BIOTECHNOLOGY BULLETIN 2011 7 cdna 1 1 2 1 1 361021 2 78666 Larimichthys crocea SMART switching mechanism at 5' end of RNA transcript DSN duplex-specific nuclease cdna 4. 3 10 6 PFU /ml 95. 8% 0. 5-3 kb 3 961 3 535 ESTs 33 17 16 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 361021 2 Department of Chemistry and Biochemistry Texas State University San Marcos TX 78666 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 4. 3 10 6 PFU /ml. The recombinant percentage of the normalized cdna library was 95. 8% and the cdna inserts sizes ranged between 0. 5-3 kb. A total of 3 961 clones randomly picked from the library were singlepass sequenced. Clustering and assembling of these expressed sequence tags ESTs resulted in a total of 3 535 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 1 5 6 2 5 7 8 3-5 2011-01-21 30600467 2008N0121 2010A001 E-mail yunchen@ jmu. edu. cn E-mail ylwang@ jmu. edu. cn
2011 7 cdna 181 cdna expressed 3 min 68 4 h sequence tag EST duplex-specific nuclease DSN 25 min Danio rerio ESTs 1 cdna 770 588 NCBI ESTs Advantage 2 PCR 1 1 258 2010 11 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 16 2 500 V TOP10 1 1. 1 1. 2. 3 1 μl 37 LB 10-3 10-6 10 200-250 g μl 40 μl LB RNA RDP oligotex 1. 2 1. 2. 4 ESTs M13F 1. 2. 1 RNA mrna M13R PCR RNA RDP 500 bp 3 9 RNA 961 96 5' 1 mg RNA oligotex mrna cross match Phred 1. 2. 2 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 - 5 4 μl Advantage 2 PCR 2 100 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
182 Biotechnology Bulletin 2011 7 bp 95. 8% 1 1. 2. 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 3 961 DNA 3 3 535 ESTs 1 cdna 3 535 ESTs 2 916 uni-transcript 2 1 cdna ESTs ESTs ESTs ESTs ESTs ESTs 1. PCR 2. PCR M. DNA marker 2 cdna 2. 3 cdna cdna 3 1 4. 3 10 6 PFU /ml 96 M13 PCR 4 500-3 000 LYC 3961 0 197 230 3535 ESTs ESTs ESTs 100 bp ESTs 100 bp 2 ESTs % bp LYC 3535 415 2501 2916 82. 49 355 2. 2 cdna 3 LD-PCR cd- uni- ESTs transcript Contigs > = 2 ESTs Singlets ESTs uni-transcripts Contigs Singlets uni-transcripts ESTs uni-transcripts
2011 7 cdna 183 BLAST DNA 16 3 e-value < 1e - 5 SSR simple sequence repeats 33 17 3 EST ES bp E % GO650252 Apoptosis regulator Bcl-2 Equus caballus 157 5. 0E-06 50 ref XP_001490486. 1 GO652055 G-protein coupled receptor RDC1 Salmo salar 329 4. 0E-41 75 ref NP_001134022. 1 GO651938 Interferon gamma inducible protein 30 Danio rerio 285 1. 0E-18 59 ref XP_693944. 2 GO652310 Interferon regulatory factor 3 IRF-3 Paralichthys olivaceus 490 4. 0E-43 79 gb ACY69212. 1 GO651743 Interferon regulatory factor 5 Paralichthys olivaceus 318 2. 0E-51 90 gb ACN72758. 1 GO650714 Lectin mannose-binding 2 Danio rerio 186 6. 0E-28 90 gb AAI33154. 1 GO652436 lymphoid-restricted membrane protein LRMP Danio rerio 332 3. 0E-09 92 emb CAI20727. 2 GO651441 Pokeweed agglutinin-binding protein 29 PW29 Salmo salar 396 6. 0E-12 51 ref NP_001133734. 