CN102010868A - Garrupa MHC (Major Histocompatibility Complex) IIB gene as well as cloning method and application thereof - Google Patents
Garrupa MHC (Major Histocompatibility Complex) IIB gene as well as cloning method and application thereof Download PDFInfo
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Abstract
The invention discloses a garrupa MHC (Major Histocompatibility Complex) IIB gene as well as a cloning method and application thereof. The nucleotide sequence of the garrupa MHC IIB gene is shown as SEQ ID NO:1, and the amino acid sequence of protein encoded by the gene is shown as SEQ ID NO:2. In the invention, by using a homologous cloning method, the MHC IIB gene is separated from garrupa, 12 different MHC IIB gene types are identified, the correlation between the polymorphism and the disease resistance of the MHC IIB gene are analyzed, and 6 molecular marks of the MHC IIB gene relevant to the disease resistance of the garrupa are screened; and the cloning method is suitable for screening the marks relevant to the disease resistance of the garrupa and breeding disease-resistant species.
Description
Technical field
The present invention relates to the genetically engineered field, be specifically related to a kind of cabrilla MHC IIB gene and cloning process and application.
Background technology
Major histocompatibility complex (major histocompatibility complex, MHC) be the highly polymorphic gene group of ubiquitous class coding immunoglobulin-like receptor in vertebrates, this gene group has vital role in immunity system, be one of focus of immune in recent years direction research.Mhc gene is divided into I type, II type and III type, and II type gene is divided into IIA and IIB again.MHC II genoid coded product is a MHC II quasi-molecule, and they combine with the exogenous antigen peptide and submission is given t helper cell, cause the immunne response in downstream.Many studies show that, the polymorphism of MHC II gene is closely related with individual resistance against diseases.
MHC II quasi-molecule is a heterodimer, is made up of a α polypeptide chain and a beta polypeptides chain.These two chains all stride the film district by two nuclear outskirts (α 1/ α 2 and β 1/ β 2), land, one and a cytoplasmic domain is formed.α 1/ β 1 forms the polypeptide land, and (Peptide Binding Region PBR), has the height polymorphism, and the little peptide that its effect is with antigen resolves into combines, to give the T cell with its submission; The zone that α 2/ β 2 forms does not have the height polymorphism, is MHC molecule and T cell bonded important structure territory.
MHC II quasi-molecule has the height polymorphism, and especially in its polypeptide land (peptide-bindingregion, PBR), the antigen peptide generation effect that this zone is direct and antigen is cracked into, and then cause the immune cascade reaction in downstream.The allelotrope of MHC II exists the non-synonym of higher proportion to replace (non-synonymous Substitution) usually in the PBR zone.In fact, MHC shows gene of high polymorphism in all knowns, and finding in the mankind's research only just has 500 allelotrope of surpassing on a locus.Mhc gene selects to keep its polymorphism by balance, and mhc gene has linkage disequilibrium (linkage disequilibrium) and two characteristics of haplotype (Haplotype) heredity in heredity.
The height polymorphism of MHC molecule has profound significance, and it gives the ability that population adapts to changeable envrionment conditions, realizes the Genetic Control to immune response, and makes MHC become individual lifelong genetic marker.The polymorphism of MHC is also closely bound up with individual resistance against diseases, has all delivered the article of MHC polymorphism and body disease-resistant power correlation research in chicken, mammals, fish.The MHC polymorphism all is an extremely important material at aspects such as the research of the research of phyletic evolution, population protection and genetic breedings, is a focus of current mhc gene research.
The α of fish MHC II and β gene generally are linkage inheritances.At present in surpassing 30 kinds of teleostei the clone obtain mhc gene and proved its polymorphism, as zebra fish, carp, Atlantic salmon, lefteye flounder, Tiao Wen Shi Sushi, porgy etc.
