CN101570789B - Identification and application of pig MHC II TA gene as immunity related molecular labels - Google Patents
Identification and application of pig MHC II TA gene as immunity related molecular labels Download PDFInfo
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- CN101570789B CN101570789B CN2009100626226A CN200910062622A CN101570789B CN 101570789 B CN101570789 B CN 101570789B CN 2009100626226 A CN2009100626226 A CN 2009100626226A CN 200910062622 A CN200910062622 A CN 200910062622A CN 101570789 B CN101570789 B CN 101570789B
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Abstract
The invention belongs to the technical field of livestock molecular label preparation, in particular relates to molecular label preparation and application as auxiliary selection and related to pig immunity property. The molecular label takes mRNA sequence of a pig MHCIITA gene as a template design primer to carry out PCR amplification, PCR product purification and sequencing so as to acquire a nucleic acid shown as a sequence table SEQ ID NO:2; 9 single-chain nucleic acid polymorphic sites (SNP) are found in the nucleic acid sequence shown in the SEQ ID NO:2, and a PCR-PvuII-RFLP method is developed to detect the molecular label on the 188th bp of the nucleic acid sequence, namely base mutation C188-T188. The invention discloses the sequence amplifying a partial code region and a non-translational region of the pig MHCIITA gene, the primer used in PCR amplification and a detection method for the molecular label.
Description
Technical field
The invention belongs to domestic animal molecule marker preparing technical field, be specifically related to evaluation and the application of a boar MHCIITA gene as the immunity related molecular mark.
Background technology
Pork is the topmost animal protein food source of China resident, and tame pig occupies critical role in livestock industry.Recent decades, tame swine improvement achieves noticeable achievement, but what be primarily aimed at is the production traits, i.e. growth traits and reproductive trait etc.With respect to the production traits, but comparatively weak at health and the resist the disease Research on ability of pig.With high bacon hogs is that the breeding in modern times of target reduces relevant allelic loss or frequency to cause the physique of modern commerce kind and resistance against diseases sharply to descend.Along with popularizing of intensive farm mode, the swine disease particularly transmissible disease of some respiratory systems and propagating system is given to raise pigs to produce and has been brought enormous economic loss, disease has become one of biggest threat of modern pig industry, the sound development of the Chinese pig industry of serious restriction.The good news is that increasing herding worker comes to realise and improves swinery health to keep the importance of stably manufactured.
Face the raising of the production traits in the present Swine Production and the contradiction that disease resistance descends, only rely on medicine and vaccine both to strengthen the cost of raising pigs, come hidden danger for again the food securing band, therefore, this need us to seek more effective means improves resistance against diseases and the health level of swinery.In long-term observation, it is found that domestic animal, the animal that is not all pathogenic infection microorganisms all falls ill, and existing studies show that the part pig has natural resistance to the enterotoxigenic Escherichia coli K88 that causes grice diarrhoea, and these pigs to the opposing of disease be subjected to genetically controlled.Hence one can see that, and the generation of animal infectious disease and the genetic background of animal itself are closely-related.In the long run/term, the employing genetic method improves swinery health level and resistance against diseases is the strategy that effects a permanent cure, and screening is the basic and crucial of this work with evaluation pig immunity genes involved and molecule marker.Therefore, seek the immune disease-resistance gene and carry out breeding, improving the congenital resistance against diseases of swinery is the trend of the times of current development pig breeding work.The MHCIITA gene is exactly one and body disease-resistant and the closely-related key gene of immunizing power.
MHCIITA (MHC class II transactivator) is a molecular switch of regulating major histocompatibility complex II quasi-molecule (MHCII).(Major Histocompatibility Complex MHC) is and disease resistance and the closely-related one group of gene group of immunne response major histocompatibility complex.The MHC of pig (called after SLA) is positioned on No. 7 karyomit(e)s of pig, and two types of I and II are arranged.MHC II quasi-molecule is mainly brought into play antigen presentation in vivo, plays the role of a nucleus in bringing out the acquired immune response, and the unconventionality expression of mhc class ii molecule can cause serious immune deficiency disorder usually, as lymphocyte syndrome (BLS) etc.Consistent with MHC II quasi-molecule, only antigen presenting cell can constructive expression MHCIITA, but other various cells can abduction delivering MHCIITA.
