apo-14 apolipoprotein HDL Anguilla japonica 14 kda 3 Cyprinus carpio Takifugu rubripes Epinephelus bleekeri Ctenopharyngodon idella

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1 4 1 Vol. 4 No JOURNAL OF TROPICAL BIOLOGY Mar apo Marsupenaeus japonicus RNA 3' 5'-RACE apolipoprotein-14 apo-14 ORF 3'-UTR 5'-UTR apo-14 cdna 784 bp ORF 420 bp 139 apo-14 apo-14 apo-14 Q 781 A apolipoprotein kda HDL Anguilla japonica 3 Cyprinus carpio Takifugu rubripes Epinephelus bleekeri Ctenopharyngodon idella Pimephales promelas 11 4 Marsupenaeus japonicus apolipoprotein-14 apo RNA dntp PCR Taq GeneRuler DNA Mixture ladder Trizol MBI Fermentas TaKaRa 1. 2 RNA 0. 5 g 300 μl Trizol 300 μl r min min r min min 1 ml φ = 70% 2 DEPC CARS maoyong@ xmu. edu. cn

2 RT-PCR apo-14 PCR 5' 3' cdna PCR 5 μl PCR w = 1% PCR 1. 4 T PMD18T DH5α LB 37 LB PMD18T PCR SignalP 3. 0 NetNGlyc 1. 0 NetOGlyc 3. 1 NetPhos 2. 0 Bioedit MEGA apo-14 cdna 784 bp bp ATG 57 ~ ~ cdna 3' bp 669 ~ 674 3' - poly A 1 TGGTGCAGTTGACTCCAGTCCTTTATTTCTCTCTACAGGAATTCTCACCAGCACAAG 57 ATG AAG CTG ACA TTT GCT CTC ATC CTT GCA CTC CAA GTG TCA GTG TGT TTC AGT TCT TTG 1 M K L T F A L I L A L Q V S V C F S S L CCT GAG CCC GAC AAG GAG TTA GTT GAG AAA TAT ACT GGG CTG AAG GAA GTT TTC CTT AAG 21 P E P D K E L V E K Y T G L K E V F L K AGG CTT GTC ACT GCT TAT GGA AAG TTC AAA GAC GCC ATT CAG CCA CTG CTA GAG AGC TCA 41 R L V T A Y G K F K D A I Q P L L E S S CCA ACA GGA GAA AAA GCC AAG GAG GTT TTT GAA GAA CTG AAG CAA AGC CCA AGA GTG GAA 61 P T G E K A K E V F E E L K Q S P R V E AGC GCT GTA AAA ATT ATC AGT GGT CTG GCC ACT GAG CTC GAG CCT GTC GTG GAA AAA GCT 81 S A V R I I S G L A T E L E P V V E K A CGC TTG GCT CTT CTC GGT GCT TAT GGC CAT TAT CTC CGG CCC TAC GTT GGC GAA CAC CTG 101 R L A L L G A Y G H Y L R P Y V G E H L GAC GCA GCA ATC ACC AAC GTC AAG CCA GTG CTG GAT ATC GTC CTG CCT CAT GAT GGT TAG 121 D A A I T N V R P V L D I V L P H D G 140 AGTCCGCTGCTCGTCAACCACTGACTACTTCAACCCAGCAAACATGCATTTTCACTGCTTCAATAATGGATCCAAATCTG AAACTATCTGACAGCTGTGTGTCCAGCACAGTCATCATGTCAGTACAGTCTTCTTTTGCATGTATATACTGTATCTGTGT ATTTGATACTGTTCTGTTTAGTGCAGGAAAAAAATAAAATAACATCCGGTCCAGCATTAATAGATTATGAATTATGTGAT TTTGTGTGCCTCATAAAGAAACAAGACTCATTCTAAATAAAAACCTCTGGAAAATGCAAAAAAAAAAA Fig. 1 1 apo-14 cdna Full cdna sequence of apo-14 of Marsupenaeus japonicus signalp3. 0 N apo-14 apo-14 apo-14 N- 33 codon block 5 α- 1 ~ Pro 3

1 apo-14 19 signalp 鄄 NN prediction (euk networks): seguence 1.0 C soore S soore Y soore 0.8 Score 0.6 0.4 0.2 0.

japonicus H.molitrix H.mylodon C.carpio C.gibelio C.idella P.flavascens E.coioides P.altivelis E.bruneus L.calcarifer A.japonica P.fleus P.olivaceus O.mossambicus O.fasciatus O.melastigma T.

