Sichuan Journal of Zoology Vol. 33 No DOI /j. issn cdna * 1* ( 1. 中国科学院西北高原生物研

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1 DOI /j. issn cdna * 1* ( 1. 中国科学院西北高原生物研究所, 中国科学院高原生物适应与进化重点实验室, 西宁 ; 2. 中国科学院大学, 北京 ; 3. 青海民族大学, 西宁 ; 4. 青海师范大学生命与地理科学学院, 西宁 ) Ochotona curzoniae 3000 ~ 5000 m NO inos L- NO RT- PCR cdna 3 3 RACE inos cdna inos cdna 3981 bp ORF 3450 bp % 87% 82% 78% 78% 82% 83% inos inos inos inos 3 32T 33Y 46R inos inos inos cdna Q Q78 A Cloning and Characterization of Inducible Nitric Oxide Synthase from Plateau Pika Ochotona curzoniae XIE Ling 1 2 GUO Xinyi 1 2 ZHANG Xuze 3 PANG Bo 1 2 DU Yurong 4 ZOU Xiaoyan 1 GUO Songchang 1* ZHAO Xinquan 1* ( 1. Key Laboratory of Adaptation and Evolution of Plateau Biota,Northwest Institute of Plateau Biology,Chinese Academy of Sciences,Xining ,China; 2. University of Chinese Academy of Sciences,Beijing ,China; 3. Qinghai University for Nationalities,Xining ,China; 4. School of Life and Geography Science,Qinghai Normal University,Xining ,China) Abstract Plateau pika Ochotona curzoniae a native species living at 3000 ~ 5000 m above sea-level on the Qinghai- Tibetan Plateau is a typical hypoxia-tolerant mammal. Nitric oxide NO which has been proved to be a vasodilator plays important roles in preventing hypoxia-induced hypoxic pulmonary vasoconstriction and pulmonary hypertension under hypoxia. Inducible nitric oxide synthase inos is a key enzymes that catalyzing L-arginine to NO and regulated by hypoxia stress. In this study the cdna of inos was cloned by RT-PCR and 3 -rapid amplification PCR of cdna ends 3 -RACE from the lung of plateau pika. The results showed that the length of inos cdna of plateau pika was 3981 bp containing an open reading frame ORF of 3450 bp and encoding 1149 amino acid residues. The deduced amino acid sequence was highly identical to those of the American pika 98% followed by rabbit 87% human 82% rat 78% mouse 78% dog 82% and pig 83%. The typical inos domains including oxidase domain reductase domain and FAD binding site were identified in the inos of plateau pika. The molecular phylogenetic trees based on ML and Bayesian methods showed that pika has a close relationship with rabbit. Three positive selection sites 32T 33Y and 46R were identified by the PAML analysis. However the modeling results showed that the evolution of inos was mainly under negative selection suggested that there was no adaptive evolution in plateau pika inos. This study laid a foundation for further exploration KSCX2-EW-J Z ~ * Corresponding author guo@ nwipb. cas. cn xqzhao@ nwipb. cas. cn 511

2 on the expression pattern function and regulatory mechanism of inos in plateau pika adapting to high altitude environment. Key words plateau pika inducible nitric oxide synthase inos cdna clone nitric oxide NO HRE inos Melillo et al Vargiu et al NO Furchgott & Ochotona curzoniae Zawadzki 1980 Palmer et al Lagomopha Ochotonidae O dell et al Vincent ~ 5000 m Nathan & Hibbs Jr 1991 Bogdan 2001 NO Klinger 2007 NO Archer Ge et al. et al NO 1998 Sheafor 2003 NO Manukhina et al Hampl & Herget 2000 inos NO NO NO synthases guanosine 3 5 -cyclic monophosphate cgmp NOSs NAD- DNA PH FAD FMN BH 4 1 L- Stuehr 1999 Alderton et al NOS 1. 1 C N Ca 2 + /Ca 2 + CaM Stuehr 1999 N 3 mg /kg BH 4 L- NO C FAD FMN NADPH Nishida et al Andrew & Mayer 1999 Stuehr 1999 NOS NOS endothelial NOS enos NOS neuronal NOS nnos Ex Taq dntp mix TaKaRa NOS inducible NOS inos pgem-t easy Promega Stuehr 1999 Alderton 1999 soluble guanylyl cyclase SG 3700 Williams 1980 inos cdna 3200 m RNA cdna K1631 Fermentas DH5α DNA et al enos nnos NOS cnos 2 Ca 2 + CaM inos Ca 2 + CaM RNA cdna 6 -α RNA RNAVzol Stuehr 1999 Aktan mg -1α HIF-1α inos 512