MHCIITA plays a significant role in the immune response relevant with the mhc class ii molecule.There are some researches show that pathogenic agent escapes immune approach and relate to the adjusting that MHCIITA is expressed.By changing the expression that IFN-γ signal transduction pathway is reduced MHCIITA, reduced the immunostimulation of mhc class ii such as cytomegalovirus like this.When mouse macrophage was subjected to Mycobacterium bovis BCG infection, its gene product can suppress the phosphorylation of the STAT-1 of IFN-γ mediation, caused the expression of MHCIITA and mhc class ii molecule to reduce.Chlamydozoan has disturbed the MHCIITA gene transcription by making upstream stimulating factor 1 sex change.Therefore, the MHCIITA gene plays a significant role in body is resisted the process of various cause of diseases.
So far, about the research of pig MHCIITA gene seldom, and not about the correlative study report of its genetic polymorphism.Therefore, between the vital role that MHCIITA brings into play in the body disease-resistant immune response, the applicant has selected this gene and it has been carried out correlative study and immune character association analysis.The applicant passes through the partial sequence of clone MHCIITA, and utilizes software that sequencing result is analyzed, and identifies 9 SNP sites in this gene 3 ' UTR district.Choose the C188-T188 base mutation site that wherein is positioned at 188bp, it has been done association analysis with immune character, simultaneously, further detected this sudden change genetic diversity in 6 pig blood relationships at home and abroad; Thereby, successfully identified a new molecule marker relevant with pig immunity.
Summary of the invention
The objective of the invention is to clone a boar MHCIITA gene as the immunity related molecular mark, and be applied to the pig molecule mark assistant breeding, in the hope of obtaining higher society and economic benefit.
The present invention is achieved through the following technical solutions:
The applicant clone obtains a kind of molecule marker relevant with pig immunity, and it is the nucleotide sequence shown in the sequence table SEQ ID NO:2.Find the base mutation of 1 C188-T188 at the 188bp place of sequence table SEQ ID NO:2, cause the PvuII-RFLP polymorphism.
Wherein, right as follows of the coding region of clone pig MHCIITA gene (CDS) primer with nucleotide sequence:
Forward primer: 5 '-TGGTGACTGGCACTTTCCT-3 ', reverse primer: 5 '-TGAGCATTGGGTGGGG-3 '.
3 ' non-translational region of clone pig MHCIITA gene (the right nucleotide sequence of 3 ' UTR) primers is as follows:
Forward primer: 5 '-GTTCTCTGAGGACGCTGA-3 ', reverse primer: 5 '-TTTTGAAGACTGAATAAGCC-3 '.
It is as follows that PCR-PvuII-RFLP detects the right nucleotide sequence of the used primer of the described molecule marker of SEQ ID NO:2 (promptly detecting base mutation):
Forward primer: 5 '-GTTCTCTGAGGACGCTGA-3 ', reverse primer: 5 '-CCGTAAGGTCTGGCGTGTCT-3 '.
The applicant has set up a kind of preparation molecular marker method relevant with pig immunity with detection, and its step is as follows:
With pig MHCIITA gene mRNA sequence is template design primer, pcr amplification, and PCR product purification and cloning and sequencing obtain the nucleotide sequence shown in sequence table SEQ ID NO:2, use the polymorphism that the PCR-PvuII-RFLP method detects pig MHCIITA gene.
More detailed technical scheme is referring to " embodiment ".
Description of drawings
Fig. 1: be techniqueflow chart of the present invention.
Fig. 2: be the electrophoretogram of pig MHCIITA gene coding region (CDS) partial sequence amplification among the present invention, clip size is 455bp (agarose gel concentration is 2%).Among the figure: the Marker swimming lane is a dna molecular amount standard (DL2000).
Fig. 3: the pig MHCIITA Gene Partial CDS sequence (being the information shown in the sequence table SEQ ID NO:1) that cloning and sequencing of the present invention obtains.
Fig. 4: be the electrophoretogram of pig MHCIITA gene 3 ' UTR partial sequence amplification among the present invention, clip size is 719bp (agarose gel concentration is 2%).Among the figure: the Marker swimming lane is a dna molecular amount standard (DL2000).
