Microsoft Word - 10 金舒博new.doc

Size: px

Start display at page:

Download "Microsoft Word - 10 金舒博new.doc"

永武童
7 years ago
Views:

1 2013 1, 20(1): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J cdna 金舒博 1, 王宁 2, 乔慧 1, 傅洪拓 1,2, 吴滟 1, 龚永生 1, 蒋速飞 1, 熊贻伟 1.,, ; 2., 摘要 : (Macrobrachium nipponense) (crustacean hyperglycemic homone, CHH) cdna, PCR CHH CHH cdna bp, 241 bp 5 UTR, 408 bp (ORF), 355 bp 3 UTR, CHH Cys CHH C GK, ES CHH, CHH CHH I, CHH, PCR, CHH, 1, CHH , CHH : ; ES ; cdna; PCR 中图分类号 : S917 文献标志码 : A 文章编号 : (2013) (CHH ), [1 3] (crustacean hyperglycemic hormone, CHH), CHH, [4 7], CHH (Homarus americanus) [8] (Penaeus monodon) [9] (Macrobrachium rosenbergii) [10] (Litopenaeus schmitti) [11] (Callinectes sapidus) [12] (Chionoecetes bairdi) [13], CHH [14] (Macrobrachium nipponense), CHH, RACE CHH cdna, PCR CHH, CHH,, , 1, 24 h 26~28, 收稿日期 : ; 修订日期 : 基金项目 : (BG ); (2011JBFA02). 作者简介 : (1986 ),,,. jinsb@ffrc.cn 通信作者 : (1964 ),,,. Tel: ; fuht@ffrc.cn

2 1 : cdna 83 1/2, RNA RNA,, RNA RNAiso Plus cdna 3 -Full RACE Kit 5 -Full RACE Kit TaKaRa; iscript cdna Synthesis Kit SsoFast EvaGreen Supermix BIO-RAD; RNeasy Mini Kit QIAGEN; UNIQ-10 DNA Sangon; Sangon ( Microfuge 22R), ( CEQ 8000), ( BioPhotometer), PCR ( Autherized Thermal Cycler) PCR ( BIO-RAD iq5) RNA cdna mg RNA RNA RNAiso Plus RNA, RNA-freeDNaseI RNA, RNA RNA RNA TaKaRa cdna, 80, CHH cdna cdna BIO-RAD iscript cdna Synthesis Kit, CHH PCR cdna : 4 μl5 iscript reaction mix, 1 μl iscript reverse transcriptase, 1 μg RNA, Nuclease-free water 20 μl : 25 5 min; min; 85 5 min; 4 5min cdna CHH CHH (Rimicaris kairei) CHH2 cdna, FS1 RS1( 1) cdna, CHH PCR 50 μl, : 94 2 min; s; s; s; 35, 72 5 min PCR 1.2%, pmd18-t,,, CHH cdna NCBI Blastx CHH cdna, Primer RACE (FS2 FS3) 5 RACE (RS2 RS3)( 1) 3 -Full RACE 5 -Full RACE, cdna, PCR CHH cdna , PCR 1.2%, pmd18-t,, CHH cdna Tab. 1 表 1 基因克隆所用的引物及其序列 Primers and their sequences used in gene cloning primer primer sequence /bp length function FS1 5 -GYSTYGMCAGGATRGAGAAGCTC-3 23 RS1 5 -ATCTGGACWGMGKTSRCGTATTC-3 23 FS2 5 -TACAACCTCTACAGGAAGCCATACG-3 25 FS3 5 -GGGAGGGCTGCTACCAGAACTT-3 22 RS2 5 -CAAGTTCTGGTAGCAGCCCTCC-3 22 RS3 5 -CTTTGCAGGATTGGTCGAGGATG-3 23 middle fragment extension 3 3 -Full RACE 5 5 -Full RACE

