CN1810972A - Lefteye flounder disease resistance related MHC gene marker and subsidiary breeding method - Google Patents
Lefteye flounder disease resistance related MHC gene marker and subsidiary breeding method Download PDFInfo
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Abstract
The lefteye flounder disease resistance related MHC gene marker and its subsidiary breeding method are disclosed. The method includes cloning MHCIIB gene, preparing disease resisting colony and disease infecting colony, screening disease resistance related MHCIIB gene marker and establishing gene marker subsidiary breeding technology. The present invention clones whole lefteye flounder MHCIIB gene, identifies 13 kinds one MHCIIB genotypes from the disease resisting colony and the disease infecting colony, and establishes disease resistance related gene marker subsidiary breeding by using genetype g and h as the disease resistance related MHC gene markers. The method of the present invention has the features of high pertinency and high breeding efficiency, and is suitable for various kinds of fishes.
Description
Affiliated technical field:
The invention belongs to the fish molecular mark technology in the aquatic living things technical field, is a kind of fish disease-resistant related gene mark and the auxiliary breeding means thereof that can use in the fish disease-resistant variety is cultivated.
Background technology:
Disease is the bottleneck of restriction culture fishery fast development, owing to lack good disease-resistant variety, China's due to illness harmful direct economic loss that already causes to fish farming in every year is up to over ten billion Yuan Renminbi.For a long time, owing to ignore research to disease-resistant mechanism of fish and disease-resistant functional gene, lack disease-resistant relevant genetic marker and molecule marker, the progress that causes the fish disease-resistant variety to be cultivated is slow, relevant fish disease-resistant related gene mark report is few both at home and abroad, does not also see the report that the disease-resistant improved seeds of relevant fish are cultivated.
Major histocompatibility complex (MHC) genes encoding cell surface glycoprotein, the glycoprotein of coding can combine with antigenic peptide, by with the interaction of T cell, form a ternary complex, induce and regulate the immunne response of body, the excitating organism specific immune response.In general, the MHC I quasi-molecule structure of fish is similar with Mammals, is by α chain and β
2The non-covalent heterodimer that is connected to form of-microglobulin.The heterodimer that the MHC II quasi-molecule of fish is made up of α chain and β chain.In bony fish-carp, reported that mhc gene so far for the first time from nineteen ninety Hashimoto et al., MHC I, II α, the II β gene of most vertebrate carried out clone's research so far, as selachian, bony fish, batrachians, reptiles, birds and mammals.Studies show that mhc gene is a abundantest genic system of present known polymorphism, there is a large amount of locus, each locus exists a large amount of allelotrope again, this polymorphism caused each individuality can both in conjunction with and present the polypeptide that a considerable amount of and immune response have the direct function dependency, wherein pleomorphism site is mainly on exon 2.Studies show that vertebrate the MHC mixture plays an important role in organism immune response, and special MHC allelotrope is relevant with body disease-resistant power, this all had relevant report at biologies such as chicken, mouse, sheep and people.Fish, cloned rainbow trout (Grimholt et al. at present, 2000), porgy (Chen et al., 2005), ditch Nian (Liu et al., 2004) and turbot (Zhang and Chen, the mhc gene of fish such as 2005), but about the mutual relationship of seawater fish disease resistances such as mhc gene polymorphism and lefteye flounder and use the research that the disease resistance related MHC genetic marker carries out disease-resistant marker assisted selection and there is no report at present both at home and abroad.
Summary of the invention:
The objective of the invention is to set up lefteye flounder molecular mark method in order to filter out the disease-resistant relevant mhc gene mark of lefteye flounder, cultivating for the lefteye flounder disease-resistant variety provides genetic marker and technological method.
Its technology contents of the present invention is as follows: one, lefteye flounder disease resistance related MHC gene marker comprises: 1, the clone of MHC II B gene; 2, the preparation of disease-resistance population and responsive colony; 3, the screening of disease resistance related MHC genetic marker; Two, lefteye flounder disease resistance related MHC gene marker auxiliary breeding means.
One, lefteye flounder disease resistance related MHC gene marker comprises: 1, the clone of lefteye flounder MHC II B gene; 2, the preparation of disease-resistance population and responsive colony; 3, the screening of disease resistance related MHC genetic marker:
1, the clone of lefteye flounder MHC II B gene: comprise acquisition, the comparison of MHCIIB protein sequence of cDNA clone, genomic dna sequence.