Fig. 5: MHCIITA gene 3 ' UTR partial sequence of the different pig kinds that cloning and sequencing of the present invention obtains (belonging to place of china pig and external pig kind respectively).Wherein Fig. 5 A, Fig. 5 B, Fig. 5 C, Fig. 5 D are followed successively by the sequence (corresponding respectively to the sequence shown in the sequence table SEQ IDNO:2-5) of landrace (in the external pig), painted face in Beijing opera (place of china pig), Large White (external pig kind) and peaceful pig (place of china pig).
Fig. 6: be 9 mutational sites finding after the pig MHCIITA gene sequencing, recording sequence with Fig. 5 A is reference sequences (being numbered SEQ ID NO:2 in sequence table), from left to right is successively: A33-G33, A62-T62, C188-T188, G271-A271, A543-G543, G565-T565, A581-C581, C587-T587 and A632-G632.
Fig. 7: A is the electrophoretogram that is used to detect the amplification of pig MHCIITA gene C 188-T188 polymorphic site among the present invention, and clip size is 589bp (agarose gel concentration is 2%).Among the figure: the Marker swimming lane is a dna molecular amount standard (DL2000).B is three kinds of genotype (CC, CT and the TT) electrophoretogram of pig MHCIITA gene PCR-PvuII-RFLP among the present invention.C is the electrophoretogram of a family (parent and the filial generation) genotype (CC, CT and TT) of pig MHCIITA gene PCR-PvuII-RFLP among the present invention.
Embodiment
Embodiment 1:
One, the clone in MHCIITA gene C DS district
1. design of primers
Utilize primer-design software Primer 5.0, the MHCIITA gene mRNA sequence (Genebank accession number AY084053.1) that goes up pig with NCBI is the masterplate design of amplification primers.It is synthetic that primer is given birth to worker's biotechnology company limited by Shanghai, and the right nucleotide sequence of synthetic primer is as follows:
Forward primer: 5 '-TGGTGACTGGCACTTTCCT-3 ',
Reverse primer: 5 '-TGAGCATTGGGTGGGG-3 '.
2.PCR the purifying of product and order-checking
(1) pcr amplification
PCR reaction cumulative volume is 10 μ L, and wherein template DNA is 50ng, contains 10 * buffer and (contains Mg2+, Takara), the dNTP final concentration is 75 μ mol/L, and the primer final concentration is 0.3 μ mol/L, 1U Taq archaeal dna polymerase (Takara), insufficient section is supplied with distilled water.
The pcr amplification program is: 95 ℃ of pre-sex change 5min, and 94 ℃ of sex change 30s, 61 ℃ of annealing 30s, 72 ℃ are extended 25s, 35 circulations, last 72 ℃ are extended 5min.The PCR reaction product detects with 2% agarose gel electrophoresis, the (see figure 2) of taking pictures under ultraviolet lamp.
(2) purifying of PCR product and order-checking:
The quick glue of the Type B miniprep dna fragment of utilizing vast Tyke, Beijing biological gene technology company to produce reclaims test kit and carries out the purifying recovery, concrete grammar is with reference to the process specifications of this test kit, step is as follows: collect enough PCR products and carry out 1% agarose gel electrophoresis, downcut the purpose band, blob of viscose is as far as possible little; Add the ratio of 700 μ l sol solutionses in 100 μ l blob of viscoses, in 75 ℃ baking oven, gel is dissolved fully; The 3S post of after dissolving, packing into, centrifugal 1 minute of 12000rpm removes waste liquid; 500 μ l rinsing liquid rinsings, repeat rinsing once at 12000rpm centrifugal 30 seconds.After outwelling waste liquid, centrifugal 1 minute again in 12000rpm; The centrifugal 1 minute wash-out of elution buffer (the mentioned reagent box carries) 12000rmp that in the 3S post, adds 30 μ l.The purpose fragment that purifying reclaims just in the liquid of wash-out gained, send Beijing AudioCodes biotech firm directly to check order portion of product.
