4 MHC-Ⅱβ cdna 119 detected in heartbrain and muscle. Key words Cobia Rachycentron canadum MHC-Ⅱβ Full length cdna mrna expression MHC major histocompa

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1 Chinese Journal of Zoology ~ 128 MHC-Ⅱβ cdna 12 1* 2* RACE bp Rachycentron canadum MHC-Ⅱβ cdna 20 bp 5 UTR 394 bp 3 UTR 747 bp ORF ku MHC-Ⅱβ Mus musculus Gallus gallus Homo sapiens MHC-Ⅱ β 28. 5% ~ 77. 5% β1 β2 CP / TM / CYT Real-time PCR MHC-Ⅱβ MHC-Ⅱβ cdna Q781 A Full Length cdna Cloning and Tissue Expression of Major Histocompatibility Complex MHC -Ⅱβ in Cobia Rachycentron canadum MAO Li-Na 1 2 FENG Juan 1 * LI Yu-Gu 2 * HOU Yue-E 1 GUO Zhi-Xun 1 XU Hai-Dong 1 1 The South China Sea Fisheries Research InstituteChinese Academy of Fishery ScienceGuangzhou 2 South China Agriculture UniversityGuangzhou China Abstract The homology cloning and RACE PCR were employed to clone the MHC-Ⅱ β gene from cobia Rachycentron canadium. The full length cdna of MHC-Ⅱβ comprises bp with a 20 bp 5 untranslated region UTR a 394 bp 3 UTR and a 747 bp open reading frame ORF encoding a polypeptide of 248 amino acid residues with a predicted molecular weight of ku and theoretical isoelectric point of The putative protein showed homology varying from 28. 5% to 77. 5% when compared with other fishesmouse Mus musculus chicken Gallus gallus and human Homo sapiens. The protein sequence showed all the important features including leader peptideβ1β2cp / TM / CYT regions and conserved cysteines. The MHC-Ⅱ β expression was detected in all tested tissues with different expression levels. The high expression was detected in head kidneymoderate expression was detected in gillspleen and intestinewhile low expression was No. 2007ZD10 * juanf@ 21cn. com liyugu@ scau. edu. cn maolina2003@ 163. com

2 4 MHC-Ⅱβ cdna 119 detected in heartbrain and muscle. Key words Cobia Rachycentron canadum MHC-Ⅱβ Full length cdna mrna expression MHC major histocompatibility complex Perciformes Percoidei Rachycentridae MHC 8 MHC MHC MHC 9 MHC 10 - MHC 12 MHC-Ⅱβ MHC MHC-Ⅱβ cdna cdna MHC 1950 George Snell 1 MHC-Ⅱβ MHC Homo MHC-Ⅱβ sapiens HLA Mus musculus H-2 4 MHC-Ⅱ β Gallus gallus B MHC MHC- MHC Ⅱ MHC-Ⅱ MHC MHC Hashimoto 2 Ⅰα3 Ⅱβ 2 1 d Cyprinus carpio MHC MHC 1. 2 RNA cdna g Trizol Invitrogen RNA RNA Danio rerio 3-80 RNA 5 μg Oncorhynchus mykiss 4 Salmo salar 5 Ictalurus punctatus 6 Chrysophrys major 7 MHC-Ⅱβ Rachycentron oligo-dt 1 1 μl 10 pmol / L RT-PCR cdna MHC-Ⅱβ canadum MHC CLUSTAL W2

3 120 Chinese Journal of Zoology 45 MEGA4. 1 RCMC2BF UTR 3 UTR RCMC2BR 1 MHC-Ⅱβ PCR 1% MHC-Ⅱ β ORF pmd18-t 1. 4 cdna 3 RCMC32BOUT RCMC32BIN RCMC52BOUT RCMC52BIN 1 3 qpcr semi-nested PCR cdna rapid amplification of cdna ends RACE 3 5 PCR PCR PCR PCR 1% pmd18-t TOP10 SYBR Green Ⅰ Mastercyclerep realplex DNAstar M5 M3 1 4 TOP MHC-Ⅱ β Real- time PCR g bp cdna β-actinf β-actinr PCR PCR Eppendorf AGHamburgGer NCBI BLAST many 4 DNAstar MHC-Ⅱβ cdna Mastercyclerep realplex System PHDsec program 2 - ΔΔCT CLUSTAL W2 Table 1 1 Oligonuleotide primers used in this experiment Prime Nucleotide sequence 5-3 Applied range RCMC2BF AGCAGCAGTGTGGGGAAGT RCMC2BR ACTCAGGCATGGACGGATC oligo-dt AAGCAGTGGTATCAACGCAGAGTACT 30 VN SMART 3a CAGAGTACTTTTTTTTTTTTTTTT 3 RACE RCMC32BOUT GAACGCTGGAACAAAGGTC 3 RACE RCMC32BIN CCGAGATGGACAGGAAGTG 3 RACE Olido-dG GGGGGGGGGGGGGGG A / T / C 5 RACE RCMC52BOUT TACCAGTCGCCGTCAGCC 5 RACE RCMC52BIN TTGTTCCAGCGTTCTGCGTT 5 RACE M5 AGAGCTCTCAACTCACCATC M3 GGACCAGACTGAGGTGTTC β-actinf TGAGACCACCTACAACAGC RT-PCR β-actinr CTGCATCCTGTCAGCGAT RT-PCR J2BF ACCTCTGGTTATTGACTGGG RT-PCR J2BR TCCAGCCAGAGATAAGATCAGAC RT-PCR

