CN100413886C - Protein associated to pig immunity and growth trait, coding genes and application - Google Patents

Abstract

The present invention discloses pig immunity and growth trait associated protein, encoding gene and applications thereof. The protein has one of the following amino acid residue sequence: 1) SEQ ID No: 6 in a sequence table; 2) protein obtained by replacing, deleting or adding the amino acid residue sequence of the SEQ ID No: 6 in the sequence table by one to ten amino acid residues and being associated with the pig immunity and growth trait. The method for detecting the pig immunity and growth traits comprises the following steps: a pair of primers composed of nucleotide sequences of SEQ ID No: 2 and SEQ ID No: 3 in the sequence table are used for PCR amplification for genomic DNA of a pig to be measured to determine whether the 244th basic group of the 5' end of the SEQ ID NO: 1 in the sequence table is A or C, or enzyme cleavage is applied to the PCR amplification product by Csp6I to detect the size and the quantity of the obtained enzyme slice segments. The pig immunity and growth trait associated protein and the encoding gene thereof in the present invention can perform important functions in pig breeding.

Description

Pig immunity, growth traits associated protein and encoding gene and application

Technical field

The present invention relates to a kind of protein and encoding gene thereof and application, particularly relate to a boar immunity, growth traits associated protein and encoding gene thereof and utilize the single nucleotide polymorphism of this protein coding gene and restrictive fragment length polymerphism detects pig immunity, the method for growth traits.

Background technology

Pork is the topmost animal food of China, and the animal food safety problem comes into one's own day by day.The emphasis that modern herding is produced is included two aspects: be the disease resistance that improves pig on the one hand, i.e. and breeding for disease resistance, thus reduce the growth cost of unit product, and reduce drug residue, improve the quality of livestock product; Be the utilization GENERALIZATION OF MODERN BREEDING TECHNIQUE on the other hand, and the binding molecule labeling technique, the production performance of pig further improved.

Following period of time in the past, people too pay attention to the raising of the speed of growth of pig, have ignored the seed selection to its disease-resistant performance, thereby the quick growth of pig also is accompanied by the reduction of disease resistance; Recent research shows that its speed of growth of pig of a certain specific gene type and trunk composition determine its health level to a certain extent.Disease resistance is good, and healthy pig feed intake, the speed of growth and feed efficiency of conversion are all than higher; In addition, the trunk that also can change pig is formed, and makes pig deposit more muscle or body protein, reduces the deposition of fatty tissue as far as possible.Yet the mutual relationship between pig immunological competence and production performance is still not fully aware of, and therefore the mutual relationship that discloses between production performance and immunizing power controlling gene from molecular level is crucial, and also the marker assisted selection for domestic animal provides new thinking.

Individual immunity power is to weigh an important indicator of animal health condition, and it belongs to quantitative character, and related genes involved is more, and molecular mechanism is also complicated.When individuality was subjected to pathogenic agent and infects, body can be transferred the defense mechanism of three aspects, and promptly epithelium defense mechanism, nonspecific defense mechanism and specificity defense mechanism are resisted.The healthy state of pig depends on and infects and the defensive enginery results of interaction, if defensive enginery just shows the nature disease resistance by force.

Disease resistance major histocompatibility complex (Major Histocompatibility complex, MHC) be and disease resistance and the closely-related one group of gene group of immunne response, the MHC of pig (called after SLA) is positioned at (Warner on No. 7 karyomit(e)s of pig, Mapping of C2, Bf, and C4genes to the swine majorhistocompatibility complex.J.Immunology.1987,139:3388-3395), comprise I type and II type gene, wherein I type gene has extremely strong polymorphism.The research of proterties such as pig SLA gene haplotype and pig birth weight, the speed of growth, the thickness of backfat is engaged in the Rothschild laboratory for a long time, they think that there are correlationship (Rothschild MF in SLA gene haplotype and above-mentioned production performance, Identification of quantitative traitloci and interesting candidate genes in the pig:progress and prospects.Proc6th WCGALP 1998,26:403-409).Mallard etc. (1998) find that in high immunne response of selecting through 8 generations to form of pig (H system) and low immunne response (L system) strain H system reaches market weight than the Zao 10d of L system.The above-mentioned SLA that studies show that pig and the multiple production traits all have chain, and get in touch the relation of being proportionate between the SLA of pig and growth, back fat and reproductive trait more.Show that it is feasible simultaneously the production traits and immune character being selected, thereby the gene that can influence pig growth traits and immune character simultaneously just seems particularly important, this genoid also just becomes the key object of marker assisted selection research.

The albumen of RPLPO genes encoding is a kind of in the big protein subunit of ribosomal protein.(ribosmalprotein rp) has tens of kinds to ribosomal protein, is the little polypeptide class of molecular weight mostly, and the albumen that is distributed in the big subunit of ribosome is called RPL, and the albumen that is distributed in small subunit is called RPS.Prokaryotic organism and Eukaryotic ribosome are formed by the large and small subunit that is easy to depolymerization.To ribosomal 2nd/3rd of its quality, rNRA, 1st/3rd, the protein discovered of intestinal bacteria.RRNA is divided into three kinds of 5S, 16S, 23S; Small subunit is made of 16SrRNA and 21 kinds of RPS, and big subunit is made of 5S, 23S rRNA and 31 kinds of RPL.Eukaryote ribosome small subunit contains 18S rRNA and more than 30 kind of RPS, and big subunit contains 28S, 5.8S, three kinds of rRNA of 5S, nearly 50 kinds of RPL, and the rRNA in the various biological ribosome small subunits has similar secondary structure.