(3) the dna sequence dna homology search is identified
By NCBI-BLAST (Basic Local Alignment Search Tool) instrument, the reference sequences of announcing in the sequence that order-checking back is obtained and the GenBank database is compared, and the result shows and successfully clones the part fragment (see figure 3) that obtains pig MHCIITA gene C DS.
Two, MHCIITA gene SNP scanning
1. design of primers
Utilize primer-design software Primer 5.0, it is right to be with the MHCIITA gene mRNA (Genebank accession number AY084053.1) of pig that masterplate is designed the pcr amplification primer that is used for polymorphic site scanning.Primer is synthetic by Beijing AudioCodes biotech company, and the right nucleotide sequence of described primer is as follows:
Forward primer: 5 '-GTTCTCTGAGGACGCTGA-3 ',
Reverse primer: 5 ' TTTTGAAGACTGAATAAGCC-3 '.
2.PCR the purifying of product and order-checking
(1) pcr amplification
4 pig kinds of pcr amplification DNA is respectively plum mountain pig and peaceful pig (place of china pig blood relationship), Large White and landrace (external pig kind blood relationship, down together).
Add dna profiling 0.5 μ L in the reaction system of 10uL, distilled water 4.1 μ L, each 0.2 μ L before and after Mix (buffer, dNTP and the Taq enzyme mixture) 5ul, 10mM primer.The PCR reaction conditions is: behind 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 30s, 54.5 ℃ of annealing 40s, 72 ℃ of extension 30s, 35 circulations, last 72 ℃ are extended 5min.
The PCR product detects through 2% agarose gel electrophoresis, and amplification has obtained 719bp specific amplification fragment (see figure 4), is used for reclaiming order-checking.
(2) purifying of PCR product and order-checking:
The quick glue of the Type B miniprep dna fragment of utilizing vast Tyke, Beijing biological gene technology company to produce reclaims test kit and carries out the purifying recovery, concrete grammar is with reference to the process specifications of this test kit, step is as follows: collect enough PCR products and carry out 1% agarose gel electrophoresis, downcut the purpose band, blob of viscose is as far as possible little; Add the ratio of 700 μ l sol solutionses in 100 μ l blob of viscoses, in 75 ℃ baking oven, gel is dissolved fully; The 3S post of after dissolving, packing into, centrifugal 1 minute of 12000rpm removes waste liquid; 500 μ l rinsing liquid rinsings, repeat rinsing once at 12000rpm centrifugal 30 seconds.After outwelling waste liquid, centrifugal 1 minute again in 12000rpm; The centrifugal 1 minute wash-out of elution buffer (this test kit carries) 12000rmp that in the 3S post, adds 30 μ l.The purpose fragment that purifying reclaims is just in the liquid of wash-out gained; Send Beijing AudioCodes biotech firm directly to check order portion of product.
(3) the determining of mutational site in the dna sequence dna
By NCBI-BLAST (Basic Local Alignment Search Tool) instrument, the reference sequences of announcing in sequence that order-checking back is obtained and the GenBank database is compared, and the result shows 4 cloning and sequencings in kind (see figure 5) of all succeeing.
The order-checking peak figure of 4 product boars is imported SeqMan software to compare, the result shows that sequencing quality is very high, there have 9 sites to occur at pig MHCIITA gene 3 ' UTR to be significantly bimodal, and this illustrates that this gene in these 9 site the base mutation (see figure 6) has taken place.These 9 sites are respectively: the base mutation that 1 A33-G33 is arranged at the 33bp place of sequence table SEQ ID NO:2; The base mutation that 1 A62-T62 is arranged at the 62bp place of sequence table SEQ ID NO:2; The base mutation that 1 C188-T188 is arranged at the 188bp place of sequence table SEQ ID NO:2; The base mutation that 1 G271-A271 is arranged at the 271bp place of sequence table SEQ ID NO:2; The base mutation that 1 A543-G543 is arranged at the 543bp place of sequence table SEQ ID NO:2; The base mutation that 1 G565-T565 is arranged at the 565bp place of sequence table SEQ ID NO:2; The base mutation that 1 A581-C581 is arranged at the 581bp place of sequence table SEQ ID NO:2; The base mutation that 1 C587-T587 is arranged at the 587bp place of sequence table SEQ ID NO:2; The base mutation that 1 A632-G632 is arranged at the 632bp place of sequence table SEQ ID NO:2.Wherein, the base mutation at the C188-T188 of 188bp place of sequence table SEQ ID NO:2 causes the PCR-PvuII-RFLP polymorphism.