3 0. 1% DEPC 1. 5 ml 20 /μl RNA 1 μl DEPC 7 μl RNAVzol 10 min 65 5 min min min 75% cdna % DEPC RNA 0. 1% DEPC GenBank XM _ A 260 /280 1% NM _ XM _ RNA cdna RevertAid First Strand cdna Synthesis Kit Fermentas 20 μl 1 μg /μl RNA 4 μl 5 reaction buffer 4 μl 10 mm dntp Mix 2 μl 100 μm oligo dt 18 1 μl 200 /μl M-MLV 1 μl NM _ NM _ inos cdna Primer Primer 5. 0 Primer Biosoft USA 3 RACE 1 Name of primers inos-1-sense inos-1-antisense inos-2-sense inos-2-antisense inos-3-sense inos-3-antisense inos-4-sense inos-4-antisense inos-5-sense inos-5-antisense dt-ap AP 3 -GSP1 3 -GSP2 1 inos cdna Table 1 Primers for cdna cloning of plateau pika inos 3 5 bp Sequence of primers Products Annealing temperature CACTGAGCTAGAATCCACAGAAG CGTAGCGGATGAGTTGAGTG CAAAGGAAATAGAAACAACAGGAAC GTAGTAGAAAGGGGACAGGACG GATGCCCARCGCTACAACA GTGATGTCCAGGAAGTAGGTGAG ACCCTGGATGAGAGTGGCA CCCGAAGTCCTGTGTTTGC CTGCTTTGTGCGGAGTGC CACCTGCTCCTCGCTTAG CTGATCTAGAGGTACCGGATCCTTTTTTTTTTTTTTT CTGATCTAGAGGTACCGGATCC CTGCTTTGTGCGGAGTGC AGCTAGCCGATGAGGTGCTC 2. 3 PCR cdna Tm RevertAid First Strand cdna Synthesis Kit Fermentas PCR 50 μl cdna 1. 5 μl 10 Ex PCR buffer 5 μl dntp mix 2. 5 mm each 4 μl Sense /Antisense primer 10 μm 1 μl Ex Taq DNA Polymerase 5 U /μl 0. 5 μl 37 μl PCR GSP1 3 -GSP2 AP PCR Ex Taq DNA Polymerase TaKaRa Tm PCR 3 RACE pgem-t easy 3 min s 58 ~ s 72 1 min min PCR 1% DNA Ladder 2. 5 cdna DNA pgem-t easy DNAMAN 6. 0 LB cdna cdna RACE dt-ap oligo dt 18 www. ncbi. nlm. nih. gov www. expasy. org inos 513

4 PAUP4. 0 Swofford 2002 MrBayes Huelsenbeck & Ronquist 2001 ML 3450 bp 1149 Bayes ML GenBank KC Stepwise-addition Bootstrap test % G % T % C % 1000 Bayes A + T % G + C % Markov chain Monte Carlo process 97% 87% 84% % % d N /d S d N nonsynonymous substitution rate d S synonymous substitution rate d N /d S neutral selection negative selection ω = 1 < 1 > positive selection Yang et al Phe 47 Trp 22 Val PAML 165 Pro 74 Met 26 Yang Polar 367 CODEML inos Asn 33 Cys 24 Gln 62 Ser 75 Thr 61 Gly foreground branch background branch branch model branch site model site model Site model M0 M1a M7 3 M3 M2a M8 Anisimova et al likelihood ratio test LRT 2 α- Helix 35. 5% colil 47. 2% 2Δ Anisimova et al ~ 499 β- extended-beta 17. 3% 538 ~ 669 Bayes 722 ~ empirical Bayes BEB Anisimova et al ω > 1 P positive selection site AATAAA cdna A 80% 81% 83% 83% http / /web. expasy. org 1149 inos C 5826 H 9070 N 1620 O 1674 S 50 MW kda pi Hydrophobic Ala 80 Ile 41 Leu Tyr 37 Strongly Basic 168 Arg 74 His 38 Lys 56 Strongly Acidic 128 Asp 50 Glu Expasy inos ~ 1127 C 1 98% 87% 82% 78% 78% 3 82% 83% 83% 82% inos NP_ AAM XP_ inos inos cdna N C 3981 bp ATG TGA inos Met M 863 Gln Q 514