3 NCBI Blastx ContigExpress 3 5, CHH cdna DNAStar NCBI CHH cdna BLAST [15] ; SMART ; ClustalX DNAStar MegAlign MAGE4.0 (Neighbour- Joining) CHH [16 17] PCR(qRT-PCR) RNA 3, 3 RNA, BIO-RAD iscript cdna Synthesis Kit 3 RNA cdna, 3 cdna, qrt-pcr cdna CHH cdna 1 QAFS1 QARS1( 2); β-actin cdna 1 Actin F Actin R( 2) qrt-pcr 25 μl: 0.5 μl, Super Mix 10 μl, ddh2o 13 μl, cdna 1 μl( 50 ng) BIO-RAD SsoFast EvaGreen Supermix, PCR, 95 1 min, 40, s, s,, 55 95, BIO-RAD iq5, cdna 3 β-actin, 3 cdna, BIO-RAD iq5, CHH mrna 1, 2 Ct mrna [18 19] ; Excel ; SPSS13. ; Turkey, P< CHH cdna CHH cdna 304 bp, 507 bp bp 5 BLASTx 97% 98% 97%, CHH cdna CHH cdna 97%, CHH cdna ContigExpress 3 5, CHH cdna bp, 241 bp 5 UTR, 408 bp ORF, 355 bp 3 UTR, 3 UTR, Poly(A) ( 1) ( 1), kd, pi 8.250, 17, 14, 49, 40 Signal 3.0 Server Ala26 Trp27, CHH CHH 表 2 荧光定量 PCR 所用的引物及其序列 Tab. 2 Primers and their sequences used in quantitative real-time PCR primer primer sequence /bp length function QAFS1 5 - CTCCCCGGAAGCCTTAAAAG-3 20 QARS1 5 - TTGACTAGCTTGGGACCTCTGTAGT-3 25 Actin F 5 - TATGCACTTCCTCATGCCATC Actin R 5 - AGGAGGCGGCAGTGGTCAT RT-PCR primer RT-PCR control primer

4 1 : cdna 85 (CHH-precursor related peptide, CPRP) K60R61, CHH CPRP KR 74 6 CHH Cys (Cys68, Cys84, Cys87, Cys100, Cys104, Cys113), C Gly134, 1 Cys 5 CHH Ⅱ Gly 1 CHH cdna ; CHH ; ; (Gly 134 ) ; *. Fig. 1 Nucleotide and deduced amino acid sequences of CHH cdnas Signal peptide is shaded; CHH-precursor related peptide is underlined; the dibasic cleavage site (KR) is italic; the putative amidation site (Gly 108 ) is in bold; the asterisk indicates stop codon. 2.3 CHH ClustalW1.83 CHH CHH[ PO(pericardial organ) ES (eyestalk), PO CHH-PO ] ( 3) ( 2), ClustalW1.83 CHH CHH ( 4), CHH CHH Macrobrachium lanchesteri CHH (98%, 96%), CHH-PO(PO ) (80%), CHH (34%~56%) CHH CHH M. lanchesteri CHH (99%), CHH- PO 66%, (Orconectes limosus)chha (Procambarus clarkia) CHH (Nephrops norvegicus) (Homarus gammarus) (Homarus americanus)chhb 62%~69%, CHH (Rimicaris kairei) (Gecarcoidea natalis) (Pachygrapsus marmoratus) (Carcinus maenas) (Scylla olivacea) 5 ES CHH CHH 5 PO CHH 2.4 CHH MEGA4.0 NJ, Bootstrap CHH (ion-transport peptide, ITP), CHH ITP CHH ES PO, CHH ES PO, ES CHH PO CHH CHH CHH ITP ( 4), MEGA 4 NJ ( 3), CHH ITP, CHH, ITP CHH, CHH M. lanchesteri, 100%, PO-CHH