Three, the foundation of PCR-RFLP genotype detection method
1. design of primers
According to the result of cloning and sequencing, the applicant utilizes the increase primer of this polymorphic site of Primer 5.0 software designs near the sequence at MHCIITA gene 188bp place, and its dna sequence dna is as follows:
Forward primer: 5 '-GTTCTCTGAGGACGCTGA-3 ',
Reverse primer: 5 '-CCGTAAGGTCTGGCGTGTCT-3 '.
2.PCR amplification
Add dna profiling 0.5 μ L in the reaction system of 10uL, distilled water 4.1 μ L, each 0.2 μ L before and after Mix (buffer, dNTP and the Taq enzyme mixture) 5ul, 10mM primer.The PCR reaction conditions is: behind 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ of extension 30s, 35 circulations, last 72 ℃ are extended 5min.2 μ L PCR products detect through 2% agarose gel electrophoresis, and positive is used for next step enzyme and cuts detection, the results are shown in Figure 7-A.
3.PCR-PvuII-RFLP detection molecules mark C188-T188
With PCR product 5 μ L, restriction enzyme PvuII is 0.25 μ L (2U), adds distilled water and mends to 10 μ L, centrifugal behind the sample mixing, 37 ℃ of incubators are placed 10h, then, detect enzyme with 2% agarose gel electrophoresis and cut the result and take pictures under ultraviolet lamp, the record genotype the results are shown in Figure 7-B.Simultaneously, the applicant has also made a family gene type and has differentiated collection of illustrative plates, its objective is for further confirmatory experiment result's reliability, and the results are shown in Figure 7-C, genotype detection result meets mendel's law.
Molecular marker gene type analysis: shown in Fig. 7-B, in three kinds of genotype: the homozygous individuality (two DNA bands of 501bp and 88bp are arranged during electrophoresis detection) that the CC type is cut for enzyme does not take place, the TT type is the homozygous individuality (occurring three DNA bands of 227bp, 224bp and 88bp during electrophoresis detection) that the dna double chain enzyme all takes place is cut, and CT is the heterozygous individuality (occurring 501bp, 227bp, 224bp and four DNA bands of 88bp during electrophoresis detection) that has only a DNA chain to be cut by the PvuII enzyme.
Embodiment 2:
Detect the pig MHCIITA gene PCR-distribution of PvuII-RFLP polymorphism in different varieties
(1) PCR-PvuII-RFLP detects
Utilizing PCR-PvuII-RFLP to detect the DNA sample of 6 product boars, 3 Chinese native pig breed blood relationships are wherein arranged, is respectively painted face in Beijing opera pig, Arabescato and Tibetan pig; 3 external pig blood relationships are respectively landrace, Large White and duroc.
(2) detected result analysis
Genotype number of individuals and the gene frequency of sudden change C188-T188 in different pig kinds is as shown in table 1.The result is presented in the place of china pig allele C and preponderates, promptly in 3 ' UTR district 188 mainly exist with the forms of base C; Only CT genotype individuality occurs in hiding pig, all the other all are the CC type.Allele C is also preponderated in the pig abroad, but the frequency of occurrences of T is apparently higher than domestic local pig breed; Three kinds of genotype all can detect in the pig blood relationship abroad, and in landrace and Du Luoke, the frequency of allelotrope T is very high.Chi square test shows that the distribution of MHCIITA gene PCR-PvuII-RFLP polymorphism in different pig kinds there are differences, and reaches utmost point conspicuous level.The result of the chi square test in twos of six pig kinds is as shown in table 2.