1) amplification of .cDNA and clone: utilize the TRIzol test kit from fish liver, to extract total RNA, utilize the M-MLV reverse transcription to obtain cDNA article one chain, and utilize Smart RACE cDNA amplification kit (Clontech) synthetic respectively 5 '-and 3 '-RACE-Ready-cDNA, the RACEPCR reaction is standby.According to the homology design degenerated primer of the aminoacid sequence of different plant species MHC IIB, obtain the cDNA fragment of one section MHC II B gene.Carry out RACE PCR according to gained fragment design special primer.Obtain the MHCIIB full length cDNA sequence of 1182 bases, comprise the coding region of 741 bases, 3 ' the terminal untranslated region of the 5 ' untranslated region of 33bp and 407bp, and have the PolyA tail of a typical tailing signal AATAAA of vertebrates and 27bp.
2). the acquisition of genomic dna sequence: carry out the amplification of intron according to the known MHC II B exon of the close fish of other sibship and the boundary site design special primer of intron.The result shows that lefteye flounder MHC II B genome and other vertebrate MHC II B genome structure are similar, include 5 exons and 4 introns, pcr amplification has obtained whole 4 introns, wherein introne 1s, 2,3 and 4 length is respectively 96/84bp, 392bp, 85bp and 109bp, and the length of 5 exons is respectively 85bp, 279bp, 274bp, 114bp and 430bp.Measure the sequence of whole 5 exons and 4 introns, therefore obtained lefteye flounder mhc gene full length sequence.
3) .MHCIIB protein sequence comparison: infer the protein sequence that obtains the coded 246bp in 741bp coding region by DNAMAN, its molecular weight is 27.78KD, and iso-electric point is 6.69.Protein sequence through the MHC II B of this protein sequence of MEGA2.0 software analysis and known other vertebrates (Atlantic salmon, carp, beautiful fish, people, mouse, shark, rainbow trout, porgy, perch and zebra fish), the result shows that the sibship of lefteye flounder MHCIIB protein sequence and porgy, perch and beautiful fish is nearest, take second place with the relation of Atlantic salmon, carp and rainbow trout, with shark, mouse and people's sibship farthest.
2, the preparation of disease-resistance population and responsive colony: 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 two basic populations of disease-resistance population not according to the resistivity size to pathogenic bacterial infection.
3, the screening of disease resistance related MHC genetic marker:
411 positive colonies to 84 individualities check order altogether, and the result shows, has 13 kinds of different nucleotide sequences, the 13 kinds of different aminoacid sequences (Paol-allele*aPaol-allele*m) of encoding respectively.The exogenous array of 280/268bp in 411 positive colony plasmids, remove the intermediary intron sequences, 61 amino acid of encoding altogether, by comparing with the aminoacid sequence of human MHC II B, the corresponding site of amino acid section of determining the MHCIIB gene that this institute gets is 22～82, and wherein antigen binding site is respectively the 35th, 37,39,56,58,60,65,66 and the 75th.In total coding region, 13 of pleomorphism sites, wherein the polypeptide binding site is 6, accounts for 46.15% of polymorphic site.In 13 kinds of different aminoacid sequences (Paol-allele*a-Paol-allele*m), Paol-allele*a-Paol-allele*f and Paol-allele*j, Paol-allele*k, Paol-allele*i, Paol-allele*m are that disease-resistance population and susceptible colony are common.Be genotype a, b, c, d, e, f, i, j, k and m come across disease-resistance population and disease-resistance population not simultaneously, and genotype g and h only appear in the disease-resistance population, and genotype l only appears in the susceptible colony.The not distribution of isoallele in disease-resistance population and susceptible colony.Therefore, genotype g is considered to disease-resistant relevant mhc gene mark with h, and it may be the responsive relevant mhc gene mark of disease that genotype l then is considered to.
Two, lefteye flounder disease resistance related MHC gene marker auxiliary breeding means
With the MHCIIB genotype that only in disease-resistance population, occurs as the disease resistance related MHC genetic marker, the fish of carrying these disease-resistant gene marks is bred, cultivate the offspring, separate the MHCIIB gene among the offspring, the polymorphism of research MHCIIB gene, carry out the cause pathogeny imcrobe infection experiment simultaneously, study disease-resistant MHCIIB genetic marker genetic development and with the relation of fish body disease resistance, therefrom filter out and both contained disease-resistant MHCIIB 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 present invention and prior art contrast are characterized in: the present invention adopts functional genome's technology, with the lefteye flounder is that material has been set up China's cultured fishes disease resistance related MHC genetic marker first, tentatively set up the disease-resistant mhc gene marker-assisted breeding of lefteye flounder technology, characteristics such as this technology is pointed by force, seed selection efficient height, seed selection cycle weak point, for new molecular breeding technological approaches has been opened up in the cultivation of fish disease-resistant variety, suit in all cultured fishes, to apply, the cultured fishes disease-resistant variety is cultivated significant and using value.
