Genomic Structure,Polymorphism And Expression Analysis Of The Spotted Scat(Scatophagus Argus) MHCⅡ Gene

The spotted scat(Scatophagus argus) is eurythermic and euryhaline, and it is widely distributed in the brackish water and marine habitats of the Indo-Pacific region.For its strong environmental tolerance, the spotted scat has become popular economically in southern China in recent years. To explore the gene structure and properties of MHC II gene in spotted scat(Scatophagus argus), we got the gene’s c DNA complete sequence by homology-based cloning and rapid amplification of c DNA ends polymerase chain reaction(RACE).Then, we further analyzed the introns,gene polymorphism and tissue expression of MHC II gene. These results may lay the foundation for screening of resistance genes and breeding for disease resistance of Scatophagus argus in the future.The main results were as follows:1 Genomic structure, polymorphism and expression analysis of MHC II α geneIn terms of homology-based cloning and RACE, we got the complete sequence of MHC II α gene c DNA of spotted scat with the length of 1319 bp. It was here designated Scar-DXA*01.Analysis showed that:The MHC II α gene is composed of signal peptide(SP), two structure domains(α1,α2), connecting peptide, transmembrane domain and cytoplasmic domain. In this way, it was similar to the general structure of other teleosts.The ORF molecule encodes 242 amino acids. It is here speculated that its molecular structure is C2145H3563N729O895S177, with a molecular weight of 59.56 k Da and isoelectric point(PI) of 5.12. The MHC II α gene including four exons and three introns. Exon 1 encodes 58 bp 5′-UTR and 73 bp signal peptide; exon 2 encode α1, exon3 encode α2, Exons 4 encode connecting peptide, transmembrane domain and cytoplasmic domain and 537 bp 3′-UTR. Exon 1 and exon 2 are separated by intron 1,which is 246 bp; intron 2 is 590 bp, followed by exon 2; intron 3 is 91 bp; All the matching sites of introns and exons are gt/ag.The main locus of function of the MHC IIα protein gene predicted using Predict Protein show that: N-glycosylation sites(NVSW)were found in the α2 domain, six N-nutmeg acetoxylation loci(GCSDSD,GVQVTE,GTSTNV,GVGPAV,GLGLTD, and GVAAGT) were identified inα1 domain, α2 domain and CP/TM/CYT region; three protein kinase C phosphorylation site(SFR,TSR,TVR)was observed in the α2 domain; and four tyrosine kinase II-phosphorylation sites(SQHE,SDSD,SFID, and SRLE) were found in the α1domain and α2 domain.From 209 positive clones in 43 individual spotted scat, 209 different nucleotide sequences were obtained. These contained complete domains, such as SP, α1, α2, CP,TM, and CYT. Coding 163 different amino acid sequence, respectively.According to standard naming rules, these alleles can be classified into 40 main types and 94 subtypes, here called Scar-DXA*0101~Scar-DXA*4001(Gen Bank accession nos.KT176102~KT176141).Analysis showed that, the spotted scat MHC II α gene nucleotide sequence had143 points of variation, and the mutation rate could be 20.46%, of which 112 were parsimony informative sites. Amino acids showed 69 points of variation and the mutation rate was 29.61%, including 54 parsimony informative sites. Analysis of all sequences showed the HD, K,and PI of spotted scat MHC II α gene to be 1.000, 45.219 and 0.065.RT-PCR analysis showed that the MHC II α gene expressed in all eleven tissues,with high level in skin, intestine, spleen, muscle, and gill; moderate level in stomach,brain, heart, and kidney; low level in liver, and eye. After infected Aeromonas hydrophila, the m RNA expression level of MHC II α gene varied in intestine, skin,liver, kidney, gill, and spleen: intestine, spleen, skin, gill, and kidney were fluctuated in different time-points, and are lower than the control group; the expression in liver are lower than the control group in 24h~48h, while, higher than the control group in 12h and 72 h.2 Genomic structure, polymorphism and expression analysis of MHC II β geneIn terms of homology-based cloning and RACE, we got the complete sequence of MHC II β gene c DNA of spotted scat with the length of 1172 bp. It was here designated Scar-DXB*01. Analysis showed that: The MHC II β gene is composed of signal peptide(SP), two structure domains(β1,β2), connecting peptide, transmembrane domain and cytoplasmic domain. In this way, it was similar to the general structure of other teleosts. The ORF molecule encodes 249 amino acids. It is here speculated that its molecular structure is C2239H3730N750O934S198, with a molecular weight of 62.45 k Da and isoelectric point(PI) of 5.09. The MHC II β gene including six exons and five introns. Exon 1 encodes 34 bp 5′-UTR and 55 bp signal peptide; exon 2 encode β1domain, exon 3 and 4 encode β2 domain, exons 5 and 6 encode connecting peptide,transmembrane domain and cytoplasmic domain and 388 bp 3′-UTR. Exon 1 and exon2 are separated by intron 1, which is 216 bp; intron 2 is 1010 bp, followed by exon 2;intron 3 is 85 bp and separate β2 domain; intron 4 and 5 are 258 bp and 94 bp,respectively. All the matching sites of introns and exons are gt/ag. The main locus of function of the MHC II β protein gene predicted using Predict Protein show that: one N-glycosylation sites(NSTE) were found in the β1 domain; four N-nutmeg acetoxylation loci(GNFVGY, GLMYAE, GQEVTS, and GLILGL) were identified inβ1 domain, β2 domain and CP/TM/CYT region; three protein kinase C phosphorylation site(TPR, SLK, SER) was observed in the β2 domain and CP/TM/CYT region; five tyrosine kinase II-phosphorylation sites(SQHE, SDSD,SFID, and SRLE) were found in the β2 domain and CP/TM/CYT region; one Tyrosine kinase phosphorylation site(KVEDVGY) was existed in the β1 domain.From 209 positive clones in 43 individual spotted scat, 209 different nucleotide sequences were obtained. These contained complete domains, such as SP, β1, β2, CP,TM, and CYT. Coding 179 different amino acid sequence, respectively. According to standard naming rules, these alleles can be classified into 48 main types and 96 subtypes, here called Scar-DXB*0101~Scar-DXB*4801(Gen Bank accession nos.KT176142~KT176189). Analysis showed that, the spotted scat MHC II β gene nucleotide sequence had 183 points of variation, and the mutation rate could be25.74%, of which 139 were parsimony informative sites. Amino acids showed 83 points of variation and the mutation rate was 35.02%, including 62 parsimony informative sites. Analysis of all sequences showed the HD, K,and PI of spotted scat MHC II β gene to be 1.000、57.480 and 0.081.RT-PCR analysis showed that the MHC II β gene expressed in all eleven tissues,with high level in spleen, gill, intestine, and skin; moderate level in kidney, stomach,and heart; low level in eye, brain, liver, and muscle. After infected with Aeromonas hydrophila, the m RNA expression level of MHC II β gene varied in intestine,skin,liver, kidney, gill, and spleen: the expression of gill, intestine, spleen are lower than the control group in 12h~48h, while, apparently higher than the control group in 72h; skin,and kidney were fluctuated in different time-points, and are lower than the control group; the expression in liver are lower than the control group in 12h~24h, while,higher than the control group in 48h~72h.