Studies show that the difference that some ribosomal protein puts in order in same boundary can reflect the sibship between species, sibship is near more, and the sequence in the gene order is approaching more.So just can study Phylogenetic Relationships between species by putting in order of icp gene.

Studies show that rRNA must combine with ribosomal protein could carry out its function-synthetic protein, therefore, scientist begins to launch the research to the ribosomal protein function.Tong Kezhong researcher is engaged in the isolating research of ribosomal protein mutant always, and study of the influence of these mutant to other genetic expression, separated and identified 51 kinds of ribosomal protein mutant altogether such as Bacillus subtilus and intestinal bacteria ribosomal protein mutant, occupied first place in the world.The sudden change of above-mentioned ribosomal protein is found the influence of bacterial gene and phage gene expression after deliberation: 1) ribosomal protein of some sudden change is different with the translation specificity of original ribosomal protein; 2) same ribosomal protein mutant is to the difference that influences of different genes or genomic expression; 3) different ribosomal protein mutant is to the difference that influences of same gene or genomic expression.The difference of these genetic expression height is accepted or rejected to some extent by the different mRNA of ribosomal protein confrontation just and is caused the intestinal bacteria ribosomal protein S of this conclusion and department of biochemistry of California, USA university (Berkeley) report ₁MRNA is had selectivity to coincide.The scholars of U.S. west Wei Jiniya university and University of California medical college have checked the crystalline mRNA of cataract patient, warp and contrast crystalline mRNA make comparisons, find that the genetic expression aspect between patient and the contrast exists than big-difference, L21 in patient's body, L15, these four kinds of proteic expression amounts of L13a and L7a are relative less.Infer thus, synthetic and other function of the albumen of ribosomal protein regulation and control, relevant as the cataract pathogenesis relevant with aging.Another discovers that ribosomal protein L 6 has and T chronic myeloid leukemia virus (HTLV)-I long segment tumor-necrosis factor glycoproteins bonded activity, this leukemoid treatment of behaving provides new information (Wang J etc., Cloning of mouse genomic ribosomal protein L6gene and analysisof its promoter.Biochim Biophys Acta.2002,1576 (1-2): 219-224).

In addition, RPLPO also can be used as housekeeping gene, is used for expression of gene research.People's RPLPO gene is located in people HAS12q24.2 (http://www.ncbi.nlm.nih.gov/LocusLink/).The RPLPO gene is by 7 exons, and 6 introns are formed.But so far, the research of relevant RPLPO gene function also is not very thorough, the polymorphism of research mutational site in colony, and carry out the strong means that the proterties association analysis is the research gene function.

(single nucleotide polymorphism SNP), mainly is meant on genomic level by the caused dna sequence polymorphism of the variation of single Nucleotide single nucleotide polymorphism.The polymorphism that SNP showed only relates to the variation of single base, and this variation can be caused by the conversion (transition) or the transversion (transversion) of single base, also can be by due to the insertion or disappearance of base.But usually said SNP does not comprise back two kinds of situations.This variation may be that (C → T then is G → A) on its complementary strand, also may be transversion (C → A, G → T, C → G, A → T) in conversion.The incidence of conversion is always apparently higher than other several variations, and the SNP with conversion hysteria variation accounts for 2/3, and the occurrence probability of other several variations is similar.The SNP detection method often adopts some existing mature technologies, as dna sequencing, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP), allele specific oligonucleotide oligonucleotide hybridization (ASO) etc.No matter adopt any method, all at first must increase, just can carry out other detection then target sequence.

The PCR-RFLP full name is that (restrictionfragment length Polymorphism RFLP) analyzes polymerase chain reaction,PCR (PCR) restriction fragment length polymorphism.Mainly comprise three links: (1) target gene pcr amplification; (2) Kuo Zeng dna fragmentation restriction map (length polymorphism), this art is used PCR target gene fragment is increased more than 1,000,000 times, even sample nucleic acid amount is less than the pg level, also can be amplified out, this has not only improved the sensitivity of genetic analysis greatly, and need not mark or radioactive probe crossover process .PCR-RFLP is mainly used in nucleic acid Variability Analysis and comparison, when DNA or RNA molecule since single base mutation or sequence reset, certain restriction enzyme site is increased, reduce or disappearance, cause restriction fragment length to change, just produced restriction fragment length polymorphism, because restriction enzyme has special restriction enzyme site on dna molecular, according to the position and the quantity of its recognition sequence, can cut into different fragments to the similar and variant DNA of sequence of size by nucleic acid electrophoresis, can

The DNA sheet that is uneven in length separated and show its length polymorphism (segmental size) .PCR-RFLP and be used for genetic analysis and have the resolution height, good reproducibility, advantages such as easy quicklook, be mainly used in the somatotype branch hypotype of hiving off of microorganism, oncogene is analyzed, the diagnosis of inherited disease, the analysis of HLA somatotype and lipophorin gene etc.

Summary of the invention

The purpose of this invention is to provide a boar immunity, growth traits associated protein and encoding gene thereof.

Pig immunity provided by the present invention, the growth traits associated protein, name is called RPLPO, derives from pig, is the protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 5;

2) with SEQ ID № in the sequence table: 5 amino acid residue sequence through replacement, disappearance or the interpolation of one to ten amino-acid residue and with pig immunity, the protein that growth traits is relevant.

Wherein, the SEQ ID № in the sequence table: 5 are made up of 318 amino-acid residues.