Fish mhc gene function is identical with mammals, participates in antigen presentation and causes immune cascade reaction.Studies show that the gene fragment (being generally second exon on the mhc gene) of coding PBR structural domain also has the height polymorphism in the fish mhc gene, and closely related with the immune disease-resistance ability of fish.
Screen disease-resistance population Deng with the Atlantic salmon of 7 about 2000 tails of family with 5 kinds of MHC I allelotrope and 4 kinds of MHC II allelotrope combinations and in conjunction with traditional family selective breeding mode at ISA (infections anaemia), successfully filter out effective disease-resistant gene type combination, this has started and has used MHC to carry out the beginning of the disease-resistant assistant breeding of fish.After this, in lefteye flounder, carp, also there is similar research to be in the news.At present, still not having any research about the cabrilla mhc gene both at home and abroad is in the news.
Summary of the invention
The objective of the invention is to according in the prior art to the still incomplete problem of the research of mhc gene, a kind of and the disease-resistant relevant cabrilla MHC IIB gene of cabrilla are provided.
Another purpose of the present invention is to provide the allelotrope on the above-mentioned cabrilla MHC IIB gene.
Another purpose of the present invention is to provide the albumen of above-mentioned cabrilla MHC IIB genes encoding.
Another purpose of the present invention is to provide the cloning process of above-mentioned cabrilla MHC IIB gene.
Another purpose of the present invention is to provide the detection method of the sequence polymorphism of above-mentioned cabrilla MHC IIB gene.
A further object of the invention is to provide the application of above-mentioned cabrilla MHC IIB gene.
Above-mentioned purpose of the present invention is achieved by the following technical programs:
A kind of cabrilla MHC IIB gene, its sequence are shown in SEQ ID NO:1, and this gene is to separate from the Epinephelus coioide spleen by the method for homologous clone to obtain.At least have 12 different allelotrope in the described gene, its nucleotide sequence is shown in SEQ ID NO:3～14.
The albumen of above-mentioned cabrilla MHC IIB coded by said gene, shown in its aminoacid sequence SEQ ID NO:2, totally 249 amino acid, iso-electric point is 6.53, molecular weight is 28.31 kilodaltons, cabrilla MHC IIB albumen mainly is divided into 4 structural domains, be respectively Exon1 encoded signals peptide (SP), Exon2 encoded polypeptides land (be β 1 structural domain, PBR), IGc1 district (being β 2 structural domains) and the Exon4 and the part Exon5 of Exon3 coding encode stride the film district.74.0%, 79.1%, 58.4% the albumen of cabrilla MHC IIB coded by said gene and turbot, porgy, rainbow trout, zebra fish, mouse and the proteic homology of people MHC IIB coded by said gene are respectively:.55.2%, 32.0% and 31.5%.
The cloning process of cabrilla MHC IIB gene of the present invention comprises the steps:
(1) extract the total RNA of cabrilla, reverse transcription obtains cDNA article one chain, preparation RACE PCR reaction template;
(2), obtain the cDNA fragment of cabrilla MHC IIB gene according to the homology design degenerated primer of the aminoacid sequence of different plant species MHC IIB; The sequence of described degenerated primer is shown in SEQ ID NO:15～16;
(3) carry out RACE PCR, described primer sequence such as SEQ ID according to gained fragment design primer
Shown in NO:17～22, obtain the MHC IIB full-length cDNA of 1338bp;
(4) according to designing primer with the known MHC IIB exon of the close fish of cabrilla sibship and the boundary site of intron, carry out the intron amplification, obtain the complete genome sequence of cabrilla MHC IIB, described primer is shown in SEQ ID NO:23～30.
