BIOTECHNOLOGY BULLETIN cdna. Construction and Preliminary Analysis of Normalized cdna Library from the Gonad of Larimichthys crocea

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1 BIOTECHNOLOGY BULLETIN cdna Larimichthys crocea SMART switching mechanism at 5' end of RNA transcript DSN duplex-specific nuclease cdna PFU /ml 95. 8% kb ESTs cdna ESTs Construction and Preliminary Analysis of Normalized cdna Library from the Gonad of Larimichthys crocea Chen Yun 1 Zhou Peng 1 Zhang Ziping 2 Wang Yilei 1 1 The Key Laboratory of Science and Technology for Aquaculture and Food Safety Fisheries College of Jimei University Xiamen Department of Chemistry and Biochemistry Texas State University San Marcos TX USA Abstract A normalized gonad cdna library was constructed using the combination of SMART and DSN duplex-specific nuclease techniques. The content of cdna library was PFU /ml. The recombinant percentage of the normalized cdna library was 95. 8% and the cdna inserts sizes ranged between kb. A total of clones randomly picked from the library were singlepass sequenced. Clustering and assembling of these expressed sequence tags ESTs resulted in a total of high quality sequences including 17 acquired reproductive-immunization-response related genes and 16 innate immune response-related genes. Key words Gonad development cdna library Larimichthys crocea ESTs Reproductive-immunization Larimichthys crocea N A001 yunchen@ jmu. edu. cn ylwang@ jmu. edu. cn

2 cdna 181 cdna expressed 3 min 68 4 h sequence tag EST duplex-specific nuclease DSN 25 min Danio rerio ESTs 1 cdna NCBI ESTs Advantage 2 PCR μl cdna M1 M2 PCR Wizard SV Geland cdna PCR Clean-Up System 2 ESTs μg cdna Sfi I 50 2 h 500 bp 150 ng Sfi I pdnr-lib V TOP μl 37 LB g μl 40 μl LB RNA RDP oligotex ESTs M13F RNA mrna M13R PCR RNA RDP 500 bp 3 9 RNA ' 1 mg RNA oligotex mrna cross match Phred cdna cdna rrna mito 0. 5 μg mrna SM- CPA3 EST ART TM cdna 1 μl ESTs BLAST GenBank DNA SMART Ⅳ 1 μl CDS-3M Super- e-value Scriptse cdna 1e μl Advantage 2 PCR μl 18 cdna 5 μl cdna 50 μl cdna Tris 1 RDP cdna 300 ng 4 μl 4 2 μl 98 LB 3 37 QIAGEN SMART TM cdna = / Clontech Advantage 2 PCR Clontech DSN Invitrogen 96 M13F M13R Wizard SV Geland PCR Clean-Up System Promega PCR 5 μl PCR 2. 1 RNA mrna cdna RNA 18S rrna 28S rrna RNA

3 182 Biotechnology Bulletin bp 95. 8% RNA NA 2 cdna Sfi I 1. Sfi I M. DNA marker cdna cdna Sfi I GGCCNNNNNGGCC DNA 4 PCR 2. 4 ESTs 1% Sfi I DNA ESTs 1 cdna ESTs uni-transcript 2 1 cdna ESTs ESTs ESTs ESTs ESTs ESTs 1. PCR 2. PCR M. DNA marker 2 cdna 2. 3 cdna cdna PFU /ml 96 M13 PCR LYC ESTs ESTs ESTs 100 bp ESTs 100 bp 2 ESTs % bp LYC cdna 3 LD-PCR cd- uni- ESTs transcript Contigs > = 2 ESTs Singlets ESTs uni-transcripts Contigs Singlets uni-transcripts ESTs uni-transcripts