5 86 20 表 3 用于序列分析的 CHH 家族神经肽 Tab. 3 CHH family neuropeptide for sequence analysis sequence name GenBank GenBank access no resource species CHH Macrobrachium nipponense CHH AAL40915 Macrobrachium rosenbergii CHH O77220 Macrobrachium lanchesteri CHH-PO AAL40916 Macrobrachium rosenbergii CHH2 ACS35347 Rimicaris kairei CHHB AAQ22392 Nephrops norvegicus CHHA Q25589 Orconectes limosus CHHB ABA42180 Homarus gammarus CHHB Homarus americanus CHH Q25683 Procambarus clarkia 蟳 CHH ACN87216 Charybdis japonica CHH1-PO ACS35346 Rimicaris kairei CHH ABQ41269 Cancer productus CHH P14944 Carcinus maenas CHH AAQ75760 Scylla olivacea CHH2 Q9U5D2 Marsupenaeus japonicas CHH-PO ABA70561 Potamon ibericum CHH1 O97383 Penaeus monodon CHHB-PO AAO27806 Pachygrapsus marmoratus CHHB AAO27805 Pachygrapsus marmoratus CHH-PO ABL09571 Gecarcoidea natalis CHH ABL09570 Gecarcoidea natalis CHH-PO ABP88270 Scylla olivacea CHH1-PO AAG29432 Carcinus maenas CHHB Q9NGP0 Metapenaeus ensis ITP AAD20820 Locusta migratoria ITP Q26491 Schistocerca gregaria ITPE NP_ Drosophila melanogaster ITPB ACZ96371 Rhodnius prolixus IPT EFA07585 Tribolium castaneum IPT AAY29658 Manduca sexta 71%, CHH, CHH, CHH 2.5 CHH PCR CHH, CHH, 1,, 500 ; 250 ; ;, , 3, 3 t, (P<0.05, 4) CHH CHH cdna Ala26 Trp27,

6 1 : cdna 87

7 88 20 Fig. 2 2 CHH CHH. Alignment of precursor amino acide sequence of Macrobrachium niponense CHH with other crustecean preprochhs The single peptide and dibasic cleavage sites are indicated by the vertical line. Tab. 4 表 4 青虾 CHH 与其他甲壳动物 CHH 成熟肽和前体的相似性 The identity of prochh/mature CHH between Macrobrachium niponense and other crusteceans sequence name CHH mature CHH of M.niponense CHH prochh of M.niponense M. rosenbergii CHH M. lanchesteri CHH Rimicaris kairei CHH Orconectes limosus CHHA Gecarcoidea natalis CHH Procambarus clarkia CHH Nephrops norvegicus CHHB Pachygrapsus marmoratus CHHB Macrobrachium rosenbergii CHH-PO Homarus gammarus CHHB Homarus americanus CHHB Carcinus maenas CHH Scylla olivacea CHH Rimicaris kairei CHH1-PO Carcinus maenaschh1-po Gecarcoidea natalis CHH-PO Pachygrapsus marmoratus CHHB-PO Scylla olivacea CHH-PO % CHH CHH CHH Ⅰ CHH (CPRP) KR 6 CHH Cys, 1 Cys 5 CHH Ⅱ Gly, CHH cdna CHH C- GK, ES CHH CHH, CHH, M. lanchesteri CHH-ES, CHH CHH-PO 80%, CHH-ES (98%),,, CHH 5 ES CHH CHH 5 PO CHH, CHH ES, CHH CHHA CHHB, 3.2 CHH CHH CHH ITP, CHH, ITP ITP CHH, CHH, 6 Cys, 1 Cys 5 CHH Gly

8 1 : cdna 89 3 CHH NJ Fig. 3 Neighbor-Joining phylogenetic tree analysis of preprochh 4 qrt-pcr CHH Fig 4 Expression of CHH mrna in different tissues of Macrobrachium niponense by qrt-pcr,, CHH [20 21] ITP CHH Ⅰ, ITP,

