鲤蛋白酶体激活因子PA28α亚单位基因组DNA的克隆及序列分析

Cloning and squence analysis of common carp(Cyprinus carpio L.) proteasome activator PA28α subunit genomic DNA

PA28α can activate the latent 20S proteasome together with PA28β,playing an important role in the processing of MHC class I antigen.PMSE1 gene encoding this activator has been characterized and documented in mammals,whereas,few reported among piscine.In the present study,two pairs of primer were designed and synthesised according to the full-length cDNA sequence of proteasome activator PA28α subunit which we had found in common carp.Using PCR two specific gene fragments of PA28α subunit were amplified from total genomic DNA extracted from the spleen of common,PCR products cloned into pMD18-T vector.The recombinant plasmids were verified by sequencing.The PA28α subunit gene(PSME1) of common carp had been successfully cloned.The sequence results were analysized with DNAStar,DNAMAN and BLAST software.Result indicated that carp PSME1 gene encompassed 3 602 nucleotides,11 exons,10 introns,which was very similar to the known PSME1 genes of other species with the same exon/intron arrangement.Three forms are shown at intron/exon boundaries of carp PSME1 gene,exon 5/intron 5 boundary belongs to class 1(GAA/G),exon 8/intron 8 boundary belongs to class 2(TCC/AA),the rest belong to class 0.The splice sites have been well conserved through evolution,and observe the regulation of GT-AG completely.A phylogenetic analysis using PA28α and PA28β protein sequences from the GenBank verifies the presumed orthologous relationships of the carp gene to their mammalian counterparts,and reveales a closer relationship between carp PA28α and zebrafish PA28α than between carp PA28α and mammalian PA28α.Comparing with human,pig,mouse,zebrafish,the structure of carp PMSE1 gene has been also well conserved through evolution.The base number of all exons is almost stable,althongh few introns(introns 1,5,7 in carp;introns 1, 4,7,8 in zebrafish) more variable than other three mammals,especially,the base number of intron 8 in zebrafish PSME1 gene.Our studies have demonstrated the carp PMSE1 gene,moreover,we have done a further work on the distinctions of PMSE1 genes among different species.However,further extensive study on this kinds of subunit genes will be necessary for more information about their molecular properties and functions.

PA28α can activate the latent 20S proteasome together with PA28β, playing an important role in the processing of MHC class 1 antigen. PMSE1 gene encoding this activator has been characterized and documented in mammals, whereas, few reported among piscine. In the present study, two pairs of primer were designed and synthesised according to the full-length cDNA sequence of proteasome activator PA28α subunit which we had found in common carp. Using PCR two specific gene fragments of PA28α subunit were amplified from total genomic DNA extracted from the spleen of common, PCR products cloned into pMD18-T vector. The recombinant plasmids were verified by sequencing. The PA28α subunit gene （PSME1） of common carp had been successfully cloned. The sequence results were anaiysized with DNAStar, DNAMAN and BLAST software. Result indicated that carp PSME1 gene encompassed 3 602 nucleotides, 11 exons, 10 introns, which was very similar to the known PSME1 genes of other species with the same exon/intron arrangement. Three forms are shown at intron/exon boundaries of carp PSME1 gene, exon 5/intron 5 boundary belongs to class 1 （GAA/G）, exon 8/intron 8 boundary belongs to class 2 （TCC/AA）, the rest belong to class 0. The splice sites have been well conserved through evolution, and observe the regulation of GT-AG completely. A phylogenetic analysis using PA28α and PA28β protein sequences from the CJenBank verifies the presumed orthologous relationships of the carp gene to their mammalian counterparts, and reveales a closer relationship between carp PA28α and zebrafish PA28α than between carp PA28α and mammalian PA28α. Comparing with human, pig, mouse, zebrafish, the structure of carp PMSE1 gene has been also well conserved through evolution. The base number ofall exons is almost stable, althongh few introns （introns 1, 5, 7 in carp; introns 1, 4, 7, 8 in zebrafish） more variable than other three mammals, especially, the base number of intron 8 in zebrafish PSME1 gene. Our studies have demonstrated the carp PMSE1 gene, moreover, we have done a further work on the distinctions of PMSE1 genes among different species. However, further extensive study on this kinds of subunit genes will be necessary for more information about their molecular properties and functions.