Cabrilla MHCIIB Study on gene polymorphism of the present invention, according to synthetic specificity upstream primer EpcoMHC2bPl-F on first exon of cabrilla MHC IIB gene, sequence is shown in SEQ ID NO:31, at the end and the synthetic specificity downstream primer EpcoMHC2bPl-R of the second intron front end of second exon, sequence is shown in SEQ ID NO:32; In 45 different cabrilla individualities, carry out pcr amplification, 145 positive colonies of picking and send order-checking after, obtain the raw data of MHC IIB gene PBR zone polymorphism.Sequencing result shows that there are two different loci in MHC IIB gene in cabrilla, and its molecular weight difference in size is the length difference of Intron 1.The Intron1 size in one of them site is 138bp, called after DAB2; The Intron1 size in another site is widely different at different fingerlings, but always greater than 138bp, called after DAB 1.Co-exist in 12 different sequences in 145 positive colonies of 45 fishes, this shows in cabrilla MHC IIB gene and has 12 different allelotrope at least that its nucleotide sequence is shown in SEQ ID NO:3～14.Wherein 10 allelotrope belong to the DAB1 site, and 2 allelotrope belong to the DAB2 site.With 10 allelotrope called after EpcoDAB*1-01 that belong to the DAB1 site to EpcoDAB*1-10, with 2 allelotrope called after EpcoDAB*2-01 and EpcoDAB*2-02 that belong to the DAB2 site.
The screening of the disease-resistant mark of correlation of above-mentioned cabrilla MHC IIB adopts natural selection and artificial challenge's approach to obtain disease-resistance population and disease-resistance population not.In the morbidity busy season, collect the individuality of morbidity back survival from plant, the fry of collecting with pathogenic bacterial infection simultaneously obtains disease-resistance population and disease-resistance population not according to the opposing survival ability size to pathogenic bacterial infection.321 positive colonies to 81 individualities check order altogether, the result shows, there are 6 kinds of mhc gene type high frequencies to appear in the disease-resistant individuality, EpcoDAB*1-01 wherein, EpcoDAB*1-02, EpcoDAB*1-04, EpcoDAB*1-05, EpcoDAB*2-01 and EpcoDAB*2-02, the frequency that is occurred is respectively 14.81%, 7.41%, 7.41%, 14.81%, 7.41%, 11.11% and 37.04%.
The method of the disease-resistant mark of correlation assistant breeding of above-mentioned cabrilla MHC IIB: the MHC IIB genotype that will occur at the disease-resistance population high frequency is as disease resistance related MHC IIB genetic marker, the parent population of carrying these disease-resistant gene marks is bred, cultivate the offspring, separate the MHC IIB gene among the offspring, research MHC IIB gene pleiomorphism, carry out the cause pathogeny imcrobe infection experiment simultaneously, study disease-resistant MHC IIB genetic marker genetic development and with the relation of fish body disease resistance, therefrom filter out and both contained disease-resistant MHC IIB genetic marker, the fry that disease resistance improves again carries out can setting up disease-resistant varieties after many generation breedings and the cultivation.
Compared with prior art, the present invention has following beneficial effect:
The present invention adopts functional genome's technology, in cabrilla, screen disease-resistant relevant mhc gene mark, tentatively set up the disease-resistant mhc gene marker-assisted breeding of cabrilla technology, this technological operation is simple, seed selection efficient height, suit on all marine fishs, to apply, the cabrilla disease-resistant variety is cultivated significant and using value.
Description of drawings
Fig. 1 is the distribution frequencies of 12 kinds of different genotype of cabrilla MHC IIB in disease-resistance population.
Embodiment
Further explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.
Embodiment 1
1. the extraction of the total RNA of cabrilla spleen
Get healthy Epinephelus coioide (Epinephelus coioides), in 2 ages, the about 750g of body weight with the about 1min of MS-222 (tricaine methanesulphonate) anesthesia, isolates spleen immediately after the tail vein is got blood.Adopt Trizol reagent method to extract and obtain the total RNA of cabrilla spleen, its OD260/280=1.85, electrophoresis result shows, 28S rRNA, 18S rRNA band is clear, and the brightness of 28S band is the twice of 18S, show that the total RNA of gained is not polluted the purity height by protein, phenol and genomic dna.