Above-mentioned pig immunity, the encoding gene of growth traits associated protein (RPLPO) also belongs to protection scope of the present invention.

Above-mentioned pig immunity, the encoding gene of growth traits associated protein comprises the pig immunity, the cDNA sequence of growth traits associated protein and pig immunity, the genomic fragment of growth traits associated protein.Its cDNA gene can have one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 4 polynucleotide;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 5 protein sequences;

3) under the rigorous condition of height can with the SEQ ID № in the sequence table: the nucleotide sequence of the 4 dna sequence dnas hybridization that limit.

The rigorous condition of described height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.

SEQ ID № in the sequence table: 4 by 1293 based compositions, and the encoding sequence of this cDNA sequence is SEQ ID № in the sequence table: 5 from 5 ' end 177-1133 bit base.

Its genomic gene has one of following nucleotide fragments:

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

2) SEQ ID № in the code sequence tabulation: the DNA of 5 protein sequences;

3) under the rigorous condition of height can with the SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.

The rigorous condition of described height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.

Sequence 1 in the sequence table is by 595 based compositions, 1-56 bit base from 5 ' end is the 4th exon of this genomic gene, 194-340 bit base from 5 ' end is the 5th exon of this genomic gene, 447-595 bit base from 5 ' end is the 6th exon of this genomic gene, from 5 ' the 57-193 bit base of end be the 4th intron of this genomic gene, from 5 ' the 341-446 bit base held is the 5th intron of this genomic gene.

Contain pig immunity of the present invention, the carrier of growth traits associated protein encoding gene, clone and host bacterium all belong to protection scope of the present invention.

Second purpose of the present invention provides the immunity of a kind of detection pig, the method for growth traits.

Detection pig provided by the present invention immunity, the method of growth traits, be to use by SEQ ID № in the sequence table: 2 and SEQ ID №: a pair of primer that 3 nucleotide sequence is formed carries out pcr amplification to the genomic dna of pig to be measured, then pcr amplification product is carried out following at least a detection:

1) described pcr amplification product is carried out single nucleotide polymorphism and detect, determine that from 5 of SEQ ID NO:1 ' end the 244th bit base be C or A;

2) cut described pcr amplification product with the Csp6I enzyme, detect in the endonuclease bamhi that obtains whether contain a 595bp band and/or a 353bp band and a 242bp band.

If the single nucleotide polymorphism detected result of pcr amplification product is from 5 of SEQ ID NO:1 ' end the 244th bit base, when promptly 5 of sequence 4 ' end the 545th bit base was C in sequence table, its homozygotic genotype was CC; From 5 of SEQ ID NO:1 ' end the 244th bit base, when promptly 5 of sequence 4 ' end the 545th bit base was A in sequence table, its homozygotic genotype was AA; Their heterozygote genotype is AC.

The Csp6I enzyme is cut described pcr amplification product, if the endonuclease bamhi that obtains is the band of a 595bp, determines that then the genotype of pig to be measured is CC; If the endonuclease bamhi that obtains is two bands of a 353bp and a 242bp, determine that then the genotype of pig to be measured is AA; If the endonuclease bamhi that obtains is the band of a 595bp, three bands of a 353bp and a 242bp determine that then the genotype of pig to be measured is AC.

Wherein, the estimation eye muscle area (cm of CC genotype individuality ²) value is significantly higher than the genotypic respective value of AA, the genotypic respective value of AC is placed in the middle; And the respective value lipid content of AA genotype intramuscular fat content is the highest, the respective value of AC genotype intramuscular fat content secondly, the respective value minimum of CC genotype intramuscular fat content, the respective value of AA genotype intramuscular fat content is significantly higher than the genotypic respective value of CC; The genotypic IgG2 content of AA is the highest, and is significantly higher than the AC genotype; CC genotype respective value is taken second place, and the respective value of heterozygosis AC genotype individuality is minimum, shows that the immune character of AA genotype individuality is better; The preliminary demonstration do not have related with other immune character.

Described single nucleotide polymorphism detects and can adopt dna sequencing, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP), the hybridization of allele specific oligonucleotide oligonucleotide (ASO), the little sequencing according to the DNA array, dynamic allele specific oligonucleotide hybridization, special connection, DNA chip and the TaqMan system etc. of oligonucleotide to detect.

Pig immunity of the present invention, growth traits associated protein and encoding gene thereof can be used for detecting pig IgG 2 content immune characters, for the molecular breeding of pig provides a new genetic marker, will play a significant role in the breeding of pig.

Description of drawings

Fig. 1 is for detecting the schema of pig RPLPO partial dna sequence polymorphism

Fig. 2 is the amplification of the genome partial dna sequence of pig RPLPO gene

Fig. 3 detects the agarose gel electrophoresis detected result of the genome partial dna sequence polymorphism of pig RPLPO gene for the PCR-RFLP method

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment.

The acquisition of embodiment 1, pig RPLPO

1, the acquisition of the cDNA sequence of pig RPLPO gene

Selected clone order-checking from pig embryo skeletal muscle library (making up) by Shanghai associating genome company, then sequencing result is searched for homologous sequence in NCBI, with people RPLPO gene order similarity degree the highest (NM_001002,93%), confirm as the cDNA of pig RPLPO gene.

