3 : 1 53 software, and sequence analysis suggested that GPx1 was homotetrameric. Key words: Oreochromis niloticus; glutathione peroxidase1; gene cloni

Transcription

1 South China Fisheries Science Vol. 6, No. 3 Jun., 2010 doi: / j. issn , 2, 1 1 1,,, 1 ( 1.,, ; 2., ) : cdna ( rapid amplification of cdna ends, RACE), ( Oreochromis niloticus) 1 ( glutathione peroxidase1, GPx1) ( complete coding se- quence, CDS) GPx1 984 bp, 5 UTR 56 bp, CDS 576 bp, 3 UTR 352 bp, PolyA 20 bp; ( 3 UTR) TGA ( UGA) 1 ( selenocysteine insertion sequence, SECIS), ( Sec) GPx1 191, kda, 8. 04, N-, GPx1 SecTrpGlnAsn GPx1 GPx1, 43. 2% 58. 2%, 58. 1% 80. 6%, GPx1 Swiss-Model GPx1 3D,, GPx1 1 : ; 1; : Q 785 : A : ( 2010) Molecular cloning and characterization analysis of glutathione peroxidase 1 from Nile tilapia ( Oreochromis niloticus) CAO Yanlin 1, 2, KE Hao 1, LIU Zhenxing 1, ZHANG Jianfei 1, LIN Min 1 ( 1. Guangdong Public Lab. of Veterinary Public Health, Institute of Veterinary Medicine, Guangdong Academy of Agricultural Sciences, Guangzhou , China; 2. College of Life Sciences, South China Agricultural University, Guangzhou , China) Abstract: The complete coding sequence of glutathione peroxidase 1 ( GPx1) of Nile tilapia ( Oreochromis niloticus) rapid amplification of cdna ends ( RACE). was cloned by The GPx1 contains 984 bp, including a56 bp 5 -untranslated region, a 576 bp coding sequence ( CDS), a 352 bp 3 -untranslated region and a 20 bp PolyA tail. A selenocysteine ( Sec) was encoded by the unusual stop codon, TGA, with the selenocysteine insertion sequence ( SECIS) located at 3 UTR. The GPx1 was predicted to encode 191 amino acids, and its molecular weight was kda with a pi of 8. 04; neither signal peptide nor N-Glycosylation site was found. The analysis showed that GPx1 was a non-transmembrane protein, and it possessed classic catalytic tetrad comprising selenocysteine, tryptophan, glutamine and asparagine. We compared GPx1 between Nile tilapia and other vertebrate animals, and found that the similarity of nucleotide acid sequence was 43. 2% 58. 2%, and that of amino acid sequence was 58. 1% 80. 6%. Phylogenic analysis indicated that GPx1s of different classes of vertebrates split into different clusters. The 3D structure of tilapia GPx1 was predicted with Swiss-Model : ; : : ( 2006D ) ; ( 2008B ) : ( ),,, yanlincao@ yahoo. cn :, keha@ tom. com