Description of drawings:
Fig. 1: lefteye flounder MHC II B complete genome sequence and deduced amino acid thereof
Fig. 2: 13 kinds of different genotype of lefteye flounder MHC II B
Fig. 3: distribution frequency and the corresponding chi square test of 13 kinds of different genotype of lefteye flounder MHC II B in disease-resistance population and susceptible colony;
Embodiment:
With the example that is established as of the screening of lefteye flounder disease resistance related MHC gene marker and assistant breeding technology thereof, technology contents of the present invention is elaborated below.
One, the screening of lefteye flounder disease resistance related MHC gene marker comprises: 1, the clone of lefteye flounder MHC II B gene; 2, the preparation of lefteye flounder disease-resistance population and responsive colony; 3, the screening of lefteye flounder disease resistance related MHC gene marker:
1. the clone of lefteye flounder MHC II B gene: comprise acquisition, the comparison of MHCIIB protein sequence of cDNA clone, genomic dna sequence.
1), the amplification of cDNA and clone: utilize the TRIzol test kit from the lefteye flounder liver, to extract total RNA, utilize the M-MLV reverse transcription to obtain cDNA article one chain, and utilize Smart RACE cDNA amplification kit (Clontech) synthetic respectively 5 '-and 3 '-RACE-Ready-cDNA, the RACEPCR reaction is standby.According to the homology design degenerated primer dfMHCF of the aminoacid sequence of different plant species MHC II B (5 '-GGTWTGTSGGWTACACTGA-3 ') dfMHCR (5 '-TCAGTGGYYGTSACATC AGA-3 ') (W=A/T; S=G/C; Y=C/T), amplification obtains the cDNA fragment of lefteye flounder MHCII B gene, and its length is 295bp.According to gained fragment design gene specific primer GSP5 ' (5 '-GTGACTTCCTG TCCGTCTCTTTGCCA-3 ') and GSP3 ' (5 '-GGAATCAAGAACGCTGAGAGGTGGAA-3 '), carry out 5 ' and 3 ' RACE PCR respectively, obtain the special band of the about 500bp of 5 ' RACE and the special band of the about 950bp of 3 ' RACE.After analyzing removal overlap (260bp) and joint sequence, Dnaman obtains the full length cDNA sequence of 1182bp, (its molecular weight is 27.78KD to this MHC II B cDNA by the 741bp coding region, PH is 6.69), 5 ' the UTR district of 33bp and 3 ' the UTR district of 407bp form, and have the PolyA tail of a typical tailing signal AATAAA of vertebrates and 27bp.
2), the acquisition of genomic dna sequence: carried out the amplification of intron according to the known MHC II B exon of the close fish of other sibship and the boundary site design special primer of intron.Similar with other vertebrate MHC II B genome structure, lefteye flounder MHCIIB genome includes 5 exons and 4 introns.Pcr amplification has obtained whole 4 introns, introne 1 wherein, and 2,3 and 4 length is respectively 96/84bp, 392bp, 85bp and 109bp, and the length of 5 exons is respectively 85bp, 279bp, 274bp, 114bp and 430bp.Measure the sequence of whole 5 exons and 4 introns, therefore obtained lefteye flounder mhc gene full length sequence (Fig. 1).
3), MHCIIB gene order comparison: infer the protein sequence that has obtained the coded 246bp in 741bp coding region wherein by DNAMAN, its molecular weight is 27.78KD, and PH is 6.69.Analyzing this protein sequence through MEGA 2.0 compares with the protein sequence of the MHC II B of known other vertebrates (Atlantic salmon, carp, beautiful fish, people, mouse, shark, rainbow trout, porgy, perch and zebra fish), its homology is respectively 57.6%, 55.7%, 66.4%, 34.4%, 34.0%, 28.3%, 55.3%, 72.5%, 72.1% and 51.0%.This result shows that the sibship of the MHC II B sequence of lefteye flounder and porgy, perch and beautiful fish is nearer, take second place with the relation of Atlantic salmon, carp and rainbow trout, with shark, mouse and people's sibship farthest.Lefteye flounder MHC II B amino acid of inferring and fish, mouse and people's MHC II B aminoacid sequence comparative result, 24 with antigenic peptide bonded related amino acid in, wherein 61 tyrosine (Y), 63 tyrosine (Y), 78 phenylalanines (F), 87 Methionins (K), 99 leucines (L), 105 L-glutamic acid (E), 109 tyrosine (Y) are conservative substantially in fish, and the aspartic acid (N) on 113 all exists in fish, mouse and people.