9 90 20 CHH, CHH, [22] MF [1] [23] [24], ITP [25], ITP CHH, ITP Shirley [26] ITP,,, RNAi, ITP, ITP, ITP CHH, ITP CHH CHH booststrap 50%, CHH CHH 3.3 CHH, CHH CHH [14] [9] [11] (C. bairdi) [13] [27], CHH CHH PCR PCR CHH 8 PCR, PCR, CHH,,,,,, CHH 250 (P<0.01),, CHH, CHH cdna,, PCR, PCR, CHH CHH, CHH 参考文献 : [1] Liu L, Laufer H, Wang Y J, Hayes T. A neurohomone regulating both mehtylfarnesoate synthesis and glueose metabolism in a crustacean[j]. Biochem Biophys Res Commun, 1997, 237: [2] Tang C H, Lu W, Wainwrihgt G, et al. Molecular characteriaztion and expression of amndibular organ-inhibiting homrone, a recently discovered neuropeptide involved in regulation of growth and reproduction in the crab, Cancer pagurus[j]. Biochem, 1999, 343: [3] Keller R. Curstacean neuropeptides:sturctures, structure, functions and comparative aspects[j]. Experientia, 1992, 48: [4] Cooke I M, Sullivan R E. Hormones and neurosecret ion [M]//Bliss D E, Atwood H L, Sandeman D C, ed. The biology of crustacean. neurobiology: structure and function. New York: Academic Press, 1982, 3: [5] Keller R. Crustacean neuropeptides: structures, functions and comparative aspects [J]. Experientia, 1992, 48: [6]. [J]., 1998, 22(2): [7] Lacombe C, Greve P, Martin G. Overview on the sub2- grouping of the crustacean hyperglycemic hormone family [J]. Neuropeptides, 1999, 33: [8] de Kleijn D P, de Leeuw, E P, van den Berg M C, et al. Cloning and expression of two mrnas encoding structurally different crustacean hyperglycemic hormone precursors in the lobster Homarus americanus[j]. Biochim Biophys, 1995, 1260 (1): [9] Wiwegweaw A, Udomkit A, Panyim S. Molecular structure and organization of crustacean hyperglycaemic hormone genes of Penaeus monodon[j]. Biochem Mol Biol, 2004, 37(2):

10 1 : cdna 91 [10] Chen S H, Lin C Y, Kuo C M. Cloning of two crustacean hyperglycemic hormone isoforms in freshwater giant prawn (Macrobrachium rosenbergii): Evidence of alternative splicing[j]. Mar Biotechnol, 2004, 6 (1): [11] Lugo J M, Morera Y, Rodriguez T, et al. Molecular cloning and characterization of the crustacean hyperglycemic hormone cdna from Litopenaeus schmitti. Functional analysis by double-stranded RNA interference technique[j]. FEBS, 2006, 273 (24): [12] Choi C Y, Zheng J, Watson R D. Molecular cloning of a cdna encoding a crustacean hyperglycemic hormone from eyestalk ganglia of the blue crab, Callinectes sapidus[j]. Gen Comp Endocrinol, 2006, 148 (3): [13] Chung J S, Bembe S, Tamone S, et al. Molecular cloning of the crustacean hyperglycemic hormone (CHH) precursor from the X-organ and the identification of the neuropeptide from sinus gland of the Alaskan Tanner crab, Chionoecetes bairdi[j]. Gen Comp Endocrinol, 2009,162 (2): [14] Choi C Y, Zheng J, Watson R D. Molecular cloning of a cdna encoding a crustacean hyperglycemic hormone from eyestalk ganglia of the blue crab, Callinectes sapidus[j]. Gen Comp Endocrinol, 2006, 148(3): [15] Jacob A, Lancaster J, Buhler J. Mercury BLASTP: accelerating protein sequence alignment[j].assoc Comput Mach, 2008, 1(2): 9. [16] Tamurak K, Dudley J. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0[J]. Mol Biol Evol, 2007, 24 (8): [17] Thompson J D, Gibson T J, Plewniak F, et al. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools[j]. Nucl Acid Res, 1997, 25(24): [18] Davey M, Duve H, Thorpe A, et al. Characterisation of the helicostatin peptide precursor gene from Helicoverpa armigera (Lepidoptera:Noctuidae)[J]. Insect Biochem Mol Biol, 1999, 29(12): [19] Lenz C, Williamson M, Grimmelikhuijzen C J. Molecular cloning and genomic organization of an allatostatin preprohormone from Drosophila melanogaster[j]. Biochem Biophys Res Commun, 2000, 273(3): [20] Lacombe C, Greve P, Martin G. Overview on the subgrouping of the crustacean hyperglycemic hormone family[j]. Neuropeptides, 1999, 33(1): [21] Endo H, Nagasawa H, Watanaba T. Isolation of a cdna encoding a CHH-family peptide from the silkworm Bombyx mori[j]. Insect Bioehem Mol Biol, 2000, 30: [22] Santos E A, Nery L E, Keller R, et al. Evidence for the involvement of the crustacean hypergly- cemic hormone in the regulation of lipid metabolism[j]. Physiol Zool, 1997, 70: [23] Tensen C P, Janssen K P C, van Herp F. Isolation, characterization and physiological specificity of the crustacean hyperglycemic factors from the sinus gland of the lobster Homarus americanus (Milne-Edwards)[J]. Invert Reprod Dev, 1989, 16: [24] Huberman A, Agular M B, Brew K, et al. Primary structure of the major isomorphs of the crustacean hyperglycemic homone from the sinus gland of the Mexican crayfish peptides[j]. Peptides, 1993, 14: [25] Pienot C, Soyez D, Van Herp F, et al. Involvement of crustacean hyperglycemic hormone in the control of gill ion transport in the crab Pachygrapsus marmoratus[j]. Gen Comp Endocrinol, 2000, 119: [26] Shirley H K, Tiu He J G, Chan S M. The LvCHH-ITP gene of the shrimp (Litopenaeus vannamei) produces a widely expressed putative ion transport peptide (LvITP) for osmoregulation [J]. Gene, 2007, 396: [27] Sithigorngul P, Panchan N, Vilaivan T, et al. Immunochemical analysis and immunocytochemical localization of crustacean hyperglycemic hormone from the eyestalk of Macrobrachium rosenbergii[j]. Comp Biochem Physiol Part B: Biochem Mol Biol, 2011, 124(1):