4 cdna 183 BLAST DNA 16 3 e-value < 1e - 5 SSR simple sequence repeats EST ES bp E % GO Apoptosis regulator Bcl-2 Equus caballus E ref XP_ GO G-protein coupled receptor RDC1 Salmo salar E ref NP_ GO Interferon gamma inducible protein 30 Danio rerio E ref XP_ GO Interferon regulatory factor 3 IRF-3 Paralichthys olivaceus E gb ACY GO Interferon regulatory factor 5 Paralichthys olivaceus E gb ACN GO Lectin mannose-binding 2 Danio rerio E gb AAI GO lymphoid-restricted membrane protein LRMP Danio rerio E emb CAI GO Pokeweed agglutinin-binding protein 29 PW29 Salmo salar E ref NP_ GO Programmed cell death 2 Danio rerio E gb AAH GO programmed cell death 6 interacting protein Gallus gallus E ref NP_ GO sarcoma antigen NY-SAR-48 isoform b Xenopus laevis E ref NP_ GO serine hydrolase-like Danio rerio E emb CAM GO Signaling lymphocyte-activation molecule precursor CD48 Salmo salar E ref NP_ GO TNF ligand superfamily Danio rerio E ref NP_ GO TNF receptorof ovarian Danio rerio E gb ABG GO TNF receptor-associated protein 1 Danio rerio E ref NP_ GO zinc finger protein Danio rerio E ref XP_ GO Antigen peptide transporter 2 TAP2 Oncorhynchus mykiss E gb AAD AF115537_1 GO autoantigen RCD8 Danio rerio E emb CAF GO Fc fragment of IgG binding protein Pan troglodytes E ref XP_ GO immunodominant MHC-associated peptides Ctenopharyngodon idella E-55 83% gb AAS GO immunoglobulin D heavy chain Hippoglossus E-12 68% gb AAL GO immunoglobulin mu binding protein 2 Gallus gallus E-09 76% ref NP_ GO MHC class II antigen alpha chain Larimichthys crocea E-42 77% gb ABV GO MHC class II antigen gene cluster Ovis aries E-07 56% ref NP_ GO MHC class II antigen gene cluster Oryzias latipes E-53 88% ref NP_ GO MHC class II antigen gene cluster Danio rerio E-22 49% ref NP_ GO MHC class II region expressed gene KE2 Oncorhynchus mykiss E-50 89% gb ACO GO MHC class II regulatory factor RFX1 Danio rerio E-57 88% ref NP_ GO MHC class II regulatory factor RFX1 Danio rerio E-60 86% ref NP_ GO squamous-cell carcinoma T-cell-recognized antigen Tetraodon nigroviridis E-29 78% gb AAG AF299060_1 GO T-cell immunomodulatory protein Canis familiaris E % ref XP_ GO transcription factor 3 Gallus gallus E-40 85% ref NP_

5 184 Biotechnology Bulletin TNF tumor necrosis factor receptor EST cdna MHC 10 DSN MHC T cdna % 19 cdna 20 Thalassophryne nattereri cdna Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ % 11 Astatotilapia burtoni cd- I II NA 30% 12 Singlets ESTs 20 MHC MHC % MHCⅡ uni-transcripts % Oncorhynchus mykiss Danio cdna rerio MHC MHCII Penaeus monodon cdna 29. 3% 13 Litopenaeus vannamei 15. 7% MHC Ⅰ 29. 5% cdna 14 ESTs cdna NK T IL-1 TNF-α IL-1 TNF-α IFN-α IFN-γ major histocompatibility complex MHC interferon IFN interferon regulatory factor IRF Interleukin IL lectin

6 cdna Zheng WB Liu GZ Ao JQ et al. Expression analysis of immune-relevant genes in the spleen of large yellow croaker Pseudosciaena crocea stimulated with poly I C. Fish and Shellfish Immunology Beer AE Billingham RE. Host responses to intra-uterine tissue cellular and fetal allografts. Journal of Reproduction and Fertility 1974 suppl Hellberg P Thomsen P Janson PO et al. Leukocyte supplementation increase the luteinizing hormone-induced ovutution rate in vitro perfused ovary. Biology of Reproduction and polymorphonuclear leukocytes in cattle. Theriogenology Pollard JW Bartocci A Arcecl R et al. Apparent role of the macrophage growth factor CSF-1 in placental development. Nature Oraka M Voutilainen R Vihko R. Interferon inhibits steroidogenesis and accumulation of mrna of the steroidogenic enzymes P Chomczynski P. A reagent for the single-step simultaneous isolation of RNA DNA and proteins from cell and tissue samples. Biotechniques Hillier LD Lennon G Becker M et al. Generation and analysis of human expressed sequence tags. Genome Research Magalhaes GS Junqueira-de-Azevedo ILM Lopes-Ferreira M et al. Transcriptome analysis of expressed sequence tags from the venom glands of the fish Thalassophryne nattereri Biochimie Salzburger W Renn SCP Steinke D et al. Annotation of expressed sequence tags for the East African cichlid fish Astatotilapia burtoni and evolutionary analyses of cichlid ORFs. BMC Genomics Preechaphol R Leelatanawit R Sittikankeaw K et al. Expressed sequence tag analysis for identification and characterization of sex-related genes in the giant tiger shrimp Penaeus monodon. Journal of Biochemistry and Molecular Biology Gross PS Bartlett TC Browdy CL et al. Immune gene discovery by expressed sequence tag analysis of hemocytes and hepatopancreas in the pacific white shrimp Litopenaeus vannamei and the Atlantic White Shrimp L. setiferus. Developmental and Comparative Immunology cdna EST Lander Chacin MF Hansen RJ Drost M. Effects of stage of the estrous cycle and steroid treatment on uterine immunoglobulin content Gogolin-Ewens KJ Lee CS Mercer WR et al. Site- directed differences in immune response to the fetus. Immunology Oscc and P45 Oc17 in cultured procine Leydig cells. Molecular Endocrinology M Esteban MA Cuesta A Ortuno J et al. Immunomodulatory effect s of dietary intake of chitin on gilt head seabream Sparus aurata L. innate immune system. Fish Shellfish Immunology Samuel CE. Antiviral actions of interferons. Clinical Microbiology Reviews

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