Microsoft Word - 02 舒妙安.doc

Size: px

Start display at page:

Download "Microsoft Word - 02 舒妙安.doc"

昱龄鄂
7 years ago
Views:

1 43 4 Vol.43, No 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 ( ) RT-PCR RACE, CHH ( C1 C2)cDNA : C1 1859bp, 420bp, 139, kDa, 5.540; C2 1739bp, 426bp, 141, kDa, CHH, CHH CHH (92%), (80%) (78%), CHH, CHH,, : 24h, C2 C1 30 ; C2 5 (P>0.05), 15 (P<0.05), (P<0.01);, C2 CHH, CHH,, Q75 (Scylla paramamosain) (Arthropoda) (Crustacea) (Decapoda) (Portunidae) (Scylla),, 4 (S. serrata) (S. olivacea) (S. tranquebarica) (, 2007),, (Fanjul-Moles, 2006) X- (X-organ-sinus gland, XO-SG) (crustacean hyperglycemic hormone, CHH) (gonad-inhibiting hormone, GIH) (molt-inhibiting hormone, MIH) (mandibular organ-inhibiting hormone, MOIH)(Keller, 1992; De KIeijn et al, 1995), (De KIeijn et al, 1995; Garefa et al, 1998), (crustacean hyperglycemic hormone family, CHH ), (Mettulio et al, 2004; Okumura et al, 2005) (crustacean hyperglycemic hormone, CHH), (, 2006) CHH (Thannumalaya et al, 1998), (Spaning-Pierrot et al, 2000) (Chang et al, 1998) (Chang et al, 1998) (Lorenzon et al, 2000) (Webster et al, 1996) (Stentiford et al, 2001) CHH *, 2008C12008, shuma@zju.edu.cn : , :

2 696 43,, cdna (, 2003),, CHH cdna, CHH, CHH, (Scylla paramamosain) , [(200±20)g] [(10±1.2)cm] [(8±0.8)cm], 80cm 80cm PVC,, (25±0.4), 8±0.2, ph 7.5±0.3,, 1/3, 18 6,, 3 10, ,, 24h,, : 7, 1.2 RNA,, 0.05g 1ml Invitrogen Trizol Reagent, RNA RNA Nanodrop2000, TBE SMART TM RACE cdna Amplification Kit cdna, CHH cdna GenBank CHH cdna, CHH-F, CHH-R( 1) TaKaRa Taq TM PCR PCR TaKaRa Taq (5U/μl), 0.2μl; 10 PCR Buffer (Mg 2+ Plus), 2.5μl; dntp Mixture( 2.5 mmol/l), 1μl; cdna, 1μl; CHH-F (10 μmol/l), 1μl; CHH-R (10μmol/L), 1μl; ddh 2 O 20μl PCR 94 5min; 94 30s, 55 30s, 72 1min, 30 ; 72 10min; 4 PCR Axygen, pmd-18t (TaKaRa) Top10 (Invitrogen) PCR, Invitrogen BLASTX, CHH CHH, 3 5 RACE ( 1) SMART TM RACE cdna Amplification Kit (Clontech) cdna, 表 1 本研究所用的引物 Tab.1 Primer sequences used in this study (5 3 ) CHH-F CGGCGCACCCCTTAGAAAAAAGAC Gene CHH-R CCCTTTAACCCTTGCTAAGCTGCC Cloning GSP1 TCC GTT GAA GGG GCG ACT ATG GTG 5'Race GSP2 CTA CAG CAA CGT GGT CAT CCG CC 3'Race GSP3 GTA GGA CCC AGA CCA AGA CCC ACA 3'Race UPM-long CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT Race UPM-short CTAATACGACTCACTATAGGGC Race NUP AAGCAGTGGTATCAACGCAGAGT NestRace OligoG AAGCAGTGGTATCAACGCAGAGTACGCGGG 5'Race 3'-CDS AAGCAGTGGTATCAACGCAGAGTAC(T)30VN 3'Race CHH-SF CTG CTG CAC AAT CCT GAC RT-PCR CHH-SR CTT CGA AGT TGT CCA TCA GC RT-PCR CHH-DF CTA CCT TAA AGC CAC CAC RT-PCR CHH-DR ATC AGA TAG GCT CGT TTC RT-PCR -actin-f GCCCTTCCTCACGCTATCCT -actin-r GCGGCAGTGGTCATCTCCT