2, the location of pig RPLPO gene

With pig * rodents somatic cell hybrid clone panel RH clone plate (INRA-Minnesota porcine radiationhybrid panel, ImpRH, 118 hybrid cell systems) DNA in is (available from French Academy of Agricultural Sciences, Laboratoirede G é n é tique Cellulaire, INRA) be template, under the guiding of forward primer: 5 '-AGCAGATCCGCATGTCTCTC-3 ' and reverse primer: 5 '-GCAGGTACAGTGACTTCGCA-3 ', carry out pcr amplification.Amplification condition is: 94 ℃ of sex change 4min, and 94 ℃ of sex change 20s, 59 ℃ of annealing 20s, 72 ℃ are extended 20s, 30 circulations.Extend 5min at 72 ℃ at last.Reaction system wherein consists of: PCR reaction cumulative volume is 10 μ l, and wherein the about 25ng of RH clone plate DNA contains 1 * damping fluid (Promega), 2mmol/L MgCl ₂, the dNTP final concentration is 150 μ mol/L, the primer final concentration is 0.2 μ mol/L, 1U Taq archaeal dna polymerase (Promega).The PCR reaction product is carried out the pcr amplified fragment somatotype with 1.5% agarose gel electrophoresis, wherein the 6th, 8, and 10,12,13,21,22,27,29,49,51,59,61,62,73,74,75,82,86,94,100,101,103,105,107,109,114,117, obtain positive amplification among the DNA during No. 118 hybrid cell is.PCR somatotype data are submitted to HybWeb (http://imprh.toulouse.inra.fr/) carry out statistical study to obtain zone location information, the data analysis result shows that the pig RPLPO assignment of genes gene mapping is on No. 14 karyomit(e)s of pig, with microsatellite marker SW1321 close linkage, the LOD value is 14.54.

The single nucleotide polymorphism of embodiment 2, the genomic partial dna sequence of pig RPLPO gene and RFLP polymorphism detect

The flow process that the single nucleotide polymorphism of the genomic partial dna sequence of pig RPLPO gene and RFLP polymorphism detect as shown in Figure 1, concrete steps are as follows:

One, the detection of the single nucleotide polymorphism of the genomic partial dna sequence of gene of pig RPLPO

Detailed process comprises the steps:

1, design of primers

CDNA sequence (the GenBank number of including: NM_001002) be the information probe of personnel selection RPLPO, utilize the BLAST instrument of NCBI in GenBank pig est database, to do the homologous sequence retrieval, obtain a series of homologys and reach ESTs (fragment length is greater than 100bp) more than 90%, corresponding sequence is inquired about in the number of including of these ESTs in the ENTREZ of NCBI (http://www.ncbi.nlm.nih.gov/Web/Search/index.htmL), use the EST contig of the ASSEMBLY program construction pig among the software GeneTool then and in conjunction with the sub-sequencing result of library clone, according to the sequences Design amplimer of splicing, primer sequence is as follows at last:

Primer 1 (forward): 5 '-TTGTGTTCACCAAGGAGGAC-3 ' (SEQ ID №: 2)

Primer 2 (oppositely): 5 '-GTGATGTCAAGCACTTCAGG-3 ' (SEQ ID №: 3)

2, the purifying of pcr amplification and amplified production thereof, clone and order-checking

Respectively to greatly enhance 23 of logical combinations (Da Bai * length in vain * Tongcheng pig), grow up and lead to combination (in vain long * Da Bai * Tongcheng pig) 26,15 of Large Whites, 15 of landraces, total DNA of the poba gene group that 15 of Du Luoke and Tongcheng pig are 70 is a template, under the guiding of primer 1 and primer 2, pcr amplification pig RPLPO partial dna sequence.PCR reaction cumulative volume is 20 μ l, wherein, and the about 100ng of pig genomic dna, 2 μ l, 10 * PCR damping fluids (Promega), MgCl ₂Final concentration is 1mmol/L, and the dNTP final concentration is 150 μ mol/L, and primer 1 and primer 2 final concentration respectively are 0.2 μ mol/L, 2U Taq archaeal dna polymerase.The PCR response procedures is: 94 ℃ of 4min of elder generation; 94 ℃ of 20s then, 59 ℃ of 20s, 72 ℃ of 20s, 30 circulations; Last 72 ℃ are extended 5min.The PCR reaction product is carried out 1.5% agarose gel electrophoresis, and electrophoresis result shows that the PCR product size of acquisition is about 595bp as shown in Figure 2.Among Fig. 2,2 swimming lanes are pcr amplification product, and the M swimming lane is dna molecular amount mark (100-1000bp ladder).Carry out purifying, clone and the order-checking of PCR product then as follows:

(1) purifying of PCR product: under ultraviolet lamp, contain the segmental gel of purpose from the sepharose cutting-out, put into 1.5ml Ependorff pipe, bathe to gel in 70 ℃ of temperature and to melt fully, use PCR product purification test kit (Promega) purified pcr product then, according to the operation of test kit specification sheets, concrete steps are to add 1ml resin (Resin), mixing 20s in the gel that per 300 μ l melt, with the Resin/DNA mixture syringe of packing into, make slurries pass through microtrabeculae (Minicolumn) and extrude.In syringe, add 80% Virahol 2ml again, touch piston Virahol is extruded by Minicolumn, take off Minicolumn and pack in the 1.5ml Ependorff pipe 10 into, the centrifugal 2min of 000g is with dry Resin, Minicolumn is packed in another clean 1.5ml Ependorff pipe, add 30-50 μ l sterilized water, leave standstill 1min, 10, the centrifugal 20s of 000g abandons supernatant, and eluted dna is stored in the Ependorff pipe.