11 92 20 Molecular cloning and expression of a full length cdna encoding crustacean hyperglycemic hormone (CHH) in oriental river pawn (Macrobrachium nipponense) JIN Shubo 1, WANG Ning 2, QIAO Hui 1, FU Hongtuo 1,2, WU Yan 1, GONG Yongsheng 1, JIANG Sufei 1, XIONG Yiwei 1 1. Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi , China; 2. WuXi Fishery College Nanjing Agricultural University, Wuxi , China Abstract: Crustacean hyperglycemic hormone (CHH) is a neuropeptide hormone that is secreted by a subset of cells in the X-organ of the eyestalk. CHH plays a role in the regulation of ions and energy metabolism, molting, reproduction, and other important physiological processes in crustaceans. The CHH gene has been cloned in a variety of crustacean species, However, little is known about the pattern of expression in different tissues. We cloned the full-length CHH cdna sequence of the oriental river prawn, Macrobrachium nipponense and used quantitative real-time PCR to detect the expression of CHH in different tissues. We cloned a 1017 bp cdna of the CHH gene, containing a 241 bp 5 -untranslated region (UTR), a 355 bp 3 UTR, and 408 bp open reading flame (ORF). The ORF encoded a 135 amino acid CHH precursor polypeptide. The mature peptide contained six conserved Cys residues in the CHH family. The C-terminus mature peptide was GK, which has the same characteristics as ES-CHH. Based on protein similarity comparison, the separated CHH gene was classified into the type I CHH family of peptides. Phylogenetic tree analysis revealed that oriental river prawn CHH was most closely related to that of Macrobrachium rosenbergii. The CHH gene was expressed in all tissues tested in oriental river prawn. Expression was significantly higher in the eyestalk and testis than in the ovary (500 and 250 higher, respectively). CHH gene expression was very high in the intestine and ventral nerve cord, but very low in the ovary, liver, brain, and heart. The pattern of expression suggests that the CHH peptide plays a role in male reproduction. Our results provide insight into the role of CHH peptide in the oriental river prawn, and further our understanding of the CHH gene in crustacean species. Key words: Macrobrachium nipponense; ES-type crustacean hyperglycemic hormone; full-length of cdna; quantitative PCR Corresponding author: FU Hongtuo. fuht@ffrc.cn

Microsoft Word - 02 舒妙安.doc

Microsoft Word - 02 舒妙安.doc 43 4 Vol.43, No.4 2012 7 OCEANOLOGIA ET LIMNOLOGIA SINICA July, 2012 拟穴青蟹 (Scylla paramamosain) 两种型高血糖 * 激素基因全长 cdna 的克隆及组织表达分析 ( 310058) RT-PCR RACE, CHH ( C1 C2)cDNA : C1 1859bp, 420bp, 139, 15.326kDa,