4 4 MHC-Ⅱβ cdna bp 5 UTR 394 bp 3 UTR 3 RACE PCR 5 RACE PCR 704 bp 325 bp MHC-Ⅱβ MHC-Ⅱβ cdna 2 MHC-Ⅱβ cdna 747 bp 20 bp AATAAA 16 bp Poly A 3 UTR RNA 1 DNAstar ATTTA RNA instability motifs bp Fig. 1 1 RACE PCR Electrophoresis pictures of homology cloning and RACE PCR A. 1. PCR B. 3 RACE C. 5 RACE M. DL 2000 DNA A. Product of homology cloning1. Homology cloning product B. 3 RACE PCR product1. The first round PCR product 2. The second round PCR product C. 5 RACE PCR product1. The first round PCR product 2. The second round PCR product. M. DL 2000 DNA ladder MHC-Ⅱ β N- 69 GVKNAE 98 MHC-Ⅱ β cdna 248 GLDYTA 149 GQEVAS 220 GLILGL 2 DNAstar CP / TM / CYT ku leader peptide β1 β2 CP / TM / CYT β1 32 N- NSSD β2 4 C- 112 SVR 178 TPR 193 SLK 209 SER 5 Ⅱ 132 SVYD 157 SSTE 173 SHLE 193 SLKE 205 GXXGXXXGXXXXXXG CLUSTAL W2 MHC-Ⅱβ MHC-Ⅱβ 3 β2 MEGA Cyphotilapia frontosa SMPE β1 β2 4 DNA star

5 122 Chinese Journal of Zoology 45 Fig. 2 2 MHC-Ⅱβ cdna cdna sequence of cobia MHC-Ⅱβ and deduced amino acid sequence atg tga aataaa RNA attta < > β1 β2 The start and stop codons or the putative polyadenylation signal aataaa are shown in bold print. RNA instability motif attta in the 3 UTR is represented by box. The start amino acid of different region is underlined. Lowercase letters show nucleotide sequencecapital letters show amino acid sequence < indicates the starting position of the region > indicates the termination position of the regionthese regions include the leader peptideβ1 regionβ2 regioncp / TM / CYT region connecting peptidetransmembrane and cytoplasm. Epinephelus akaara 77. 5% 77. 1% 77. 1% 75. 5% 75. 1% 73. 5% Dicentrarchus labrax Morone 70. 3% 69. 9% 69. 9% 68. 7% saxatil Stizostedion vitreum Larimichthys crocea 63. 1% 61. 4% 51. 8% 51. 8% Gasterosteus aculeatus Scophthalmus MHC-Ⅱ β maximus Paralichthys olivaceus 28. 5% 33. 7% 30. 1%