2. the preparation of lefteye flounder disease-resistance population and responsive colony:
Adopt natural selection and artificial challenge's approach to obtain disease-resistance population and disease-resistance population not.In the lefteye flounder morbidity busy season, collect the individuality of morbidity back survival from plant, infect the fry of collecting with Vibrio anguillarum simultaneously, obtain disease-resistance population and two basic populations of disease-resistance population not according to resistivity size to pathogenic bacterial infection.
3. the screening of lefteye flounder disease resistance related MHC gene marker:
Altogether to from 42 disease-resistant individualities and 42 not 411 mhc genes clones of disease-resistant individuality check order, the result shows, have 13 kinds of different nucleotide sequences, the 13 kinds of different aminoacid sequences (Paol-allele*a Paol-allele*m) of encoding respectively.In 13 kinds of different nucleotide sequences, 5 kinds of different introne 1 sequences are arranged, 12 Nucleotide insertion/deletion mutantions are wherein arranged, 2 Nucleotide conversions and 2 nucleotide transversions.In 84 individualities, existing two or more the different sequences of 59 each and every one body surfaces are arranged, account for 70.2% of total individual number; There are 5 each and every one body surfaces to reveal different sequences more than 3 in 59 individualities, account for 8.47%; The existing 5 kinds of different sequences of 1 each and every one body surface are arranged in 5 individualities, show to have 3 different MHCIIB sites at least.The exogenous array of 280/268bp in 411 positive colony plasmids, remove the intermediary intron sequences, 61 amino acid of encoding altogether, by comparing with the aminoacid sequence of human MHC IIB, the corresponding site of amino acid section of determining the MHCIIB gene that this institute gets is 22～82, and wherein antigen binding site (PBR) is respectively the 35th, 37,39,56,58,60,65,66 and the 75th.In the nucleotide sequence of 280/268bp, 32 Nucleotide are in exons 1, and 10 amino acid of encoding wherein find no nucleotide diversity; The introne 1 sequence of 96/84bp; The exon 2 sequence of 152bp, 51 amino acid of encoding have 25 nucleotide diversity sites, wherein 14 transversion, 10 conversions, 1 existing conversion has transversion again.In total coding region, 13 of pleomorphism sites, wherein the polypeptide binding site is 6, accounts for 46.15% of polymorphic site.
In 13 kinds of different aminoacid sequences (Paol-allele*a Paol-allele*m), Paol-allele*a Paol-allele*f and Paol-allele*j, Paol-allele*k, Paol-allele*i, the Paol-allele*m genotype is that disease-resistance population and susceptible colony are common.Be genotype a, b, c, d, e, f, i, j, k and m come across disease-resistance population and disease-resistance population not simultaneously, and genotype g and h only appear at (its sequence is seen Fig. 2) in the disease-resistance population, and genotype l only appears in the susceptible colony.Fig. 3 is not seen in the distribution of isoallele in disease-resistance population and susceptible colony.After Chi-square Test analyzed, the frequency that genotype d and l occur in susceptible colony was apparently higher than the frequency that occurs in disease-resistance population, and the frequency that genotype g and h occur in disease-resistance population is apparently higher than the frequency (table 1) that occurs in susceptible colony.Therefore, genotype g is considered to disease-resistant relevant mhc gene mark with h, and it may be the responsive relevant mhc gene mark of disease that genotype l then is considered to.
Table 1: the chi square test of 13 kinds of genotype of lefteye flounder MHC II B distribution frequency in disease-resistance population and susceptible colony
| Paol-allele | a | b | d | e | f | g | h | i | j | k | l | m |
| Resistance stock Susceptibility stock Chi-square(p) | 11.90 19.05 0.366 | 23.80 16.67 0.415 | 7.14 23.80 0.035 | 23.80 21.43 0.794 | 26.19 14.29 0.175 | 21.4 0.0 0.0015 | 9.52 0.0 0.040 | 9.52 21.43 0.1315 | 16.67 7.14 0.178 | 7.14 2.38 0.306 | 0.0 19.05 0.003 | 2.38 4.76 0.38 |
Two, lefteye flounder disease resistance related MHC gene marker auxiliary breeding means:
With the MHCIIB genotype g that only in the lefteye flounder disease-resistance population, occurs and h as the disease-resistant related gene mark, the fish of carrying these disease-resistant gene marks is bred, cultivate the offspring, separate the MHCIIB gene in offspring's individuality, the polymorphism of research MHCIIB gene, carry out the cause pathogeny imcrobe infection experiment simultaneously, the genetic development of research disease resistance related MHC IIB genetic marker and with the relation of fish body disease resistance, therefrom filter out and both contained disease resistance related MHC IIB genetic marker, and the fry that disease resistance improves again can be set up disease-resistant improved seeds after cultivating and breeding.