(2) ligation: the PCR product and the pGEM-T carrier (Promega) of step (1) purifying are used T ₄Dna ligase connects, and obtains recombinant vectors.Linked system is: 2.5 μ L, 2 * damping fluid, 0.5 μ L pGEM-T carrier, the PCR product of 0.5 μ L purifying, 0.5 μ L T ₄Dna ligase, 1 μ L aqua sterilisa, 16 ℃ of water-bath 12-24h.

(3) preparation of competent cell: with bacillus coli DH 5 alpha (TaKaRa) on the LB solid medium 37 ℃ cultivate 16-20h after, picking list colony inoculation is in 2mL LB liquid nutrient medium, 37 ℃ of shaking culture 3h, getting 1mL bacterium liquid then transfers in containing the shaking in the bottle of 30mL LB liquid nutrient medium, continue the about 4h of shaking culture at 37 ℃, treat bacterium liquid OD ₆₀₀Value will shake bottle when reaching 0.3-0.4 and take out from shaking table, ice bath 10-15min makes it cooling, then bacterium liquid is changed in the centrifuge tube in 4 ℃, 4, and the centrifugal 10min of 000g collects somatic cells, centrifuge tube is inverted to abandon clean nutrient solution, is iced the CaCl of the 0.1mol/L of precooling with 10mL ₂The resuspended precipitation of solution, i.e. somatic cells, behind the ice bath 30min, 4 ℃ 4, the centrifugal 10min of 000g is again with the CaCl of the 0.1mol/L of 4mL ice precooling ₂The resuspended precipitation of solution, it is standby to put 4 ℃ of preservations.

(4) transform: the competent cell of getting 100-120 μ l step (3) preparation under the sterile state is in the 1.5mLEpendorff pipe, add the connection product that 5 μ L steps (2) obtain again, mixing, ice bath 30min, 42 ℃ of heat shock 90s (not shaking the Ependorff pipe in the above-mentioned conversion process), take out back ice bath 3-4min, add 400 μ l again and do not contain antibiotic LB liquid nutrient medium, at 37 ℃ of shaking culture 45min, get 100 μ L bacterium liquid at last and coat the 200mg/mLIPTG (Isopropylthio-β-D-galactoside that contains 4 μ L, isopropylthio-) and on the LB flat board of the 20mg/mL X-gal of 40 μ L, is inverted under the same conditions behind 37 ℃ of pre-1h of cultivation earlier and cultivates 12-24h;

(5) a small amount of of plasmid preparation: the single colony inoculation that grows on the picking agar plate is in 2-3mL LB liquid nutrient medium, and 37 ℃ of 300r/min cultivate 12-24h, collects thalline with the centrifugal several seconds of 1.5mL EP pipe 12000r/min again.Every pipe adds the ice-cold solution I of 100 μ l (50mM glucose, 25mM Tris-HCl (pH8.0), 10mMEDTA (pH8.0)), and vortex vibrates to thalline and fully suspends.Add new preparation solution II (0.2M NaOH, 1%SDS) 200 μ l put upside down mixing fast, ice bath 5min adds solution III (5M potassium acetate, glacial acetic acid 11.5ml, the H of precooling then ₂O 28.5ml) 150 μ l, ice bath 5min behind the mixing, the centrifugal 5min of 12000r/min, supernatant is gone in another EP pipe, add phenol: chloroform: primary isoamyl alcohol 500 μ l, vortex vibration, the careful upper strata water of drawing in centrifugal back, the dehydrated alcohol that adds 2 times of volumes,-20 ℃ of precipitation 30min, the centrifugal 5min of 12000r/min, precipitation is with 70% washing with alcohol 2 times, drain, add the TE 20 μ l that contain the RNA enzyme.

(6) enzyme of recombinant plasmid is cut evaluation: get 3 μ l plasmid DNA and distilled water mixing, making its cumulative volume is 15 μ l, add restricted endoenzyme EcoRI of 2-3U and the corresponding 10 * restriction enzyme reaction damping fluid of 2 μ l, flick tube wall mixing and centrifugal, put 37 ℃ of water-bath 1-2 hours, get 2-3 μ l reaction solution and detect, obtain enzyme and cut the result and estimate identical purpose recombinant plasmid in agarose gel electrophoresis.

(7) order-checking: recombinant plasmid adopts the terminal cessation method of two deoxidations to check order on automatic dna sequencer, and sequencing is finished by Shanghai Bo Ya Bioisystech Co., Ltd.Sequencing result shows that the length of this PCR product is 595bp, nucleotide sequence with SEQ ID NO:1 in the sequence table, wherein, the 1-56 bit base from 5 ' end is the partial sequence (5 of sequence 4 ' end 439-494 bit base in sequence table) of the 4th exon of RPLPO genomic gene; 57-193 bit base from 5 ' end is the 4th an intron complete sequence of this genomic gene; 194-340 bit base from 5 ' end is the complete sequence (5 of sequence 4 ' end 495-641 bit base in sequence table) of the 5th exon of RPLPO genomic gene; 341-446 bit base from 5 ' end is the complete sequence of the 5th intron of RPLPO genomic gene; 447-595 bit base from 5 ' end is the partial sequence (5 of sequence 4 ' end 642-790 bit base in sequence table) of the 6th exon of RPLPO genomic gene.Locate to exist two allelotrope of C, A from 5 of SEQ ID NO:1 ' end the 244th bit base (5 of sequence 5 ' end the 545th bit base in sequence table), be pleomorphism site.As when being C, its homozygotic genotype is CC from 5 of SEQ ID NO:1 ' end the 244th bit base (5 of sequence 4 ' end the 545th bit base in sequence table); When being A, its homozygotic genotype is AA from 5 of SEQ ID NO:1 ' end the 244th bit base (5 of sequence 4 ' end the 545th bit base in sequence table); Their heterozygote genotype is AC.