6 4 MHC-Ⅱβ cdna MCH-Ⅱβ ORF PCR 4 β1 β % β1 MHC-Ⅱβ % β1

7 124 Chinese Journal of Zoology 45 3 Fig. 3 MHC-Ⅱβ MHC-Ⅱβ Alignment of the predicted cobia MHC-Ⅱβ amino acid sequence with other known MHC class Ⅱβ amino acid sequences β1 β2 CP / TM / CYT *. β1 β2 4 GXXGXXXGXXXXXXG Dare AAA87891Icpu U77597 Paol L04805 Scma DQ Dila AM Mosa L33967 Chma AY Lacr ABV48908 Epak ACA63828 Stvi AY Gaac BT Cyfr L13232 Onmy AF Sasa X70167 Homo CAA47028 Mumu AAC05286 Gaga NP Regions corresponding to the putative leader peptideβ1 domainβ2 domain connecting peptide transmembrane region and cytoplasmic tail are shown above the sequences. * means that the residues or nucleotides in that column are identical in all sequences in the alignment means that conserved substitutions have been observed. means that semi-conserved substitutions are observed four conserved cysteine is shaded conserved GXXGXXXGXXXXXXG box is represented by box. Sequence accession numbers are as follows Raca Rachycentron canadum Dare Danio rerio AAA87891 Icpu Ictalurus punctatus U77597 Paol Paralichthys olivaceus L04805 Scma Scophthalmus maximus DQ Dila Dicentrarchus labrax AM Mosa Morone saxatilis L33967 Chma Chrysophos major AY Lacr Larimichthys crocea ABV48908 Epak Epinephelus akaara ACA63828 Stvi Stizostedion vitreum AY Gaac Gasterosteus aculeatus BT Cyfr Cyphotilapia frontosa L13232 Onmy Oncorhynchus mykiss AF Sasa Salmo salar X70167 Homo Homo sapiens CAA47028 Mumu Mus musculus AAC05286 Gaga Gallus gallus NP

8 4 MHC-Ⅱβ cdna Clustal W2 MEGA4. 1 MHC-Ⅱβ β2 Fig. 4 Phytogenetic tree of MHC-Ⅱβ gene β2 region amino adds sequences in different groups with program Clustal W2 and MEGA 4. 1 using neighbor-join method bootstrap The numbers were the percentages of bootstrap values supporting each node from replicas. Rachycentron canadum Danio rerio Ictalurus punctatus Paralichthys olivaceus Scophthalmus maximus Dicentrarchus labrax Morone saxatilis Chrysophos major Larimichthys crocea Epinephelus akaara Stizostedion vitreum Gasterosteus aculeatus Cyphotilapia frontosaoncorhynchus mykiss Salmo salar Homo sapiens Mus musculus Gallus gallus 2. 4 MHC-Ⅱβ MHC-Ⅰ MHC-Ⅱ MHC-Ⅲ ⅡMHC cdna β-actin J2BF J2BR Real-time PCR MHC-Ⅱ β γ ⅡMHC ⅡMHC- MHC-Ⅱβ ± ± ± ± ± ± ± α β γ MHC-Ⅱ T ThCD4 + MHC MHC 13 MHC-Ⅱ MHC α1 α2 β1 β2

9 126 Chinese Journal of Zoology 45 Fig. 5 5 Alignment of amino acid sequences among different Cobias β1 β2 CP / TM / CYT - Regions corresponding to the putative leader peptideβ1 domainβ2 domainconnecting peptidetransmembrane region and cytoplasmic tail are shown above the sequences. The same amino acids is represented by -. MHC-Ⅱ β 33 β1 β bp 20 bp 5 UTR 394 bp 4 3 UTR 747 bp ORF UTR RNA ATTTA RNA instability motifs mrna PCR 17 β1 GXXGXXXGXXXXXXG 92 β2 94 CP / Cosson 21 MHC-Ⅱ α TM / CYT 45 β1 β2 4 αβ 2 MHC-Ⅱβ MHC-Ⅱα 3 32 ~ 35 N- 22 NSSD MHC-Ⅱ CD4 3 MHC MHC 22 β1 3 β

10 4 MHC-Ⅱβ cdna 127 β MHC β1 PBR β2 MEGA Glamann J MHC-Ⅱβ 24 - MHC-Ⅱ β % ~ 77. 5% Real-time PCR MHC-Ⅱβ MHC-Ⅱ β 10 Ⅱ MHC MHC Zhang 33 MHC-Ⅱβ MHC-Ⅱβ MHC MHC-Ⅱβ cdna 1 Klein J. George Snell's first foray into the unexplored territory of the major histocompatibility complex. Genetics 2 Hashimoto KNakanishi TKurosaw Y. Isolation of carp genes encoding major histocompatibility complex antigens. PNAS Ono HKlein DVincek Vet al. Major histocompatibility complex class Ⅱ genes of zebrafish. PNAS Complete coding sequence of rainbow trout Mhc Ⅱ beta chain. Scand J Immunol Hordvik IGrimholt UFosse V Met al. Cloning and sequence analysis of cdnas encoding the MHC class Ⅱ β chain in Atlantic salmon Salmo salar. Immunogenetics Godwin U BAntao AWilson M Ret al. MHC class Ⅱ B genes in the channel catfish Ictalurus punctatus. Dev Comp Immunol Chen S LZhang Y XXu M Yet al. Molecular polymorphism and expression analysis of MHC class Ⅱ B gene from red seabream Chrysophrys major. Dev Comp Immunol Rene J M. Inference of structure and function of fish major histocompatibility complex MHC molecules from expressed genes. Fish & Shellfish Immunology Bakheet TFrevel MWilliams B R Get al. ARED human AU-rich element-containing mrna database reveals an unexpectedly diverse functional repertoire of encoded proteins. Nucleic Acids Res Shaw GKamen R. A conserved AU sequence from the 3 untranslated region of GM-CSF mrna mediates selective mrna degradation. Cell Prapansak STsuyoshi OIkuo Het a1. Cloning characterization and expression of cdna containing major