Sequence table
＜110〉Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science
＜120〉lefteye flounder disease resistance related MHC gene marker and auxiliary breeding means thereof
<160>2
<170>PatentIn version 3.1
<210>1
<211>1864
<212>DNA
＜213〉lefteye flounder (Paralichthys olivaceus)
<400>1
acgcggggag tgttcgtcaa gtgaacagag gaacatggct tcattcatcc tcagcttctc 60
cctcttcttc atcacggtct gcacagcagg taggatcaga acactgatca ctgatcaata 120
cactgatcaa tacactgatc aatacatgga tcattctctg actcgttttc ttgtctgctc 180
ttcagatgga tttctgcatt atacggtgac agactgtgag tttaactcct cgaagctgaa 240
cgacatcgag tacacacagt cttactatta caacaaactg gagatcgtca ggttcagcag 300
cagtgtgggg aagtacgttg gatacactga gtttggaatc aagaacgctg agaggtggaa 360
caatggtcca gaggtgatct ctagaagagg tgagaaggag agctactgct ttcacaacgt 420
tggaatcttc acagaatctg ctctgacgaa gtcaggtgag tgtgtgtgtg ttgtatgtgt 480
gtgagtgtgt gttgtatgtg tgtgtgtgtg tgtgtgtgtg tgtgtttgga ttgtatgtgt 540
gagtgtgtgt tgtatgtgtt gtatgtgtgt gtgttgtgtg tgttcttcat gttccagatg 600
aaattcataa agaatctgat gagtcaaact tttatttatt gtctttgttt cctggaaata 660
gactcagctc ggtgtcacca cctgctggtc acacgctgta actacagccg ctgacactga 720
agcttctctt tgcttcttta tgaaataaaa taaaggatga aacaatcctc tgatgttttc 780
agttgatcaa tcgaaccaca gactaagatc cagttcacac ctgacgtgac caacatctgt 840
cttccagcca aaccctacgt caggcttaac tctgtggcgc ccccagctgg caaacatgcc 900
atgttggtct gcagcgtctt tgacttctac cccaaacgga tcaaagtgag ctggcaaaga 960
gacggacagg aagtcacctc tgatgtcact tccactgatg agctggcaga cggtgattgg 1020
tactaccaga tccactccca cctggagtac atgcccaagt ctggagagaa gatttcctgt 1080
gtggtggaac atgccagcct gagtaaacct ctgatcactg actggggtaa atacctgtct 1140
gtcacttgtc tgtctgcccc ctcgtctgtg tgctcacctg ctcagctgtc tttctccctt 1200
tctgtgctca gatccatcca tgcctgagtc agaaagaaac aagatcgcta ttgggacctc 1260
aggactgatc ctgggtctga ccttatctct ggctggattc atctactaca agaggaaggc 1320
ccaaggtcag agccagaaac ccatttcaca ccagtttata cagaccaaat ccagtttaga 1380
ccagttttaa tggtgtgtat ttgttttgac ccttgtctct tctctttaaa ccaggacggg 1440
atcctggttc ccactaagag tcttgtcctg gacctggact tggtcctgga tctatttctg 1500
ttttcctgat ggaagtaaaa ttagctgctg cttcaacctg ctttctgttt tcagtccagc 1560
agactcagcg ctgccacctg ctggacttgc tgaacaccaa attctctgat ggtctgtgct 1620
ggtctgaacc tgatctagaa ccagtcctgt tctcactgtg gatccagact ctggtacatc 1680
agctggatca caaacacctg atttgttgct tcacggtgtg aaatctatat tttttttctc 1740
atcctcctgt tgttaaacag ttttaaatcc tctttgattc agatctcagg tgtctatact 1800
tttactgttt tctaatcaaa ataaactttg aacccaaaaa aaaaaaaaaa aaaaaaaaaa 1860
aaaa 1864
<210>2
<211>246
<212>PRT
＜213〉lefteye flounder (Paralichthys olivaceus)
<400>2
Met Ala Ser Phe Ile Leu Ser Phe Ser Leu Phe Phe Ile Thr Val Cys
1 5 10 15
Thr Ala Asp Gly Phe Leu His Tyr Thr Val Thr Asp Cys Glu Phe Asn
20 25 30
Ser Ser Lys Leu Asn Asp Ile Glu Tyr Thr Gln Ser Tyr Tyr Tyr Asn
35 40 45
Lys Leu Glu Ile Val Arg Phe Ser Ser Ser Val Gly Lys Tyr Val Gly
50 55 60
Tyr Thr Glu Phe Gly Ile Lys Asn Ala Glu Arg Trp Asn Asn Gly Pro
65 70 75 80
Glu Val Ile Ser Arg Arg Gly Glu Lys Glu Ser Tyr Cys Phe His Asn
85 90 95
Val Gly Ile Phe Thr Glu Ser Ala Leu Thr Lys Ser Ala Lys Pro Tyr
100 105 110
Val Arg Leu Asn Ser Val Ala Pro Pro Ala Gly Lys His Ala Met Leu
115 120 125
Val Cys Ser Val Phe Asp Phe Tyr Pro Lys Arg Ile Lys Val Ser Trp
130 135 140
Gln Arg Asp Gly Gln Glu Val Thr Ser Asp Val Thr Ser Thr Asp Glu
145 150 155 160
Leu Ala Asp Gly Asp Trp Tyr Tyr Gln Ile His Ser His Leu Glu Tyr