2.cDNA first chain is synthetic
Get the total RNA sample of 5 μ g Epinephelus coioide spleens and carry out the DNA enzyme and handle, mix, carry out reverse transcription with RNA Oligo dT (sequence is shown in SEQ ID NO:33) to remove the pollution of genomic dna, product place-20 ℃ of preservations standby.
3. design of primers
From the GenBank database, download and obtain nearly source species, as hyoid tooth perch (DQ821113), lefteye flounder (AY848955) and porgy MHC II B homologous gene CDS sequences such as (AY190711), utilize ClustalX software to carry out the multisequencing comparison, determine conserved regions, according to definite conserved regions design degenerated primer, the amplified fragments size is about 300bp.
Primer sequence is shown in SEQ ID NO:15～16.
4. the clone of Epinephelus coioide MHC IIB gene cDNA complete sequence
As template, carry out pcr amplification with degenerated primer with the embodiment 2 synthetic first chain cDNA, sample is to the 1.8wt% sepharose on the gained PCR product, and with low voltage electrophoretic separation dna fragmentation, purifying reclaims the purpose product from gel.Purpose product behind the purifying is connected to
The Easy carrier transforms DH5 α intestinal bacteria, selects the positive colony order-checking.Blast homology analysis revealed, purpose product are the intermediate segment of MHC IIB gene.
According to the cDNA fragment sequence that obtained design Auele Specific Primer, utilize the terminal rapid amplifying technology of cDNA (Rapid Amplification ofcDNA ends, RACE) to 3 of goal gene '-and 5 '-end carries out pcr amplification.
Total RNA carries out dephosphorization acid according to RACE test kit GeneRacerTM Kit specification sheets epinephelus coioides spleen, removes mRNA 5 ' cap sequence, is connected with RNA Oligo, finally by synthetic cDNA first chain of reverse transcription.With 3 of Epinephelus coioide MHC IIB cDNA '-and 5 '-RACE special primer (MHC-F2, MHC-F3 and MHC-R2, MHC-R3) and the universal primer of GeneRacerTM Kit, the first chain cDNA is a first round pcr template with GeneRacerTM Kit reverse transcription synthetic, first round PCR product with dilution is second to take turns pcr template, amplification MHC IIB cDNA 3 ' end and 5 ' end fragment are identified and dna sequencing recovery purifying, connection T carrier, transformed into escherichia coli, the positive colony of resulting PCR product.Order-checking institute calling sequence utilizes Clustal X software to splice with the sequence of degenerated primer amplification gained again.
The special primer sequence is shown in SEQ ID NO:17～22.
5. the acquisition of Epinephelus coioide MHC IIB genomic dna complete sequence
From the cabrilla liver, extract its total DNA, use the marine animal genome to extract test kit, extract genomic dna with the method for chromatography column.Carry out the amplification of intron according to the known MHC IIB gene extron of the close fish of other sibships and the dividing point design Auele Specific Primer (sequence such as SEQ ID NO:23～30) of intron.Similar with other vertebrate MHC IIB genome structure, cabrilla MHC IIB genome includes 5 exons and 4 introns, wherein the length of 5 exons is respectively 74bp, 273bp, 282bp, 114bp and 595bp, and introne 1,2,3 and 4 length is respectively 192bp, 273bp, 118bp and 108bp; Measure the sequence of whole 5 exons and 4 introns, obtained cabrilla MHC IIB complete genome sequence.
