the genotypic hemoglobin concentration of CT (HGB) is significantly higher than CC genotype (p＜0.05), and the genotypic hemoglobin concentration of CT (HGB) is for the highest.
The association analysis of table 4miR-155 gene polymorphism sites different genotype and 35 age in days part-blood conventional indexs detects
* represent significant difference, P＜0.05; * represents extremely significantly P＜0.01 of difference
As shown in Table 4, basophilic granulocyte (BA%) and the mean corpuscular hemoglobin (MCH) of the SNP site of miR-155 gene to 35 ages in days, all have remarkably influenced (p＜0.05), and this site has no significant effect other routine blood test index.The least squares means of the SNP loci gene type of the miR-155 gene that basophilic granulocyte (BA%) and the mean corpuscular hemoglobin (MCH) of 35 ages in days had remarkably influenced is in Table 5:
The least squares means of table 5SNPs site (miR-155) genotype 35 ages in days (BA%) and MCH
* represent significant difference, P＜0.05; * represents extremely significantly P＜0.01 of difference
As shown in Table 5, the genotypic basophilic granulocyte of CC (BA%) utmost point is significantly higher than CT genotype (p＜0.01), and the genotypic mean corpuscular hemoglobin of CT (MCH) is significantly higher than CC genotype (p＜0.05).The genotypic basophilic granulocyte of CC (BA%) is for the highest, and the average hemoglobin content of the genotypic cell of CT (MCH) is for the highest.
The association analysis of table 6miR-155 gene polymorphism sites different genotype and 80 age in days part-blood conventional indexs detects
* represent significant difference, P＜0.05; * represents extremely significantly P＜0.01 of difference
As shown in Table 6, the eosinophilic granulocyte number (EO) of the SNP site of miR-155 gene to 80 ages in days, the eosinophilic granulocyte (EO%) of utmost point remarkably influenced (p＜0.01) to 80 ages in days arranged, remarkably influenced (p＜0.05) is arranged, and this site has no significant effect other routine blood test index.On the eosinophilic granulocyte number (EO) of 80 ages in days and eosinophilic granulocyte (EO%) has respectively extremely significantly and the least squares means of the SNP loci gene type of the miR-155 gene of remarkably influenced in Table 7:
The least squares means of table 7SNPs site (miR-155) genotype 80 age in days EO and EO%
* represent significant difference, P＜0.05; * represents extremely significantly P＜0.01 of difference
As shown in Table 7, the genotypic eosinophilic granulocyte number of TT (EO) utmost point is significantly higher than CC genotype (p＜0.01), and the genotypic eosinophilic granulocyte number of TT (EO) utmost point is significantly higher than CT genotype (p＜0.01).The genotypic eosinophilic granulocyte of TT (EO) is for the highest.The genotypic eosinophilic granulocyte of TT (EO%) is significantly higher than CC genotype (p＜0.05), the genotypic eosinophilic granulocyte of TT (EO%) is significantly higher than CT genotype (p＜0.05), and the genotypic eosinophilic granulocyte of TT (EO%) is for the highest.Therefore T allelotrope is the favourable mark of eosinophilic granulocyte number (EO).
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Claims (2)
1. molecule marker relevant to pig blood parameter proterties, its sequence is SEQ ID NO:2, wherein the base of the 297th of this sequence is T or C, the hemoglobin concentration that described pig blood parameter proterties is 33 ages in days, the basophilic granulocyte of 35 ages in days, the mean corpuscular hemoglobin of 35 ages in days and the eosinophilic granulocyte number of 80 ages in days.
2. the primer pair of the described molecule marker of amplification claim 1, is characterized in that, the nucleotide sequence of described primer pair is as follows:
Forward primer: 5 ' CCAAAGCAACTGCAGGATAG3 ';
Reverse primer: 5 ' CTTCTTTGTCATCCTCCC3 '.
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| CN112574994B (en) * | 2020-12-17 | 2023-10-24 | 河南牧业经济学院 | Method for screening active region of pig miR-155 gene promoter and transcriptional regulatory element |
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