3 1 apo signalp 鄄 NN prediction (euk networks): seguence 1.0 C soore S soore Y soore 0.8 Score MKL TFAL ILAL QVSVCFSSLPEPDKEL VEKYTGL KEVFLKRL VTAYGKFK DAIQPLLESSPTGEKAKEVF Position Fig. 2 2 apo-14 Signal peptide prediction of amino acid sequence of apo-14 M.japonicus H.molitrix H.mylodon C.carpio C.gibelio C.idella P.flavascens E.coioides P.altivelis E.bruneus L.calcarifer A.japonica P.fleus P.olivaceus O.mossambicus O.fasciatus O.melastigma T.rubripes M.japonicus H.molitrix H.mylodon C.carpio C.gibelio C.idella P.flavascens E.coioides P.altivelis E.bruneus L.calcarifer A.japonica P.fleus P.olivaceus O.mossambicus O.fasciatus O.melastigma T.rubripes 信号肽 33 鄄 codon block M.japonicus H.molitrix H.mylodon C.carpio C.gibelio C.idella P.flavascens E.coioides P.altivelis E.bruneus L.calcarifer A.japonica P.fleus P.olivaceus O.mossambicus O.fasciatus O.melastigma T.rubripes Fig. 3 3 apo-14 Homology analysis of amino acid sequence of apo-14

4 NetNGlyc1. 0 NetOGlyc 3. 1 apo-14 N- O- NetPhos Ser 4 Netphos 2.0: predicted phosphorylation sites in sequence Phosphorylation potential 1 Serine Threonine Tyrosine Threshold Sepuence position Fig. 4 4 apo-14 Phosphorylation sites prediction of amino acid sequence of apo-14 apo-14 apo % Hemibarbus mylodon Cyprinus carpio apo % 75. 1% Tab. 1 apo-14 Identities and distances of apo-14 proteins M. japonicush. molitrix H. mylodon C. carpio C. gibelio C. idella P. flavescense. coioides P. altivelis E. bruneus L. calcarifera. Japonica P. flesus P. olivaceuso. mossambicus O. fasciatus O. melastigma T. rubripes M. japonicus H. molitrix H. mylodon C. carpio C. gibelio C. idella P. flavescens E. coioides P. altivelis E. bruneus L. calcarifer A. Japonica P. flesus P. olivaceus O. mossambicus O. fasciatus O. melastigma T. rubripes ID identity Dist distance Id Dist apo-14 apo-14 5

5 1 apo Platichthys flesus Paralichthys olivaceus Oryzias melastigma Lates calcarifer Oreochromis mossambicus Perca flavescens Oplegnathus fasciatus Epinephelus coioides Epinephels bruneus Plecoglossus altivelis Anguilla japonica Takifugu rubripes Marsupenaeus japonicus Hypophthalmichthys molitrix Ctenopharyngodon idella 100 Hemibarbus mylodon Cyprinus carpio Carassius gibelio 0.05 Fig. 5 5 apo-14 NJ Neighbor joining tree of full length amino acid sequence of apo-14 protein PUFA HDL VHDL 2 14 apolipoprotein-14 apo-14 Anguilla japonica 3 14 apo cdna 784 bp 139 N % apo-14 apo-14 C α -

6 apo-14 apo-14 apo-Ⅱ 3 α CHOUDHURY apo-14 apoa-Ⅱ 17 Epinephelus coioides Carassius auratus gibelio apo-14 YSL apo-14 apo Epinephelus coioides apo-14 apo-14 Carassius auratus gibelio cagapo-14 cagapo-14 apo-14 KONDO apo-14 apo-14 1 apolipoprotein 784 bp cdna apo-14 apoe Sinergasilus major DNT apo-14 apoa-Ⅰ apob apo-14 apo-14 apolipoprotein-14 apo-14 apo-14 1 VAN LENTEN B J NAVAB M SHIH D et al. The role of high-density lipoproteins in oxidation and inflammation J. Trends Cardiovasc Med / SAITO H LUND-KATZ S PHILLIPS M C. Contributions of domain structure and lipid interaction to the functionality of exchangeable human apolipoproteins J. Prog. Lipid. Res KONDO H KAWAZOE I NAKAYA M et al. The novel sequences of major plasma apolipoproteins in the eel Anguilla japonica J. Biochim. Biophys. Acta / KIM K Y CHO Y S BANG I C et al. Isolation and characterization of the apolipoprotein multigene family in Hemibarbus mylodon Teleostei Cypriniformes J. Comp. Biochem. Physiol. B Biochem. Mol. Biol DEERING M J FIELDER D R HEWITT D R. Growth and fatty acid composition of juvenile leader prawns Penaeus mon-