11 128 Chinese Journal of Zoology 45 histocompatibility complex class IⅡαand Ⅱβ genes of Japanese flounder Parcdichthys olivaceus. Fisheries Science Zhang Y XChen S L. Molecular Identification Polymorphismand expression analysis of major histocompatibility complex class ⅡA and B genes of turbot Scophthalmus maximus Marine Biotechnology Chen S LXu M YHu S Net a1. Analysis of imunune - relevant genes expressed in red seabream Chrysophrys major spleen. Aquaculture Buonocore FRandelli ECasani Det al. Molecular cloningdifferential expression and 3D structural analysis of the MHC class-Ⅱ β chain from sea bass Dicentrarchus labrax L.. Fish & Shellfish Immunology Sultmarm HMayer W EFigueroa Fet a1. Organization of Mhc class Ⅱ B genes in the zebrafish Brachydanio rerio. Genomics Cosson PBonifacino J S. Role of transmembrane domain interactions in the assembly of class Ⅱ MHC molecules. Science Brown J HJardetzky T SGorga J Cet al. Threedimensional structure of the human class Ⅱ histocompatibility antigen HLA-DR1. Nature Scharsack J PKalbe MSchaschl H. Characterization of antisera raised against stickleback Gasterosteus aculeatus MHC class I and class Ⅱ molecules. Fish & Shellfish Immunology Walker R BMc Connell T JWalker R A. Variability in an MHC Mosa class Ⅱ beta chain encoding gene in striped bass Morone saxatilis. Dev Comp Immunol Ono HO'huigin CVincek Vet al. Exon-intron organization of fish major histocompatibility complex class Ⅱ B genes. Immunogenetics Beck SKelly ARadley Eet al. DNA sequence analysis of 66 kb of the human MHC class Ⅱ region encoding a cluster of genes for antigen processing. J Mol Biol Acha-Orbea HScarpellino L. Nonobese diabetic and nonobese nondiabetic mice have unique MHC class Ⅱ haplotypes. Immunogenetics199l Hosomichi KMiller M MGoto R Met al. Contribution of mutationrecombinationand gene conversion to chicken MHC-B haplotype diversity. J Immunol Juul-Madsen H RGlumuno JMadsn H Oet a1. MHC class Ⅱ beta-chain expresion in the rainbow trout. Scand J Immunol Koppang E OLundin MPress C Met a1. Differing levels of Mhc class Ⅱ B chain expression in a range of tissues from vaccinated and non-vaccinated Atlantic salmon Salmo salar L.. Fish & Shellfish Immunology MHCⅡ B cdna Ono HO'Huigin CVincek Vet a1. New beta chainencoding Mhc classⅡgenes in the carp. Immunogenetics Zhang Y XChen S LLiu Y Get a1. Major histcompatibility complex class Ⅱ B allele polymorphism and its association with resistance / susceptibility to Vibrio Anguillarum in Japanese flounder Paralichthys olivaceus. Mar Biotech

AFLP % 1. 1 DNA 5 RAPD DNA 0. 1g % ~ ph 6 10 DNA 9 40μl TE SSR STS 20ng /L

AFLP % 1. 1 DNA 5 RAPD DNA 0. 1g % ~ ph 6 10 DNA 9 40μl TE SSR STS 20ng /L 2012 13 2 201-206 Journal of Plant Genetic Resources / 071001 41 SSR 21 SSR 41 52 2 ~ 4 2. 5 Shannon 0. 21 ~ 1. 40 0. 73 5. 54 3 7 9 13. 16% ~ 186. 49% 5. 48 6 11 SSR Genetic Diversity of Peanut Arachis