188 (C188T) allelotrope in table 1. different varieties pig MHCIITA 3 ' UTR district and genotype frequency
The chi square test that 188 (C188T) genotype in table 2. different varieties pig MHCIITA 3 ' UTR district distribute
Embodiment 3:
The present invention clone's the application of pig MHCIITA molecule marker in the immune character association analysis
(1) makes up the scanning of family and genotype
The immune character resource family that is used for association analysis is the purebred landrace colony (external pig blood relationship) by Hua Zhong Agriculture University and Guangdong Province Hua Nongwenshi herding limited-liability company amalgamated consolidation, the parent is 17 landrace boars and 36 landrace sows, and F1 is for totally 302 of long white piglets.F1 adopts ordinary method that 3 kinds of antiviral antibody levels (pig breeding and breathing syndrome virus antibody horizontal, Pestivirus suis antibody horizontal, Pseudorabies virus antibody horizontal) and 18 routine blood tests are measured for gathering anticoagulation and non-anticoagulation sample respectively at 0,17,32 ages in days.
Group structure and influence factor thereof according to immune character resource family, the applicant uses mixed linear model to analyze the influence of the different genotype of pig MHCIITA gene to the immune indexes of mensuration, Mixed Models program is carried out data processing and statistical study in employing SAS (Version 8.1) software, and model is as follows:
Y
ijklmn＝μ+Genotype
i+Sex
j+Parity
k+Environment
l+Sire
m+Dam
n(Sire
m)+ε
ijklmn
Wherein Y represents above-mentioned immune indexes; μ represents the average of each proterties; Genotype represents the genotype effect, (i=3:1 sudden change produces 3 kinds of genotype), sex represents sex (j=2), and parity represents parity (k=2 :), and environment represents environmental effect (l=2), and these all are fixed effects; Sire represents male animal effect (m=17), and dam represents dam effect (n=36), and these are stochastic effect; ε represents random residual.
(2) association analysis of pig MHCIITA gene molecule marker C188-T188 and immune character
Molecule marker C188-T188 (being pig MHCIITA gene PCR-PvuII-RFLP polymorphism)
Use PCR-PvuII-RFLP and detect pig MHCIITA gene molecule marker C188-T188 genotype and carry out association analysis, the result shows: in detecting the swinery body, CC genotype individuality has 147, and CT genotype individuality has 165, and TT genotype individuality has 32.This mark is remarkable to porcine reproductive and respiratory syndrome (blue otopathy) the antibody horizontal influence of the 0 age in days mean corpuscular volume (MCV) of pig, average HGB amount and 32 ages in days.
Concrete outcome is described below:
Table 3: the association analysis result of pig MHCIITA gene PCR-PvuII-RFLP polymorphism and 0 age in days immune character
*The expression significant difference, P＜0.05;
*Expression difference is P＜0.01 extremely significantly.
As shown in Table 3, pig MHCIITA gene PCR-PvuII-RFLP polymorphism and 0 age in days mean corpuscular volume (MCV) remarkable related (p=0.0231＜0.05), be extremely significantly related (p=0.0006＜0.01) with the average HGB amount of 0 age in days, this site has no significant effect other immune character of this age in days.
Table 4: the least square average of pig MHCIITA gene PvuII-RFLP different genotype piglet 0 age in days mean corpuscular volume
As shown in Table 4, the RBC average body of CC genotype piglet actively is significantly higher than CT genotype (p＜0.05), also is higher than the TT genotype.Mean corpuscular volume is to judge the important immune indexes of anaemia, and mean corpuscular volume is in error state (ERST) for a long time can cause serious blood and relative disease.The mean corpuscular volume of three kinds of genotype individualities all meets in the range of normal value in the present embodiment, and can't distinguish which kind of allelotrope is beneficial gene.For average oxyphorase (HGB) amount, CC type individuality is the highest, and the CT type is minimum; The CC type phenotypic number utmost point is significantly higher than CT type individuality (p＜0.01), thereby the lung that CC type individuality is described is transported to each tissue of whole body in conjunction with oxygen and with it, and the carbonic acid gas in will organizing to deliver to other genotype of energy force rate of lung individual strong, illustrate that also CC genotype individuality has better healthy state simultaneously.Therefore, the individuality that carries gene C has better healthy state and vitality, and allele C is a beneficial gene.