5 Sichuan Journal of Zoology Vol. 33 图１摇高原鼠兔ｉｎｏｓ基因ｃｄｎａ序列为３９８１ｂｐ起始密码子ＡＴＧ和终止密码子ＴＧＡ黑体加粗表示下划线标记的为３端非编码区多聚腺苷化信号序列用灰色阴影表示高原鼠兔ｉｎｏｓ蛋白功能域预测一氧化氮合酶氧化区域黄素蛋白结合区黄素腺嘌呤结合区和一氧化氮合酶碳末端还原域Ｆｉｇ１摇Ｎｕｃｌｅｉｃａｃｉｄｓｅｑｕｅｎｃｅｏｆｐｌａｔｅａｕｐｉｋａｉｎｏｓｉｎ３９８１ｂｐｓｉｚｅａｎｄｆｕｎｃｔｉｏｎａｌｄｏｍａｉｎｓｐｒｅｄｉｃｔｉｏｎｓＳｔａｒｔｃｏｄｏｎＡＴＧａｎｄｔｅｒｍｉｎａｔｉｏｎｃｏｄｏｎＴＧＡａｒｅｂｏｌｄｅｄ３ＵＴＲａｎｄｔｈｅｐｏｌｙａｄｅｎｙｌａｔｉｏｎｓｉｇｎａｌｓｅｑｕｅｎｃｅｉｓｓｈａｄｅｄｂｙｇｒｅｙｂａｃｋｇｒｏｕｎｄＮＯＳｏｘｙｇｅｎａｓｅｄｏｍａｉｎＦｌａｖｏｄｏｘｉｎｂｉｎｄｉｎｇｄｏＮＯＳＣｔｅｒｍｉｎａｌｒｅｄｕｃｔａｓｅｄｏｍａｉｎｍａｉｎＦＡＤｂｉｎｄｉｎｇｄｏｍａｉｎａｎｄ四川动物 2014 年第 33 卷第 4 期 No

6 OS 89Asn 110Thr 111Tyr Ser Lys M3 M0 ω Ser Sarcophilus harrisii Ornithorhynchus anatinus 32T 33Y 46R 64S 73A 2 inos Bayes ML 4 Bayes ML bootstrap 3 m /n m Bayes n ML bootstrap Leptin Yang et al Leptin Yang et al inos in- 22 in- Ge et al α HIF- 1α 189 VEGF Branch model two-ratio ω0 = ω1 = NO inos one-ratio ω = P = Branch-site model OS site model M1a M2a LRT P = T 33Y 46R M7 M8 5 Li et al NO inos cdna Table 2 2 inos Parameter estimates and likelihood ratio test statistics 2Δ for plateau pika inos evolution analysis Model Parameter estimates lnl 2Δ Positive selection site Branch model two-ratio Site model M0 one-ratio M3 M1a M2a M7 M8 Branch-site model ω0 = ω1 = ω = p0 = p1 = p2 = ω0 = ω1 = ω2 = p0 = p1 = ω0 = ω1 = p0 = p1 = p2 = ω0 = ω1 = ω2 = p = q = p0 = p1 = p = q = ω = P = P = P = P = N. A. N. A. none N. A. 32T 33Y 46R 64S 73A 511K N. A. 14Q 16C 17N 19T 32T 33Y 34V 35L 38P 39V 46R 60A 64S 73A 111Y 275Q 511K 573R 653T 760P 791S 793D 849E 850E 974L 999S 1092H Model A alternative p0 = p1 = ω0 = none ω1 = ω2 = P = null p0 = p1 = ω0 = ω1 = ω2 = lnl 2Δ lnl 2 P M0 M3 4 M1a M2a M7 M8 2 50% 95% lnl log likelihood 2Δ 2 lnl1 - lnl2 P means the probability value test by chi-square under the special degree of freedom df the df of M0 vs M3 M1a vs M2a and M7 vs M8 is 4 2 and 2 respectively the positive selection sites with the P 50% were shown and the positive selection sites with the P 95% were shown in bold 516

2 NP_000616. 3 AAM11887 XP_005963745. 1 inos inos Fig. 2 Comparison of amino acid sequences of inos among plateau pika human GenBank accession No. NP_000616. 3 mouse GenBank accession no.