3, the dna sequence dna homology search is identified

By the American National biotechnology (NCBI of information center, National Center for BiotechnologyInformation, http://www.ncbi.nlm.nih.gov) BLAST of website (Basic Local AlignmentSearch Tool) software, the known physiological function gene of announcing in the dna sequence dna (SEQ ID NO:1) that order-checking back is obtained and the GenBank database carries out sequence homology relatively, to identify and to obtain the function information of this dna sequence dna, the result shows that pig and people's sequence similarity is 83%, the ribosomal protein RPLPO of this sequence encoding pig.

Two, the detection of the RFLP polymorphism of the genomic partial dna sequence of pig RPLPO gene

1, the genomic partial dna sequence Csp6I-RFLP of pig RPLPO gene polymorphism detects: respectively to greatly enhance 23 of logical combinations (Da Bai * length in vain * Tongcheng pig), grow up and lead to combination (in vain long * Da Bai * Tongcheng pig) 26,15 of Large Whites, 15 of landraces, total DNA of the poba gene group that 15 of Du Luoke and Tongcheng pig are 70 is a template, carries out pcr amplification under the guiding of primer 1:5 '-TTGTGTTCACCAAGGAGGAC-3 ' and primer 2: 5 '-GTGATGTCAAGCACTTCAGG-3 '.PCR reaction cumulative volume is 20 μ l, wherein, and the about 100ng of pig genomic dna, 2 μ l10 * PCR damping fluid (Promega), MgCl ₂Final concentration is 1mmol/L, and the dNTP final concentration is 150 μ mol/L, and primer 1 and primer 2 final concentration respectively are 0.2 μ mol/L, 2U Taq archaeal dna polymerase.The PCR reaction conditions is: 94 ℃ of 4min of elder generation; 94 ℃ of 20s then, 60 ℃ of 20s, 72 ℃ of 20s, 30 circulations; Last 72 ℃ are extended 5min.Then the PCR reaction product is detected with 1.5% agarose gel electrophoresis, the result has obtained the electrophoretic band of 595bp size.With PCR product Csp6I digestion with restriction enzyme, endonuclease reaction system (10 μ l): 1 * enzyme cutting buffering liquid, 1 μ l, PCR product 3-5 μ l, restriction enzyme 0.5 μ l (5U) uses H ₂O supplies 10 μ l.The enzyme tangent condition is: 37 ℃ of water-bath 4h.Detecting enzyme with 2% agarose gel electrophoresis cuts the result and writes down genotype, the result is as shown in Figure 3 (among Fig. 3, swimming lane M is Marker, swimming lane AA is that genotype is the homozygote of AA, swimming lane CC is that genotype is the homozygote of CC, swimming lane AC is that genotype is the heterozygote of AC), show in the CC genotype individuality, have to the band of a 595bp; In the AC genotype individuality, obtain three bands: a 595bp, a 353bp and a 242bp band; In the AA genotype individuality, obtain two bands: a 353bp and a 242bp band.When allelotrope is A, recognition site (the G ↓ TAC) that occurs restriction enzyme Csp6I from 5 of SEQ ID NO:1 ' end 242-245 bit base, if cut this fragment with the Csp6I enzyme, then fragment of 595bp only appears in C allelotrope, 353bp and two fragments of 242bp then can appear in A allelotrope, owing to this locus is controlled by above-mentioned two allelotrope, thereby can form CC, AC, three kinds of genotype of AA.

Embodiment 3, the immunity of detection pig, growth traits

Respectively with 15 of Large Whites, 15 of landraces, total DNA of the poba gene group that 15 of Du Luoke and Tongcheng pig are 36 is a template, and totally 81 individualities are experimental subjects, detects the single nucleotide polymorphism and the RFLP polymorphism of 81 individualities according to the method for embodiment 2, determine their genotype, its result is as shown in table 1, shows in the individuality of 4 pig varieties that detected, and the genotypic number of individuals of CC is 14, the genotypic number of individuals of AC is 52, and the genotypic number of individuals of AA is 15.A in China Tongcheng pig, the frequency of two genes of C differs not remarkable, and the A gene frequency is high slightly; In 3 foreign pig varieties, the C gene frequency of Large White and landrace is apparently higher than the analog value of A gene, and the C gene frequency of Du Luoke is starkly lower than the analog value of A gene.

The distribution of each genotype of table 1 in 4 pig varieties

2, mark property association analysis

Method according to embodiment 2 detects following 83 individualities: greatly enhance 23 pigs of logical combination (Da Bai * (in vain long * Tongcheng)), 26 pigs of logical combination (in vain long * (Da Bai * Tongcheng)) grow up, single nucleotide polymorphism and the RFLP polymorphism of 34 pigs of Tongcheng pure breeding group, determine their genotype, and carry out genotype and production, immune character association analysis.

With following least square model analysis immune character (content of hemoglobin, pcv, mean corpuscular volume (MCV), blood IgG antibody content, NCHC, average content of hemoglobin, total white blood cells and red corpuscle sum) and the production traits (nascent end body weight (90KG) age in days that reaches, dressing percentage, estimation eye muscle area (cm ²), leg stern ratio, leaf fat rate, intramuscular fat, shearing rate, pH value, average daily gain, the thickness of backfat, yellowish pink, marble grain, percentage of water loss and drip loss):

y _ijk＝μ+GENOTYPE _i+SEX _j+COMBINATION _k+ε _ijk，

Wherein, y _IjkBe the character observation value, μ is a population mean, GENOTYPE _iBe genotype effect, SEX _jBe sex effect, COMBINATION _kBe the effect of various combination, ε _IjkBe random error, suppose obey N (0, σ ²) distribute.

