| Roughskin sculpin(Trachidermus fasciatus), protected in the National Order II, is one of the most four famous freshwater fishes in China. It is only restricted to the Yalu River, Qinglong River and Fuchun River because of the water conservancy projects, environmental pollution and overfishing. To protect and restore the populations of roughskin sculpin, experiments and researches of artificial breeding had been carried out and succeeded. Six important enzymes (IDH, DLST, PK, ADH, ADH5, UGE, PGM1) in glucose metabolism at the level of gene were studied at the first time in order to provide the theoretical basis of baits preparation and diease prophylaxis and treatment in the breeding of roughskin sculpin.In this study, cDNA of seven proteins were cloned with the RACE-SMART technique. Sequence analysis, homology contrast, evolutional relationship analysis and protein prediction were investigated with the application of DANman, MEGA, Genedoc, NCBI. Expressions of IDH and PK in different tissues were determined based on semi-quantitative PCR. The main results are listed as below.Full length cDNA of NADP-IDH (1836bp) was cloned. The cDNA contained a 5' UTR (130bp), a 3'UTR (350bp) and an open reading frame (ORF) of 1356bp which encoded a 452 (Mr 50.51kDa) amino-acid polypeptide with isoelectric point (pI) 6.76. The existence of a conservative domain of isocitrate dehydrogenase superfamily of NADP-IDH was predicted. Coenzyme binding sites in this domain were Leu327, His348, Gly349, Thr350, Val351, Arg353, His354, Thr367, Asn368. These amino acid residues were highly conservative in structure. Spatial structure simulation indicated that periphery of this protein wasα-helix, and inside were P-pleated sheet structure and random coil, perhaps it was the active centre of the combination of this protein and its substrate. The homology of NADP-IDH between roughskin sculpin and other species submitted to GenBank was analyzed. The similarity was 89.14%-94.25% between roughskin sculpin and other fishes, and it was 80.53%-86.34% among roughskin sculpin and amphibia, mammals. In the species whose full-length gene sequences were already submitted, the genetic relationship between roughskin sculpin and Tilapia NADP-IDH was the nearest. Result of Semi-quantitative PCR showed that IDH gene was widely expressed in heart, liver, gill, muscle and stomach. The expression was the highest in muscle, then stomach, heart, liver and gill. It showed that muscle, stomach and heart were the major aerobic metabolic organs of roughskin sculpin. The expression of IDH was little changed before and after the stimulation by Vibrio anguillarum in the liver and gills, and the expression patterns of IDH in muscle, stomach and heart were basically the same. The expression of IDH in muscle, stomach and heart of normal roughskin sculpinin had similar level until 2h after infected by Vibrio anguillarum, and reduced after 6h, resumed normal and gradually increased from12h to 24h.Full length cDNA of DLST (1731bp) was cloned. The cDNA contained a 5' UTR (59bp), a 3'UTR (298bp) and an open reading frame (ORF) of 1374bp which encoded a 458 (Mr 48.92kDa) amino-acid polypeptide with isoelectric point (pI) 8.91. Predictive analysis was that DLST had a combinative domain of coenzyme lipoic acid. It contained a lipoyl attachment site and eight dihydrolipoamide dehydrogenase interaction surfaces. The homology of DLST between roughskin sculpin and other species was analyzed. The similarity was 80.74% between roughskin sculpin and Zebra fish and it was 67.42%-73.80% among roughskin sculpin and amphibia, birds, mammals. In the species whose full-length gene sequences were already submitted, the genetic relationship between roughskin sculpin and Zebra fish DLST was the nearest.Full length cDNA of PK (2495bp) was cloned. The cDNA contained a 5'UTR (584bp), a 3'UTR (282bp) and an open reading frame (ORF) of 1629bp which encoded a 543 (Mr 58.74kDa) amino-acid polypeptide with isoelectric point (pI) 5.90. Predicted that PK contained three domains and a total of 8 active sites,39 domain interfaces.85NFSHG89,123DTKGP127,302MVARGDLG309 were conservative motifs in PK. These motifs had catalytic activity, metal cation and substrate binding sites. The homology of PK between roughskin sculpin and other