7 2 NP_ AAM11887 XP_ inos inos Fig. 2 Comparison of amino acid sequences of inos among plateau pika human GenBank accession No. NP_ mouse GenBank accession no. AAM11887 and chiru GenBank accession no. XP_ Same residues are shown in grey and mutations in plateau pika inos compared with other mammals are shown in bold 517

8 3 inos m / n m Bayes n ML Fig 3 Molecular phylogenetic tree based on cdna sequences of inos In the m / n m means posterior probability of Bayes tree and n means bootstrap value of ML tree 518

9 80% M3 inos Anisimova et al M3 N FMN FAD DADPH C Alderton et al inos Ca 2 + /CaM Hibbs et al Palmer et al Ca 2 + /CaM Palmer et al in- M2a M8 OS CaM inos CaM Anisimova RPKRREIPLKVLVKAVLFACMLMRKTMASR Chen et al inos 5 379Asp 382Asp Fischmann et al inos inos L- Zn Archer SL Tolins JP Raij L et al Hypoxic pulmonary vasoconstriction is enhanced by inhibition of the synthesis of an endothelium de- inos N rived relaxing factor J inos C FMN FAD DADPH FMN FAD DADPH FMN FAD inos NO Masters et al Stuehr 1997 inos 2 862Met M 863Gln Q inos M2a 3 32T 33Y 46R P > 95% M8 5 32T 33Y 46R 64S 73A P > 95% et al T 33Y 46R 3 inos Charles et al RPRRREIRFRVLVKV- VFFASMLM Venema et al N RRREIRFTVLVKAVFFASVLMRKVMASRVRA CaM Iwashina et al inos inos CaM inos inos inos 200Cys inos Cubberley et al inos inos in- inos inos OS Zn inos 107Cys inos 112Cys 110Cys 115Cys 115Cys inos M Biochemical and biophysical research communications Aktan F inos-mediated nitric oxide production and its regulation J. Life Sciences Alderton WK Cooper CE Knowles RG Nitric oxide synthases structure function and inhibition J. Biochemical Journal 357 Andrew PJ Mayer B Enzymatic function of nitric oxide synthases J. Cardiovascular research Anisimova M Bielawski JP Yang Z Accuracy and power of the. Mol. Nature immu- likelihood ratio test in detecting adaptive molecular evolution J Biol Evol inos cdna Evol Bogdan C Nitric oxide and the immune response J Luo et al nology inos 519 Anisimova M Bielawski JP Yang Z Accuracy and power of bayes prediction of amino acid sites under positive selection J. Mol Biol Charles IG Palmer RM Hickery MS et al Cloning characteriza-