The method of utilizing the present invention to set up has been carried out check and analysis to MHCIITA gene PCR-PvuII-RFLP polymorphism of 17 age in days piglets, and its result is as shown in table 5:
Table 5: the association analysis result of pig MHCIITA gene PCR-PvuII-RFLP polymorphism and 17 age in days immune characters
*The expression significant difference, P＜0.05;
*Expression difference is P＜0.01 extremely significantly.
As shown in Table 5, pig MHCIITA gene PCR-PvuII-RFLP polymorphism all has no significant effect piglet 17 immune characters that age in days is surveyed.
The method of utilizing the present invention to set up has been carried out check and analysis to MHCIITA gene PCR-PvuII-RFLP polymorphism of 32 age in days piglets, and its result is shown in table 6, table 7:
Table 6: the association analysis result of pig MHCIITA gene PCR-PvuII-RFLP polymorphism and 32 age in days immune characters
*The expression significant difference, P＜0.05;
*Expression difference is P＜0.01 extremely significantly.
As shown in Table 6, pig MHCIITA gene PCR-PvuII-RFLP polymorphism is remarkable related (p=0.0426＜0.05) with porcine reproductive and respiratory syndrome (blue otopathy) antibody horizontal of 32 ages in days, and is significantly not related with other immune character of survey.
Table 7: the least square average of pig MHCIITA gene PCR-PvuII-RFLP different genotype piglet 32 age in days content of hemoglobin
As shown in Table 7, the pig breeding of 32 ages in days of TT genotype individuality is 0.7442 ± 0.1420 with breathing syndrome virus (reproductive and respiratory syndrome virus) antibody horizontal, be significantly higher than CT genotype 0.5544 ± 0.1189 (p＜0.05), also be higher than CC genotype 0.6710 ± 0.1201 simultaneously.This explanation TT genotype individuality more helps body and produces high-level antibody and resist the disease, thereby plays a significant role in Acquired immunity.
＜110〉Hua Zhong Agriculture University
＜120〉pig MHCIITA gene is as the evaluation and the application of immunity related molecular mark
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<141>2009-06-11
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cactgctccc?ctggtggggt?aagcttggcg?ctcagaagat?accagccagc?accccccagc 420
gtgttgattt?cccaaacggt?gaccgacggg?gtgtccacgg?cagctgccct?ctgcctccgg 480
cacctgcggg?tttgcactca?ctttgtttgc?cgaggccaaa?gctgggcctg?gccagacacg 540
ccagacctta?gcgggggaag?agccgacagt?acactacggg?ccgaggcggg?gtggcgaggg 600
tctggaacca?catccgcctt?cttgccctca?cgtcctgtgt?cttttttcac?tacattatac 660
atggttatcg?cctctgctaa?aa 682
<210>4
<211>670
<212>DNA
＜213〉pig (Sus scrofa)
<220>
<221>3’UTR
<222>(1)..(670)
<223>
<220>
<221>mutation
<222>(188)..(188)
<223>
<400>4
atcatctgtg?gacacagctc?ttcttaggct?gtatcccgtg?agctttggcg?atctggtgcc 60
ctgccctggt?ggctcagagt?cagcccccac?tctgctgggg?aaaggaccca?cggcctgctc 120
tgtggacaga?ccccaggccc?ggccccaggc?tccttcgggg?cccagactga?tgtcagcctt 180
gctcagccgc?tgcagtcctg?gcagacaggc?gggcacccag?tggcagctag?ggtccacccg 240
ggagccctga?agcactccct?gcaggacact?gcagacagtg?gtggccaggt?cagagtgagg 300
gatgtggcgg?ccacatcacc?tgcccaggtc?ctgctggccg?ggggagaaag?cacctctcca 360
cactgctccc?ctggtggggt?aagcttggcg?ctcagaagat?accagccagc?accccccagc 420
gtgttgattt?cccaaacggt?gaccgacggg?gtgtccacgg?cagctgccct?ctgcctccgg 480
cacctgcggg?tttgcactca?ctttgtttgc?cgaggccaaa?gctgggcctg?gccagacacg 540