165 170 175
Met Pro Lys Ser Gly Glu Lys Ile Ser Cys Val Val Glu His Ala Ser
180 185 190
Leu Ser Lys Pro Leu Ile Thr Asp Trp Asp Pro Ser Met Pro Glu Ser
195 200 205
Glu Arg Asn Lys Ile Ala Ile Gly Thr Ser Gly Leu Ile Leu Gly Leu
210 215 220
Thr Leu Ser Leu Ala Gly Phe Ile Tyr Tyr Lys Arg Lys Ala Gln Gly
225 230 235 240
Arg Asp Pro Gly Ser His
245
Claims (3)
1, a kind of lefteye flounder disease resistance related MHC gene marker, 1), the clone of lefteye flounder MHC II B gene its feature comprises aspect following three at the technology contents that is it:; 2), the preparation of disease-resistance population and responsive colony; 3), the screening of disease resistance related MHC genetic marker;
1), the clone of lefteye flounder MHC II B gene: utilize the TRIzol test kit from fish liver, to extract total RNA, utilize the M-MLV reverse transcription to obtain cDNA article one chain, and utilize Smart RACE cDNA amplification kit (Clontech) synthetic respectively 5 '-and 3 '-RACE-Ready-cDNA, RACE PCR reaction is standby; According to the homology design degenerated primer of the aminoacid sequence of different plant species MHC II B, obtain the cDNA fragment of one section MHC II B gene; Carry out RACEPCR according to gained fragment design special primer; Obtain the MHCIIB full length cDNA sequence of 1182 bases, comprise the coding region of 741 bases, 3 ' the terminal untranslated region of the 5 ' untranslated region of 33bp and 407bp, and have the PolyA tail of a typical tailing signal AATAAA of vertebrates and 27bp; Carry out the intron amplification according to the known MHC II B exon of the close fish of other sibship and the boundary site design special primer of intron; The result shows that lefteye flounder MHC II B genome and other vertebrate MHC II B genome structure are similar, include 5 exons and 4 introns, wherein the length of 5 exons is respectively 85bp, 279bp, 274bp, 114bp and 430bp, and introne 1,2,3 and 4 length is respectively 96/84bp, 392bp, 85bp and 109bp; Measure the sequence of whole 5 exons and 4 introns, obtained lefteye flounder MHCIIB complete genome sequence;
Lefteye flounder MHCIIB gene and deduced amino acid thereof:
1 ACGCGGGGAGTGTTCGTCAAGTGAACAGAGGAAC
35 ATGGCTTCATTCATCCTCAGCTTCTCCCTCTTCTTCATCACGGTCTGCACAGcaggtaggatcagaacactgatcactgatcaatacact
1 M A S F I L S F S L F F I T V C T
125 gatcaatacactgatcaatacatggatcattctctgactcgttttcttgtctgctcttCAGATGGATTTCTGCATTATACGGTGACAGAC
18 A D G F L H Y T V T D
215 TGTGAGTTTAACTCCTCGAAGCTGAACGACATCGAGTACACACAGTCTTACTATTACAACAAACTGGAGATCGTCAGGTTCAGCAGCAGT
29 C E F N S S K L N D I E Y T Q S Y Y Y N K L E I V R F S S S
305 GTGGGGAAGTACGTTGGATACACTGAGTTTGGAATCAAGAACGCTGAGAGGTGGAACAATGGTCCAGAGGTGATCTCTAGAAGAGGTGAG
59 V G K Y V G Y T E F G I K N A E R W N N G P E V I S R R G E
395 AAGGAGAGCTACTGCTTTCACAACGTTGGAATCTTCACAGAATCTGCTCTGACGAAGTCAGgtgagtgtgtgtgtgttgtatgtgtgtga
89 K E S Y C F H N V G I F T E S A L T K S
485 gtgtgtgttgtatgtgtgtgtgtgtgtgtgtgtgtgtgtgtttggattgtatgtgtgagtgtgtgttgtatgtgttgtatgtgtgtgtgt
575 tgtgtgtgttcttcatgttccagatgaaattcataaagaatctgatgagtcaaacttttatttattgtctttgtttcctggaaatagact
665 cagctcggtgtcaccacctgctggtcacacgctgtaactacagccgctgacactgaagcttctctttgcttctttatgaaataaaataaa
755 ggatgaaacaatcctctgatgttttcagttgatcaatcgaaccacagactaagatccagttcacacctgacgtgaccaacatctgtcttc