10 tion and expression of a cdna encoding an inducible nitric oxide synthase from the human chondrocyte J. Proceedings of the National Academy of Sciences of the United States of America Chen PF Tsai AL Wu KK Cysteine 99 of endothelial nitric oxide synthase NOS-III is critical for tetrahydrobiopterin-dependent NOS-III stability and activity J. Biochem Biophys Res Commun Cubberley RR Alderton WK Boyhan A et al Cysteine-200 of human inducible nitric oxide synthase is essential for dimerization of haem domains and for binding of haem nitroarginine and tetrahydrobiopterin J. Biochem J Fischmann TO Hruza A Niu XD et al Structural characterization of nitric oxide synthase isoforms reveals striking active site conservation J. Nat Struct Biol Furchgott RF Zawadzki JV The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine J. Nature Ge RL Kubo K Kobayashi T et al Blunted hypoxic pulmonary vasoconstrictive response in the rodent Ochotona curzoniae pika at high altitude J. American Journal of Physiology-Heart and Circulatory Physiology Hampl VC Herget J Role of nitric oxide in the pathogenesis of chronic pulmonary hypertension J. Physiological Reviews Hibbs JB Jr Taintor RR Vavrin Z et al Synthesis of nitric oxide from a terminal guanidine nitrogen atom of L-arginine A molecular mechanism regulating cellular proliferation that targets intracellular iron C / / Nitric Oxide from L-Arginine A Bioregulatory System S Moncada and EAHiggs eds. Elsevier Amsterdam Huelsenbeck JP Ronquist F MRBAYES Bayesian inference of phylogenetic trees J. Bioinformatics Iwashina M Hirata Y Imai T et al Molecular cloning of endothelial inducible nitric oxide synthase gene from rat aortic endothelial cell J. Eur J Biochem Klinger JR The nitric oxide / cgmp signaling pathway in pulmonary hypertension J. Clinics in chest medicine Li HG Ren YM Guo SC et al The protein level of hypoxia-inducible factor-1α is increased in the plateau pika Ochotona curzoniae inhabiting high altitudes J. J Exp Zool 311 A Li HG Guo SC Ren YM et al VEGF 189 expression is highly related to adaptation of the plateau pika Ochotona curzoniae J. High Altitude Medicine & Biology Luo YJ Gao WX Gao YQ et al Mitochondrial genome analysis of Ochotona curzoniae and implication of cytochrome c oxidase in hypoxic adaptation J. Mitochondrion Manukhina E Mashina SY Smirin B et al Role of nitric oxide in adaptation to hypoxia and adaptive defense J. Physiological Research Masters BS McMillan K Sheta EA et al Neuronal nitric oxide synthase a modular enzyme formed by convergent evolution structure 520 studies of a cysteine thiolate-liganded heme protein that hydroxylates L- arginine to produce NO as a cellular signal J. FASEB J Melillo G Taylor LS Brooks A et al Functional requirement of the hypoxia-responsive element in the activation of the inducible nitric oxide synthase promoter by the iron chelator desferrioxamine J. Journal of Biological Chemistry Nathan CF Hibbs Jr JB Role of nitric oxide synthesis in macrophage antimicrobial activity J. Current opinion in immunology Nishida K Harrison D Navas J et al Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase J. Journal of Clinical Investigation O dell TJ Hawkins RD Kandel ER et al Tests of the roles of two diffusible substances in long-term potentiation evidence for nitric oxide as a possible early retrograde messenger J. Proceedings of the National Academy of Sciences Palmer RMJ Ferrige AG Moncada S Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor J. Nature Palmer RMJ Hickery MS Charles IG et al Induction of nitric oxide synthase in human chondrocytes J. Biochem Biophys Res Commun Palmer RMJ Andrews T Foxwell NA et al Glucocorticoids do not affect the induction of a novel calcium-dependent nitric oxide synthase in rabbit chondrocytes J. Biochem Biophys Res Commun Sheafor BA Metabolic enzyme activities across an altitudinal gradient an examination of pikas genus Ochotona J. Journal of experimental biology Stuehr DJ Mammalian nitric oxide synthases J. Biochimica et Biophysica Acta BBA -Bioenergetics Stuehr DJ Structure-function aspects in the nitric oxide synthase J. Annu Rev Pharmacol Toxicol Swofford DL PAUP*. Phylogenetic Analysis Using Parsimony * and Other Methods. Version 4. 0 Beta version M. Sinauer Associates Sunderland Massachusetts USA. Vargiu C Belliardo S Cravanzola C et al Oxygen regulation of rat hepatocyte inos gene expression J. Journal of hepatology Vincent SR Nitric oxide a radical neurotransmitter in the central nervous system J. Progress in neurobiology Venema RC Sayegh HS Kent JD et al Identification characterization and comparison of the calmodulin-binding domains of the endothelial and inducible nitric oxide synthases J. The Journal of Biological Chemistry Williams DR Man at high altitude J. Pulmonary Circulation in Health and Disease Yang Z Nielsen R Goldman N et al Codon-substitution models for heterogeneous selection pressure at amino acid sites J. Genetics

Yang Z. 2007. PAML 4 Phylogenetic Analysis by Maximum Likelihood J. Mol Biol Evol 24 8 1586-1591. Yang J Wang ZL Zhao XQ et al. 2008.

11 Yang Z PAML 4 Phylogenetic Analysis by Maximum Likelihood J. Mol Biol Evol Yang J Wang ZL Zhao XQ et al Natural selection and adaptive evolution of leptin in the Ochotona family driven by the cold environment stress J. PLoS ONE 3 1 e1472. Yang J Bromage TG Zhao Q et al Functional evolution of leptin of Ochotona curzoniae in adaptive thermogenesis driven by cold environmental stress J. PLoS ONE 6 6 e 檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵檵 'E 32 40'N 3250 m 'E 32 47'N 3450 m 101 4'E 32 41'N 3190 m 11 Certhia himalayana Passeriformes Certhiidae 4 2 C. h. yunnanensis 2011 C. h. taeniura * ( 1. 青海师范大学生命与地理科学学院, 西宁 ; 2. 西宁行知摄影服务有限公司, 西宁 ) * 通讯作者, qhczn@ 126. com 521

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i i ii 1 2 3 4 % N) 5 50.0 AMINO ACID #64 [modified by Administrator] IntAmp_1 nc 40.0 30.0 20.0 10.0 0.0-10.0 1 - ala - 10.483 2 - thr - 11.107 3 - gly - 11.872 4 - val - 13.458 5 - ser - 14.903 6 - pro