ccagacctta?gcgggggaag?agccgacagt?acactacggg?ccgaggtggg?gtggcgaggg 600
tctggaacca?catccgcctt?cttgccctca?catcctgtgt?ctttttttca?ctacattata 660
catggttatc 670
<210>5
<211>687
<212>DNA
＜213〉pig (Sus scrofa)
<220>
<221>3’UTR
<222>(1)..(687)
<223>
<220>
<221>mutation
<222>(188)..(188)
<223>
<400>5
atcatctgtg?gacacagctc?ttcttaggct?gtatcccgtg?agctttggcg?atctggtgcc 60
cagccctggt?ggctcagagt?cagcccccac?tctgctgggg?aaaggaccca?cggcctgctc 120
tgtggacaga?ccccaggccc?ggccccaggc?tccttcgggg?cccagactga?tgtcagcctt 180
gctcagccgc?tgcagtcctg?gcagacaggc?gggcacccag?tggcagctag?ggtccacccg 240
ggagccctga?agcactccct?gcaggacact?gcagacagtg?gtggccaggt?cagagtgagg 300
gatgtggcgg?ccacatcacc?tgcccaggtc?ctgctggccg?ggggagaaag?cacctctcca 360
cactgctccc?ctggtggggt?aagcttggcg?ctcagaagat?accagccagc?accccccagc 420
gtgttgattt?cccaaacggt?gaccgacggg?gtgtccacgg?cagctgccct?ctgcctccgg 480
cacctgcggg?tttgcactca?ctttgtttgc?cgaggccaaa?gctgggcctg?gccagacacg 540
ccagacctta?gcgggggaag?agccgacagt?acactacggg?ccgaggtggg?gtggcgaggg 600
tctggaacca?catccgcctt?cttgccctca?cgtcctgtgt?cttttttcac?tacattatac 660
atggttatcg?cctttcccta?aaaaaaa 687
Claims (1)
1. molecule marker relevant with pig immune trait, its nucleotide sequence is shown in sequence table SEQ ID NO:2, and the base mutation in that there is 1 C188-T188 at the 188bp place of sequence table SEQ ID NO:2 causes the PvuII-RFLP polymorphism.
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| CN101984058B (en) * | 2010-11-16 | 2012-06-06 | 大连大学 | Pouch pig SLA-2 genes and application thereof |
| CN103421797B (en) * | 2013-03-11 | 2014-09-17 | 华中农业大学 | Genetic marker for character of litter size of pig utilizing SLA-11 gene |
| CN111304292A (en) * | 2020-04-21 | 2020-06-19 | 江苏农牧科技职业学院 | Gene fine screening method beneficial to pig breeding |
| CN114774468B (en) * | 2022-04-20 | 2022-12-20 | 温氏食品集团股份有限公司 | Allele molecular marker and anti-blue-ear-disease pig group construction method |
| CN117409868B (en) * | 2023-12-14 | 2024-02-20 | 成都大熊猫繁育研究基地 | Panda genetic map drawing method and system |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1337469A (en) * | 2000-08-08 | 2002-02-27 | 华中农业大学 | Typing method of pig monosperm microsatellite DNA mark |
| CN1737138A (en) * | 2004-08-20 | 2006-02-22 | 李奎 | Pig ouclar muscles area character-related protein and its coding gene and uses |
| CN101092622A (en) * | 2007-05-21 | 2007-12-26 | 华中农业大学 | TEF-1 gene of weight increment per day of pig and application in assistant selecting molecule markers of pigs |
| CN101148668A (en) * | 2007-09-12 | 2008-03-26 | 华中农业大学 | Clone for pork generation character related gene BTG1 of pig and application thereof in pig molecule mark auxiliary selection |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1337469A (en) * | 2000-08-08 | 2002-02-27 | 华中农业大学 | Typing method of pig monosperm microsatellite DNA mark |
| CN1737138A (en) * | 2004-08-20 | 2006-02-22 | 李奎 | Pig ouclar muscles area character-related protein and its coding gene and uses |
| CN101092622A (en) * | 2007-05-21 | 2007-12-26 | 华中农业大学 | TEF-1 gene of weight increment per day of pig and application in assistant selecting molecule markers of pigs |
| CN101148668A (en) * | 2007-09-12 | 2008-03-26 | 华中农业大学 | Clone for pork generation character related gene BTG1 of pig and application thereof in pig molecule mark auxiliary selection |
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