The result of the proterties significant difference of proterties between the different genotype (least square mean and standard error analysis) is as shown in table 2, and other proterties does not have significant difference between different genotype.

Table 2 different genotype and part growth traits, the association analysis of immune character

Genotype	Estimation eye muscle area (cm 2)	Intramuscular fat content (%)	IgG2(mg/ml)
				AA AC CC	23.053±2.459 27.894±1.068 31.834±2.940	4.160±0.456 3.341±0.198 2.728±0.545	66.791±3.602 57.996±1.644 58.594±4.650
AA/AC AA/CC AC/CC	0.075 0.025 * 0.212	0.104 0.047 * 0.294	0.030 * 0.168 0.904

Annotate: * * represents that difference is extremely remarkable, and * represents significant difference.

Table 2 shows, the estimation eye muscle area (cm of CC genotype individuality ²) value is significantly higher than the genotypic respective value of AA, the genotypic respective value of AC is placed in the middle; And the respective value of AA genotype intramuscular fat content is the highest, the respective value of AC genotype intramuscular fat content secondly, the respective value minimum of CC genotype intramuscular fat content, the respective value of AA genotype intramuscular fat content is significantly higher than the genotypic respective value of CC; Therefore, can illustrate that the individual lean meat productivity ratio of CC genotype is higher, and the intramuscular fat content of AA genotype individuality is than higher, meat flavor is good.The genotypic IgG2 content of AA is the highest, and is significantly higher than the AC genotype; CC genotype respective value is taken second place, and the respective value of heterozygosis AC genotype individuality is minimum, shows that the immune character of AA genotype individuality is better.

Sequence table

<160>5

<210>1

<211>595

<212>DNA

＜213〉wild boar be born in the year of pig (Sus scrofa domestica Brisson)

<220>

<221>misc-feature

<222>(244)

＜223〉n=a or c

<400>1

ttgtgttcac caaggaggac ctcactgaga tcagggacat gctgctggcc aataaggtga 60

ggggagaatc agcttggctg aagaggcctt atcattaatt ttggctcctt aaaagcaaga 120

aaaaaagaag acaatctttt tcaggttgta gctgtggaaa gaagttttct cattgtttgt 180

cctttttgtt caggtgccag ctgccgcccg tgctggtgcc atagccccat gcgaagtcac 240

tgtncctgcc cagaacactg gtctggggcc tgagaagacc tccttcttcc aggctttagg 300

catcaccact aaaatctcca ggggcaccat tgaaatcctg gtgagtgggc ctggcttggc 360

ctgtgccagc caggcaggtg gcgggggctt ggttgctgtg gacctgctgg atgtctgggt 420

taactgtcac ctaccttgtt tttcagagtg atgtgcagct gattaagact ggagacaaag 480

tgggagccag tgaagccacg ttgctgaaca tgttgaacat ctcccccttc tcctttgggc 540

tgatcatcca gcaggtgttt gacaatggca gcatctacaa ccctgaagtg cttga 595

<210>2

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>2

ttgtgttcac caaggaggac 20

<210>3

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>3

gtgatgtcaagcacttcagg 20

<210>4

<211>1293

<212>DNA

＜213〉wild boar be born in the year of pig (Sus scrofa domestica Brisson)

<220>

<221>misc-feature

<222>(545)

＜223〉n=a or c

<400>4

gtacgactca ctatagggcg aattgggtac cgggcccccc ctcgaggtcg acggtatcga 60

taagcttgat atcgaattcg tatcggccat tacggcctac tggctctcgc caggcgtcct 120

cgtgtgagtg acatcgtctt taaaccctgc gtggcaatcc ctgacgcacc gccgtgatgc 180

ccagggaaga cagggcgacc tggaagtcca actacttcct taagataatc caacttctgg 240

atgattatcc gaaatgcttc attgtgggag cagacaatgt gggctccaag cagatgcagc 300

agatccgcat gtctctccgc gggaaagccg tggtgctgat gggcaagaac accatgatgc 360

gcaaggccat ccgagggcac ctggaaaaca acccagccct ggagaaactg ttgcctcaca 420

tccgggggaa cgtgggcttt gtgttcacca aggaggacct cactgagatc agggacatgc 480

tgctggccaa taaggtgcca gctgccgccc gtgctggtgc catagcccca tgcgaagtca 540

ctgtncctgc ccagaacact ggtctggggc ctgagaagac ctccttcttc caggctttag 600

gcatcaccac taaaatctcc aggggcacca ttgaaatcct gagtgatgtg cagctgatta 660

agactggaga caaagtggga gccagtgaag ccacgttgct gaacatgttg aacatctccc 720

ccttctcctt tgggctgatc atccagcagg tgtttgacaa tggcagcatc tacaaccctg 780

aagtgcttga catcaccgag gaaactctgc attctcgctt cctggagggt gtccgcaatg 840

ttgccagcgt atgtctgcag attggttacc caactgttgc atctgtaccc cattctatca 900

tcaatgggta caagcgggtc ctggctttgt ctgtggaaac tgattacacc ttcccacttg 960

ctgaaaaggt caaggccttc ttggctgatc catctgcctt tgtggctgct gcccctgtgg 1020

ctgcagccac cactgctgct cctgctgctg ctgctgcagc cccagccaag gttgaagcaa 1080

aggaggagtc ggaggagtcg gacgaggata tgggatttgg tctctttgac taaattacca 1140

aaaagcaacc aagtcagcca gctttatttg tgaaacaaag aaataagggc ttactcctaa 1200

aaaaaaaaaa aaaaaaaaaa aaaaaaacat gtcggccgcc tcggcctatg tgcggccgcc 1260

accgcggtgg agctccagct tttgttccct tta 1293

<210>5

<211>318

<212>PRT

＜213〉wild boar be born in the year of pig (Sus scrofa domestica Brisson)