Embodiment 2 Epinephelus coioide MHC IIB nucleotide sequence variations
According to synthetic specificity upstream primer EpcoMHC2bPl-F on first exon of cabrilla MHC IIB gene, sequence is shown in SEQ ID NO:31, at the end and the synthetic specificity downstream primer EpcoMHC2bPl-R of the second intron front end of second exon, sequence is shown in SEQ ID NO:32; In 45 different cabrilla individualities, carry out pcr amplification, 145 positive colonies of picking and send order-checking after, obtain the raw data of MHC IIB gene PBR zone polymorphism.Sequencing result shows that there are two different loci in MHC IIB gene in cabrilla, and its molecular weight difference in size is the length difference of Intron 1.The Intron1 size in one of them site is 138bp, called after DAB2; The Intron1 size in another site is widely different at different fingerlings, but always greater than 138bp, called after DAB1.Co-exist in 12 different sequences in 145 positive colonies of 45 fishes, this shows in cabrilla MHC IIB gene and has 12 different allelotrope at least.Wherein 10 allelotrope belong to the DAB1 site, and 2 allelotrope belong to the DAB2 site.With 10 allelotrope called after EpcoDAB*1-01 that belong to the DAB1 site to EpcoDAB*1-10, with 2 allelotrope called after EpcoDAB*2-01 and EpcoDAB*2-02 that belong to the DAB2 site.
The screening of the disease-resistant mark of correlation of embodiment 3 Epinephelus coioide MHC IIB genes
Adopt natural selection and artificial challenge's approach to obtain disease-resistance population and disease-resistance population not.In the morbidity busy season, collect the individuality of morbidity back survival from plant, the fry of collecting with pathogenic bacterial infection simultaneously obtains disease-resistance population and disease-resistance population not according to the opposing survival ability size to pathogenic bacterial infection.321 positive colonies to 81 individualities check order altogether, the result shows, there are 6 kinds of mhc gene type high frequencies to appear in the disease-resistant individuality, EpcoDAB*1-01 wherein, EpcoDAB*1-02, EpcoDAB*1-04, EpcoDAB*1-05, EpcoDAB*2-01 and EpcoDAB*2-02, the frequency that is occurred is respectively 14.81%, 7.41%, 7.41%, 14.81%, 7.41%, 11.11% and 37.04% (Fig. 1).
The disease-resistant mark of correlation auxiliary breeding means of embodiment 4 Epinephelus coioide MHC IIB genes
The MHC IIB genotype that will occur at the disease-resistance population high frequency is as disease resistance related MHC IIB genetic marker, the parent population of carrying these disease-resistant gene marks is bred, cultivate the offspring, separate the MHCIIB gene among the offspring, research MHC IIB gene pleiomorphism, carry out the cause pathogeny imcrobe infection experiment simultaneously, study disease-resistant MHC IIB genetic marker genetic development and with the relation of fish body disease resistance, therefrom filter out and both contained disease-resistant MHC IIB genetic marker, the fry that disease resistance improves again carries out can setting up disease-resistant varieties after many generation breedings and the cultivation.
The MHC IIB genotype that will occur at the disease-resistance population high frequency is as disease resistance related MHC IIB genetic marker, by parent population MHC IIB genotype is screened, pick out and contain EpcoDAB*2-01 and the genotypic male and female Epinephelus coioide of EpcoDAB*2-02 parent population breeds, cultivate the offspring.Separate the MHC IIB gene among the offspring, study their MHC IIB gene pleiomorphism, carry out the cause pathogeny imcrobe infection experiment simultaneously, the fry of collecting detects the opposing survival ability to pathogenic bacterial infection.The result shows and contains EpcoDAB*2-01 and the genotypic fry of EpcoDAB*2-02, the survival ability ratio has common fry to increase by 27%, therefore EpcoDAB*2-01 and EpcoDAB*2-02 can be used as disease-resistant MHC IIB genetic marker, the fry that the production disease resistance is high carries out can setting up disease-resistant varieties after many generation breedings and the cultivation.
Claims (7)
1. cabrilla MHC IIB gene, its nucleotide sequence is shown in SEQ ID NO:1.
2. according to the described cabrilla MHC of claim 1 IIB gene, it is characterized in that existing at least in the described gene 12 different allelotrope, its nucleotide sequence is shown in SEQ ID NO:3～14.