species was analyzed. The similarity of PK was 84.50% between roughskin sculpin and Salmo Salar, 77.68% between roughskin sculpin and Zebra fish, and it was 58.67-66.42% among roughskin sculpin and amphibia, birds, mammals. In the species whose full-length gene sequences were already submitted, the genetic relationship between roughskin sculpin and Salmo salar PK was the nearest. Result of Semi-quantitative PCR showed that PK was widely expressed in heart, liver, gill, muscle and stomach. The expression was the highest in liver. It showed Glycolytic pathway existed in five tissues of roughskin sculpin, and liver was the most major place. The expression of PK was not changed before and after the stimulation by Vibrio anguillarum in all tissues in different periods. It showed that the expression of PK was not influenced by bacterial infection.Full length cDNA of ADH (1365bp) was cloned. The cDNA contained a 5'UTR (163bp), a 3'UTR (227bp) and an open reading frame (ORF) of 975bp which encoded a 325 (Mr 36.97kDa) amino-acid polypeptide with isoelectric point (pI) 6.03. Predictive analysis showed that ADH contained a conservative domain of aldo-keto reductase superfamily. It contained 25 active sites and 4 catalytic tetrads. The homology of ADH between roughskin sculpin and other species was analyzed. The similarity was 84.26%-87.54% between roughskin sculpin and other fishes, and it was 65.75%-71.25% among roughskin sculpin and amphibia, birds, mammals. In the species whose full-length gene sequences were already submitted, the genetic relationship between roughskin sculpin and Salmo salar ADH was the nearest.Full length cDNA of ADH5 (1441bp), including the 24bp 5'UTR and the 286bp 3'UTR, was cloned with the same method. The cDNA contained an ORF of 1131 bp which encoded a 377 (Mr 40.23kDa) amino-acid polypeptide with isoelectric point (pI) 7.02. Predicted that the protein contained two domains, one was the coenzyme binding domain, another one was the catalytic domain; a total of 22 NAD binding sites,11 substrate binding sites,33 dimer interfaces, four catalytic Zn binding sites and four structural Zn binding sites was also contained in the protein. Residues 201-206 of ADH5 amino-acid sequence read GxGxxG was a highly conservative coenzyme binding motif. The Asp, residues 225, was a highly conservative amino acid residues in all the alcohol dehydrogenases which took NAD as a coenzyme. The similarity of amino-acid sequence was 88.30%-95.21% between roughskin sculpin and other fishes submitted to GenBank, and it was 82.18%-83.51% among roughskin sculpin and amphibia, birds, mammals and 79.31% between Amphioxus. The ADH5 gene of roughskin sculpin had the closest relationship with Sparus auratus, and the farthest with Amphioxus.Full length cDNA of UGE (1434bp), including the 156bp 5'UTR and the 225bp 3'UTR, was cloned. The cDNA contained an ORF of 1053bp which encoded a 351 (Mr 38.94kDa) amino-acid polypeptide with isoelectric point (pI) 6.19. Predicted that UGE contains several domains, one was predicted by SMART; This protein contained a total of 4 active sites,27 NAD binding sites,20 substrate binding sites,21 homodimer interfaces. Residues 9-15 of UGE amino-acid sequence read GGxGxxG was a conservative NAD binding motif. Residues 104-108 of UGE amino-acid sequence read YxxxN was a conservative active site motif. The similarity of UGE was 78.29%-85.47% between roughskin sculpin and other fishes, and it was 71.71%-73.71% among roughskin sculpin and amphibian, mammals. The UGE gene of roughskin sculpin had the closest relationship with Rainbow trout.Full length cDNA of PGM1 (2127bp) was cloned, including the 76bp 5' UTR and the 365bp 3'UTR. The cDNA contained an ORF of 1686bp which encoded a 562 (Mr 61.18kDa) amino-acid polypeptide with isoelectric point (pI) 5.89. Predicted that PGM1 contained four domains and a total of 14 active sites,4 metal binding sites,14 substrate binding sites,9 dimer interfaces. The similarity of PGM1 was 85.93% between roughskin sculpin and Zebra fish,82.85% between roughskin sculpin and Salmo Salar, and it was 73.44%-78.42% among roughskin sculpin and amphibia, birds, mammals. The PGM 1 gene of roughskin sculpin had the closest relationship with Zebra fish. |
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