845 cagCCAAACCCTACGTCAGGCTTAACTCTGTGGCGCCCCCAGCTGGCAaaCATGCCATGTTGGTCTGCAGCGTCTTTGACTTCTACCCCA
109 A K P Y V R L N S V A P P A G K H A M L V C S V F D F Y P
935 AACGGATCAAAGTGAGCTGGCAAAGAGACGGACAGGAAGTCACCTCTGATGTCACTTCCACTGATGAGCTGGCAGACGGTGATTGGTACT
138 K R I K V S W Q R D G Q E V T S D V T S T D E L A D G D W Y
1025 ACCAGATCCACTCCCACCTGGAGTACATGCCCAAGTCTGGAGAGAAGATTTCCTGTGTGGTGGAACATGCCAGCCTGAGTAAACCTCTGA
168 Y Q I H S H L E Y M P K S G E K I S C V V E H A S L S K P L
1115 TCACTGACTGGGAgtaaatacctgtctgtcacttgtctgtctgccccctcgtctgtgtgctcacctgctcagctgtctttctccctttct
198 I T D W D
1205 gtgctcagTCCATCCATGCCTGAGTCAGAAAGAAACAAGATCGCTATTGGGACCTCAGGACTGATCCTGGGTCTGACCTTATCTCTGGCT
203 P S M P E S E R N K I A I G T S G L I L G L T L S L A
1295 GGATTCATCTACTACAAGAGGAAGGCCCAAGgtcagagccagaaacccatttcacaccagtttatacagaccaaatccagtttagaccag
230 G F I Y Y K R K A Q
1385 ttttaatggtgtgtatttgttttgacccttgtctcttctctttaaaccagGACGGGATCCTGGTTCCCACTAACTGAGTCTTGTCCTGGA
240 G R D P G S H *
1475 CCTGGACTTGGTCCTGGATCTATTTCTGTTTTCCTGATGGAAGTAAAATTAGCTGCTGCTTCAACCTGCTTTCTGTTTTCAGTCCAGCAG
1565 ACTCAGCGCTGCCACCTGCTGGACTTGCTGAACACCAAATTCTCTGATGGTCTGTGCTGGTCTGAACCTGATCTAGAACCAGTCCTGTTC
1655 TCACTGTGGATCCAGACTCTGGTACATCAGCTGGATCACAAACACCTGATTTGTTGCTTCACGGTGTGAAATCTATATTTTTTTTCTCAT
1745 CCTCCTGTTGTTAAACAGTTTTAAATCCTCTTTGATTCAGATCTCAGGTGTCTATACTTTTACTGTTTTCTAATCAAAATAAACTTTGAA
1835 CCCAAAAAAAAAAAAAAAAAAAAAAAAAAA。
2), the preparation of disease-resistance population and responsive colony: 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 two basic populations of disease-resistance population not according to the resistivity size to pathogenic bacterial infection;
3), the screening of lefteye flounder disease resistance related MHC gene marker:
411 MHCIIB clones to 42 disease-resistant individualities and 42 susceptible individuals check order, 13 kinds of different nucleotide sequences have been found, the 13 kinds of different aminoacid sequences of encoding respectively, 13 kinds of different genotype of called after (Paol-allele*a Paol-allele*m); The exogenous array of 280/268bp in 411 positive colony plasmids, remove the intermediary intron sequences, 61 amino acid of encoding altogether, by comparing with the aminoacid sequence of human MHC IIB, the corresponding site of amino acid section of determining the MHCIIB gene that this institute gets is 22～82, and wherein antigen binding site is respectively the 35th, 37,39,56,58,60,65,66 and the 75th; In total coding region, 13 of pleomorphism sites, wherein the polypeptide binding site is 6, accounts for 46.15% of polymorphic site; In 13 kinds of different aminoacid sequences (Paol-allele*a-Paol-allele*m), Paol-allele*a-Paol-allele*f and Paol-allele*j, Paol-allele*k, Paol-allele*i, Paol-allele*m are that disease-resistance population and susceptible colony are common; Be genotype a, b, c, d, e, f, i, j, k and m come across disease-resistance population and disease-resistance population not simultaneously, and genotype g and h only appear in the disease-resistance population, and genotype l only appears in the susceptible colony; The not distribution of isoallele in disease-resistance population and susceptible colony.Therefore, genotype g is considered to disease-resistant relevant mhc gene mark with h, and it may be the responsive relevant mhc gene mark of disease that genotype l then is considered to.