1 GO651122 Programmed cell death 2 Danio rerio 389 2. 0E-34 63 gb AAH54132. 1 GO651460 programmed cell death 6 interacting protein Gallus gallus 444 1. 0E-53 70 ref NP_001026164. 1 GO650766 sarcoma antigen NY-SAR-48 isoform b Xenopus laevis 295 7. 0E-07 35 ref NP_001090302. 1 GO650665 serine hydrolase-like Danio rerio 369 2. 0E-20 59 emb CAM14020. 1 GO652726 Signaling lymphocyte-activation molecule precursor CD48 Salmo salar 459 3. 0E-22 41 ref NP_001134732. 1 GO652163 TNF ligand superfamily Danio rerio 256 2. 0E-35 85 ref NP_571918. 2 GO651383 TNF receptorof ovarian Danio rerio 384 1. 0E-22 64 gb ABG91566. 1 GO650211 TNF receptor-associated protein 1 Danio rerio 381 1. 0E-22 92 ref NP_001107097. 1 GO650673 zinc finger protein Danio rerio 390 6. 0E-08 45 ref XP_001333328. 3 GO650243 Antigen peptide transporter 2 TAP2 Oncorhynchus mykiss 452 4. 0E-50 68 gb AAD53034. 1 AF115537_1 GO652194 autoantigen RCD8 Danio rerio 387 1. 0E-39 85 emb CAF93035. 1 GO652621 Fc fragment of IgG binding protein Pan troglodytes 310 2. 0E-15 44 ref XP_001137064. 1 GO651124 immunodominant MHC-associated peptides Ctenopharyngodon idella 399 9. 0E-55 83% gb AAS57934. 2 GO650565 immunoglobulin D heavy chain Hippoglossus 203 2. 0E-12 68% gb AAL79934. 1 GO650575 immunoglobulin mu binding protein 2 Gallus gallus 121 5. 0E-09 76% ref NP_001026346. 1 GO650509 MHC class II antigen alpha chain Larimichthys crocea 318 3. 0E-42 77% gb ABV48905. 1 GO650526 MHC class II antigen gene cluster Ovis aries 279 9. 0E-07 56% ref NP_001137364. 1 GO651745 MHC class II antigen gene cluster Oryzias latipes 411 1. 0E-53 88% ref NP_001129989. 1 GO652182 MHC class II antigen gene cluster Danio rerio 329 4. 0E-22 49% ref NP_001071213. 1 GO652686 MHC class II region expressed gene KE2 Oncorhynchus mykiss 426 1. 0E-50 89% gb ACO08333. 1 GO652124 MHC class II regulatory factor RFX1 Danio rerio 354 4. 0E-57 88% ref NP_001083022. 1 GO652505 MHC class II regulatory factor RFX1 Danio rerio 515 1. 0E-60 86% ref NP_001083022. 1 GO651907 squamous-cell carcinoma T-cell-recognized antigen Tetraodon nigroviridis 384 3. 0E-29 78% gb AAG28762. 1 AF299060_1 GO651086 T-cell immunomodulatory protein Canis familiaris 445 3. 0E-61 100% ref XP_851471. 1 GO650244 transcription factor 3 Gallus gallus 323 5. 0E-40 85% ref NP_989817. 2
184 Biotechnology Bulletin 2011 7 3 TNF tumor necrosis factor receptor EST cdna MHC 10 DSN MHC T cdna 82. 49% 19 cdna 20 Thalassophryne nattereri cdna Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ 45. 81% 11 Astatotilapia burtoni cd- I II NA 30% 12 Singlets ESTs 20 MHC MHC 85. 77% 21 1 132 MHCⅡ uni-transcripts 38. 82% 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 15 19 22 7 16 NK T 7 16 17 22 23 IL-1 TNF-α 3 4 6 IL-1 TNF-α 4 6 7 IFN-α IFN-γ 18 5 19 major histocompatibility complex MHC interferon IFN interferon regulatory factor IRF Interleukin IL lectin
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