2 3 : 1 53 software, and sequence analysis suggested that GPx1 was homotetrameric. Key words: Oreochromis niloticus; glutathione peroxidase1; gene cloning GPx) ( glutathione peroxidase,, 1. 2, cdna 0. 01% H 2 O 2 ( Sec), ( reactive oxygen, GPx 0. 5 h species, ROS),,, Trizol RNA, Prime- ( SOD) Script cdna, cd- [ 1-2] ( CAT) NA GPx, GPx1 [ 3-4], GPx GPx1, Oligo 6. 0 [ 5-7 ] GPXS1 / GPXR1 ( 1),, [ 8-9] GPx, GPx1, PCR ( biomarker) : 94 2 min, s, s, ( Danio rerio) 72 1 min, 35, minpcr GPx1, ( Thunnus maccoyii) GPx1,, pmd18-t ( Oplegnathus fasiatus) GPx1, ( Ctenopharyngodon idella) GPx1, ( Anguilla japonica) GPx1 ( complete coding sequence, CDS) GPx, 1 GPx1 [ 10-11] GPx, ( 5 3 ), GPx primer sequence ( 5 3 ) [ ] ( Oreochromis niloticus) GPXS1 GPXR1 CAGTTYGGCCAYCARGA AGRAACTTYTCRAARTTCCA LGPXS2 AGAAGGTGGATGTGAATGGA, LGPXS3 TGATGCCCTGGGTCTCAT, GPx CDS LGPXR2 TATCTGCCTCGATGTCACTTGTC RT-PCR LGPXR3 AGTTCCAGGATACGTCATTC RACE FAP GGCCACGCGTCGACTAGTAC17 ( GPx1),, RAP GGCCACGCGTCGACTAGTACT17 AP GGCCACGCGTCGACTAGTAC GPx , g,, : 94 2 min, s, Trizol Invitrogen, cdna Taq TdT, LGPXS3 / AP, pmd18-t SYBR green premix TaKaRa, PCR Axy- 30 s, 72 1 min, 35, min gen, TOP10 cdna, Top10, Tab. 1 : Y. C/T; R. A /G Primers used in GPx1 cloning GPx 3 RACE, Oligo 6. 0 LGPXS2 LGPXS3, RAP AP ( 1) cdna, LGPXS2 / RAP 30 s, 72 1 min, 35, min : 94 2 min, s, 57

3 GPx 5 RACE 3 LGPXR2 LGPXR3, FAP ( 1) cdna, polyg,, FAP/ LGPXR2, : 94 2 min, s, s, 72 1 min, 35, min, AP/LGPXR3, : 94 2 min, s, s, 72 1 min, 35, min GPx1 SE- CISearchDNA Star ClustalXMEGA 4. 0SignalP 3. 0ProtScaleNetNGlycPSORTTMHMM Swiss-Model GPx1 cdna 2. 1 GPx1 GPx complete CDS GenBank, GQ Anguilla japonica GPx GPx1 cdna Thunnus maccoyii GPx1 984 bp, 5 UTR 56 bp, CDS 576 bp, 3 UTR 352 bp, PolyA 20 bp; TGA ( UGA ) ( Sec) ; ( 3 UTR) 1 Mus musculus ( selenocysteine insertion sequence, SECIS) ( 1) GPx1 191, kda, 8. 04SignalP 3. 0 NetNGlyc, GPx1 ( 2) N GPx1 ProtScalePSORT TMHMM GPx1, GPx1 GPx1 SecTrpGln, Aln 4, GPx1 ( oligomerization GPx1 loop) PGGG ( PGGGmotif) 43. 2% 58. 2%, 58. 1% 3), GPx % ( 2) GPx1 PGGG ( 4) 2 Tab. 2 Fig. 1 1 GPx1 SECIS SECIS of O. niloticus GPx1 GPx1 Comparison of nucleotide and amino acid similarities between O. niloticus GPx1 and other vertebrate animals GPx1 % 2 species nucleotide similarity amino acid similarity Ctenopharyngodon idella Danio rerio Homo sapiens Equus caballus Xenopus ( Silurana) tropicalis Taeniopygia guttata , 3D, GPx1 (

4 3 : 1 55 Fig. 2 2 GPx1 20 NJ Phylogentic tree of 20 species of vertebrate animals based on complete sequence of GPx1 gene 3 Swiss-model GPx1 3D Fig. 3 O. niloticus GPx1 3D model predicted with Swiss-model 3 ( Sec) 21,, 1 GPx UGA, Sec t-rna mrna 3 UTR ( selenocysteine insertion sequence, SECIS), trna UGA [ 14] GPx1 1 UGA, 3 UTR SECIS ( 1), 1 Sec GPx, ( Cys) ( Sec) TrpGln Asn ( N-H), [ 3] GPx GPx1 ( 3, 4) GPx, Arg Lys86 GPx [ 1 5] GPx 3,, [ 16 ] GPx1 ( 3) [ 17 ] PGGG [ 5] GPx1 2 ( 3, 4),

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