2, lefteye flounder disease resistance related MHC gene marker according to claim 1 is characterized in that 13 kinds of different genotype of described MHCIIB, and its aminoacid sequence is:
18 28 38 48 58 68
Paol-allele*a SLFFITVCTADGFRYYMVADCEFNSSKLNDIEFTLSFYYNKLEFIRFSSSVGKYVGYTEFG
Paol-allele*b ..............H...T.............Y.Q.Y......Y.................
Paol-allele*c ..............H...T.................Y......Y.........F.......
Paol-allele*d ..............H.T.T.............Y.E.Y.....KIV........F.......
Paol-allele*e .............LH.V.T.............Y.E.Y......I.........F.......
Paol-allele*f ..............H.V.NN............Y...Y......I.........F.......
Paol-allele*g .............LH.T.NS..............Q.Y......IV................
Paol-allele*h ..............H.T.GS............Y.E.Y.....KIV........F.......
Paol-allele*i .............LH.V.DS............YIY.H......Y.........F.......
Paol-allele*j ..............F.V.T.............YIY.H......Y.................
Paol-allele*k ..............H.T.DS............YIY.H......YV................
Paol-allele*l .............LH.A.NS............Y.Q.Y......IV................
PBR * * * * * * ** *
Polymorphic pp p pp ppp p ppp p。
3, a kind of mhc gene marker-assisted breeding method, the method that it is characterized in that it is: the MHCIIB genotype that will only occur in disease-resistance population is as the disease resistance related MHC genetic marker, the fish of carrying these disease-resistant gene marks is bred, cultivate the offspring, separate the MHCIIB gene among the offspring, the polymorphism of research MHCIIB gene, carry out the cause pathogeny imcrobe infection experiment simultaneously, study disease-resistant MHCIIB genetic marker genetic development and with the relation of fish body disease resistance, therefrom filter out and both contained disease-resistant MHCIIB 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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| CN103559427A (en) * | 2013-11-12 | 2014-02-05 | 高扬 | Method for identifying biological sequence and deducing species genetic relationship through digitals |
| CN104304096A (en) * | 2014-08-26 | 2015-01-28 | 中国水产科学研究院黄海水产研究所 | Method for breeding Edwardsiella tarda-resistant excellent flounder breed |
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| CN101911918B (en) * | 2010-03-18 | 2011-06-15 | 中国水产科学研究院黄海水产研究所 | Selective breeding method for disease-resistant superior strains of paralichthys olivaceus |
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| CN103559427A (en) * | 2013-11-12 | 2014-02-05 | 高扬 | Method for identifying biological sequence and deducing species genetic relationship through digitals |
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| CN104304096A (en) * | 2014-08-26 | 2015-01-28 | 中国水产科学研究院黄海水产研究所 | Method for breeding Edwardsiella tarda-resistant excellent flounder breed |
| CN105624168A (en) * | 2016-02-04 | 2016-06-01 | 中国科学院海洋研究所 | Bastard halibut primordial germ cell dead end gene and application thereof |
| CN105624168B (en) * | 2016-02-04 | 2019-02-01 | 中国科学院海洋研究所 | A kind of lefteye flounder archaeocyte dead end gene and its application |
| 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 |
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