7 1 apo odon fed different lipids J. Aquaculture / MONTA O M NAVARRO J C. Fatty acids of wild and cultured Penaeus vannamei larvae from Ecuador J. Aquaculture / SHIAU S-Y. Nutrient requirements of penaeid shrimps J. Aquaculture / MIDDLEDITCH B S MISSLER S R HINES H B et al. Metabolic profiles of penaied shrimp dietary lipids and ovarian maturation J. J. Chromatogr VAN DEN OORD A. The absence of cholesterol synthesis in the crab Cancer pagurus L. J. Comp Biochem Physiol ZANDEE D I. Absence of sterol synthesis in some arthropods J. Nature XU X L JI W J CASTELL J D et al. Essential fatty acid requirement of the Chinese prawn Penaeus chinensis J. Aquaculture KOMATSU M ANDO S. A novel low-density lipoprotein with large amounts of phospholipid found in the egg yolk of crustacea sand crayfish Ibacus ciliatus its function as vitellogenin-degrading proteinase J. Biochem. Biophys. Res. Commun SPAZIANI E WANG W L NOVY L A. Serum high-density lipoproteins in the crab Cancer antennarius Ⅳ. Electrophoretic and immunological analyses of apolipoproteins and a question of female-specific lipoproteins J. Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology YEPIZ-PLASCENCIA G M SOTELO-MUNDO R VAZQUEZ-MORENO L et al. A non-sex-specific hemolymph lipoprotein from the white shrimp Penaeus vannamei boone. Isolation and partial characterization J. Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology KONDO H MORINAGA K MISAKI R et al. Characterization of the pufferfish Takifugu rubripes apolipoprotein multigene family J. Gene BABIN P J THISSE C DURLIAT M et al. Both apolipoprotein E and A-Ⅰ genes are present in a nonmammalian vertebrate and are highly expressed during embryonic development J. Proc. Natl. Acad. Sci. U S A CHOUDHURY M YAMADA S KOMATSU M et al. Homologue of mammalian apolipoprotein A-Ⅱ in non-mammalian vertebrates J. Acta Biochim. Biophys. Sin Shanghai MONNOT M J BABIN P J POLEO G et al. Epidermal expression of apolipoprotein E gene during fin and scale development and fin regeneration in zebrafish J. Dev. Dyn LANGE S DODDS A W GUDMUNDSDOTTIR S et al. The ontogenic transcription of complement component C3 and Apolipoprotein A-Ⅰ trna in Atlantic cod Gadus morhua L. a role in development and homeostasis J. Dev. Comp. Immunol ZHOU L WANG Y YAO B et al. Molecular cloning and expression pattern of 14 kda apolipoprotein in orange-spotted grouper Epinephelus coioides J. Comp. Biochem. Physiol. B Biochem. Mol. Biol XIA J H LIU J X ZHOU L et al. Apo-14 is required for digestive system organogenesis during fish embryogenesis and larval development J. Int. J. Dev. Biol WANG R LI Z WANG Y et al. An Apo-14 promoter-driven transgenic zebrafish that marks liver organogenesis J. PLoS One e CHANG M X NIE P LIU G Y et al. Identification of immune genes in grass carp Ctenopharyngodon idella in response to infection of the parasitic copepod Sinergasilus major J. Parasitol Res WINTZ H YOO L J LOGUINOV A et al. Gene expression profiles in fathead minnow exposed to 2 4-DNT correlation with toxicity in mammals J. Toxicol Sci WALLACE K N PACK M. Unique and conserved aspects of gut development in zebrafish J. Dev. Biol STAINIER D Y. No organ left behind tales of gut development and evolution J. Science BATES J M MITTGE E KUHLMAN J et al. Distinct signals from the microbiota promote different aspects of zebrafish gut differentiation J. Dev. Biol

8 Cloning and Sequence Analysis of the apo-14 Gene in Marsupenaeus japonicus QIAO Ying WANG Jun ZHONG Shengping LIU Hongtao MAO Yong College of Ocean and Earth Sciences Xiamen University Xiamen China Abstract Full length of apolipoprotein-14 apo-14 gene was cloned from the total RNA of Marsupenaeus japonicus. Sequence analysis showed that the full cdna sequence was 784 bp in length including the coding region of 420 bp and encoded 139 amino acids. Homology analysis of the apolipoprotein-14 amino acid sequence indicated high sequence similarity in apo-14 amino acid sequences between M. japonicus and other fish. Key words Marsupenaeus japonicas apolipoprotein apo-14 cloning sequence analysis 10 Isolation and Identification of Staphylococci warneri from Diseased Tilapia Fry LI Xiaotuo 1 CHEN Qin 1 LIAO Chenghong 1 GUO Guiying 1 2 LI Xiang 1 JIA Xiaolin 1 ZHENG Jiping 1 YU Jinan 1 1. Key Laboratory of Tropical Biological Resources Ministry of Education 2. Academic Affairs Office Hainan University Haikou China Abstract Strains of Staphylococci the largest Gram positive stains were isolated by direct plating and Gram stain from the samples of livers and gallbladders from the diseased Tilapia fry collected in Haikou. Analysis of 16S rrna gene sequence showed that these isolates were highly homologous with Staphylococcus pasteuri and Staphylococcus warneri but further identified to be Staphylococcus warneri by PCR amplification of the soda gene for superoxide dismutase. Key words tilapia Staphylococcus warneri 16S rrna soda gene

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Supplemental file 3. All 306 mapped IDs collected by IPA program. Supplemental file 6. The functions and main focused genes in each network.

LIST OF SUPPLEMENTAL FILES Supplemental file 1. Primer sets used for qrt-pcr. Supplemental file 2. All 1305 differentially expressed genes. Supplemental file 3. All 306 mapped IDs collected by IPA program.