<400>5

Met Pro Arg Glu Asp Arg Ala Thr Trp Lys Ser Asn Tyr Phe Leu Lys

1 5 10 15

Ile Ile Gln Leu Leu Asp Asp Tyr Pro Lys Cys Phe Ile Val Gly Ala

20 25 30

Asp Asn Val Gly Ser Lys Gln Met Gln Gln Ile Arg Met Ser Leu Arg

35 40 45

Gly Lys Ala Val Val Leu Met Gly Lys Asn Thr Met Met Arg Lys Ala

50 55 60

Ile Arg Gly His Leu Glu Asn Asn Pro Ala Leu Glu Lys Leu Leu Pro

65 70 75 80

His Ile Arg Gly Asn Val Gly Phe Val Phe Thr Lys Glu Asp Leu Thr

85 90 95

Glu Ile Arg Asp Met Leu Leu Ala Asn Lys Val Pro Ala Ala Ala Arg

100 105 110

Ala Gly Ala Ile Ala Pro Cys Glu Val Thr Val Pro Ala Gln Asn Thr

115 120 125

Gly Leu Gly Pro Glu Lys Thr Ser Phe Phe Gln Ala Leu Gly Ile Thr

130 135 140

Thr Lys Ile Ser Arg Gly Thr Ile Glu Ile Leu Ser Asp Val Gln Leu

145 150 155 160

Ile Lys Thr Gly Asp Lys Val Gly Ala Ser Glu Ala Thr Leu Leu Asn

165 170 175

Met Leu Asn Ile Ser Pro Phe Ser Phe Gly Leu Ile Ile Gln Gln Val

180 185 190

Phe Asp Asn Gly Ser Ile Tyr Asn Pro Glu Val Leu Asp Ile Thr Glu

195 200 205

Glu Thr Leu His Ser Arg Phe Leu Glu Gly Val Arg Asn Val Ala Ser

210 215 220

Val Cys Leu Gln Ile Gly Tyr Pro Thr Val Ala Ser Val Pro His Ser

225 230 235 240

Ile Ile Asn Gly Tyr Lys Arg Val Leu Ala Leu Ser Val Glu Thr Asp

245 250 255

Tyr Thr Phe Pro Leu Ala Glu Lys Val Lys Ala Phe Leu Ala Asp Pro

260 265 270

Ser Ala Phe Val Ala Ala Ala Pro Val Ala Ala Ala Thr Thr Ala Ala

275 280 285

Pro Ala Ala Ala Ala Ala Ala Pro Ala Lys Val Glu Ala Lys Glu Glu

290 295 300

Ser Glu Glu Ser Asp Glu Asp Met Gly Phe Gly Leu Phe Asp

305 310 315

Claims (8)

1. a boar immunity, growth traits associated protein, its amino acid residue sequence is shown in SEQ ID NO:5.

2. the encoding gene of the described pig immunity of claim 1, growth traits associated protein.

3. encoding gene according to claim 2 is characterized in that: the nucleotide sequence of described encoding gene is shown in SEQ ID NO:4.

4. the fragment of the described encoding gene of claim 2, it is characterized in that: described segmental nucleotide sequence is shown in SEQ ID NO:1.

5. contain claim 2 or 3 described encoding genes or the described segmental carrier of claim 4.

6. contain claim 2 or 3 described encoding genes or the described segmental clone of claim 4.

7. contain the described segmental host bacterium of claim 2 or 3 described encoding genes or claim 4.

8. method that detects pig immunity, growth traits, be to use a pair of primer formed by the nucleotide sequence of SEQ ID NO:2 in the sequence table and SEQ IDNO:3 that the genomic dna of pig to be measured is carried out pcr amplification, then pcr amplification product carried out following at least a detection:

1) described pcr amplification product is carried out single nucleotide polymorphism and detect, determine that from 5 of SEQ ID NO:1 ' end the 244th bit base be C or A; If when 5 of SEQ ID NO:1 ' end the 244th bit base was C, its homozygotic genotype was CC, when 5 of SEQ ID NO:1 ' end the 244th bit base was A, its homozygotic genotype was AA; Their heterozygote genotype is AC;

2) cut described pcr amplification product with the Csp6I enzyme, detect in the endonuclease bamhi that obtains and contain a 595bp band, a 353bp band and a 242bp band, still a 595bp band, a 353bp band and a 242bp band; If the endonuclease bamhi that obtains is the band of a 595bp, determine that then the genotype of pig to be measured is CC; If the endonuclease bamhi that obtains is two bands of a 353bp and a 242bp, determine that then the genotype of pig to be measured is AA; If the endonuclease bamhi that obtains is the band of a 595bp, a 353bp and a 242bp band, determine that then the genotype of pig to be measured is AC.

Images