3. the albumen of the described cabrilla MHC of claim 1 IIB genes encoding is characterized in that described proteic iso-electric point is 6.53, and molecular weight is 28.31 kilodaltons, and its aminoacid sequence is shown in SEQ ID NO:2.
4. the cloning process of the described cabrilla MHC of claim 1 IIB gene is characterized in that described method comprises the steps:
(1) extract the total RNA of cabrilla, reverse transcription obtains cDNA article one chain, preparation RACE PCR reaction template;
(2), obtain the cDNA fragment of cabrilla MHC IIB gene according to the homology design degenerated primer of the aminoacid sequence of different plant species MHC IIB; The sequence of described degenerated primer is shown in SEQ ID NO:15～16;
(3) carry out RACE PCR according to gained fragment design primer, described primer sequence obtains the MHC IIB full-length cDNA of 1338bp shown in SEQ IDNO:17～22;
(4) according to designing primer with the known MHC IIB exon of the close fish of cabrilla sibship and the boundary site of intron, carry out the intron amplification, obtain the complete genome sequence of cabrilla MHC IIB, described primer is shown in SEQ ID NO:23～30.
5. the detection method of the described cabrilla MHC of claim 1 IIB nucleotide sequence variation, it is characterized in that described method comprises the steps: according to synthetic specificity upstream primer on first exon of cabrilla MHC IIB gene, sequence is shown in SEQ ID NO:31, at the end and the synthetic specificity downstream primer of the second intron front end of second exon, sequence is shown in SEQ ID NO:32; Carry out pcr amplification in different cabrilla individualities, the picking positive colony checks order, and obtains the data of MHC IIB gene PBR zone polymorphism.
6. the application of the described cabrilla MHC of claim 1 IIB gene is characterized in that described cabrilla MHC IIB gene is used for marker-assisted breeding.
7. according to the application of the described cabrilla MHC of claim 6 IIB gene, it is characterized in that the method that described cabrilla MHC IIB gene is used for marker-assisted breeding is as follows: the MHCIIB genotype that will occur at disease-resistance population is as disease resistance related MHC IIB genetic marker, the parent population of carrying these disease-resistant gene marks is bred, cultivate the offspring, separate the MHCIIB gene among the offspring, carry out the cause pathogeny imcrobe infection experiment, therefrom filter out and both contained disease-resistant MHC IIB genetic marker, the fry that disease resistance improves again carries out can setting up disease-resistant varieties after many generation breedings and the cultivation.
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Cited By (3)
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| CN105296500A (en) * | 2015-11-11 | 2016-02-03 | 中山大学 | Epinephelus lanceolatus Neuropeptide B gene and protein and application of gene |
| CN112813171A (en) * | 2020-12-17 | 2021-05-18 | 水利部中国科学院水工程生态研究所 | MHC gene primer for cupreous rotundifolia fish and application thereof |
| CN114078568A (en) * | 2020-09-14 | 2022-02-22 | 青岛欧易生物科技有限公司 | Metagenome sequencing data processing system and processing method based on IIB type restriction endonuclease characteristics |
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2010
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296500A (en) * | 2015-11-11 | 2016-02-03 | 中山大学 | Epinephelus lanceolatus Neuropeptide B gene and protein and application of gene |
| CN105296500B (en) * | 2015-11-11 | 2018-12-25 | 中山大学 | Epinephelus lanceolatus fish Neuropeptide B gene, albumen and its application |
| CN114078568A (en) * | 2020-09-14 | 2022-02-22 | 青岛欧易生物科技有限公司 | Metagenome sequencing data processing system and processing method based on IIB type restriction endonuclease characteristics |
| CN112813171A (en) * | 2020-12-17 | 2021-05-18 | 水利部中国科学院水工程生态研究所 | MHC gene primer for cupreous rotundifolia fish and application thereof |
| CN112813171B (en) * | 2020-12-17 | 2023-05-26 | 水利部中国科学院水工程生态研究所 | MHC gene primer for round-mouth copper fish and application thereof |
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