3 4 : (Scylla paramamosain) cdna 697 GSP1 GSP2 GSP3 SMART TM RACE cdna Amplification Kit, SMART TM RACE cdna Amplification Kit CHH 3' 5' PCR CHH 1.4 CHH DNAStar SeqMan NCBI BLAST ( nlm.nih.gov/blast/), DNAStar EdiSeq (ORF) ProtParam ( expasy.org/tools/protparam.html) Interpro Scan ( Tools/InterProScan/) DNAMAN CHH, MEGA4.0 (KUMAR et al, 2004) NJ 1.5 CHH, Primer 5.0 CHH-SF CHH-SR CHH-DF CHH-DR( 1), CHH, GenBank β-actin β-actin-f β-actin-r( 1) PCR(ddCt ) 20μl, : SYBR Premix Ex Taq TM (2 ), 10μl; PCR Forward Primer (10μmol/L), 0.4μl; PCR Reverse Primer (10μmol/L), 0.4μl; ROX Reference Dye (50 )*3, 0.4μl; cdna, 2.0μl; dh 2 O, 6.8μl 95 30s; 95 5s, 60 34s, 40 ; 95 15s, 60 1min, 95 15s PCR, 2 ΔΔCt, SPSS11.0 CHH-SF CHH-SR C1, CHH-DF CHH-DR C1 C2, 2 ΔΔCt C CHH CHH-F CHH-R 398bp GSP2 3 RACE 1269bp, AATAAA, GSP3 3 RACE 505bp, GSP2 384bp, poly(a), AATAAA, 3 GSP1 5 RACE 704bp 584bp,, bp 1739bp, C1( : JQ421462) C2( : JQ421463) cdna C1 mrna 75bp 5 (UTR), 420bp (ORF), 1364bp 3 (UTR) poly(a), 139 ; C2 mrna 75bp 5 (UTR), 426bp (ORF), 1238bp 3 (UTR) poly(a), AATAAA 2.2 DNASTAR Edisequce, C1 139, kDa, (K, R) 17 (D, E) 48 (A, I, L, F, W, V) 37 (N, C, Q, S, T, Y) C2 141, kDa, (K, R) 15 (D, E) 47 (A, I, L, F, W, V) 40 (N, C, Q, S, T, Y) Motif Scan,, CHH (CHH, precursor-related peptide, CPRP) 2.3 DNAMAN (Scylla olivacea) (AAQ ) 蟳 (Charybdis japonica) (ACN ) (Portunus trituberculatus) (ACB ) (Callinectes sapidus) (AAS ) (Discoplax hirtipes) (AEM ) (Grapsus tenuicrustatus) (AER ) (Cancer productus) (ABQ )7,,

4 MEGA4.0 NJ 17 ( 3),, 2.4 CHH -actin, PCR CHH C1 C2 7, C1 C2 7,, 6, (P<0.01)( 4) 5 30, 24h, C1 10, C , C , C2 C1 30 C2 5 (P>0.05), 15 (P< 0.05), (P<0.01)( 5) 3, cdna, CHH cdna CHH (CHH precursor-related peptide, CPRP), MIH GIH MOIH cdna (Mettulio et al, 2004), 1 (C1 C2) cdna CHH Fig.1 cdna sequences and deduced amino acid sequences of two genes (C1 and CHH MIH GIH MOIH, C2) encoding CHH from the mud crab S. paramamosain CHH CHH (Garefa et al, :, (*) ; ( 1998) CHH 1 18); CHH (CPRP), CHH RSAXG; HPXEKRQIXD; CKGVYXR; (Hsu et al, LEHVCXDCYNLYR; VXSXCRXNC( 2) 2008)

5 4 : (Scylla paramamosain) cdna 699 cdna C1 C2, C1 1859bp, 139 ; C2 1739bp, 141 C1 C2 CHH, C1 C2 (Scylla olivacea) (AAQ ) 蟳 (Charybdis japonica) (ACN ) (Portunus trituberculatus) (ACB ) : 92% 80% 78%, CPRP, CHH CHH CHH, (Sedlmeier, 1998), (Keller, 1992), CHH Okumura (2005), CHH, CHH, CHH (Fanjul-Moles, 2006) RT-PCR C1 C2 mrna,, CHH 2 CHH 7 CHH, Fig.2 Amino-acid sequences alignment of S. paramamosain with different animals, CHHs : Scylla olivacea (AAQ ); Portunus trituberculatus, Tsai (ACB ); 蟳 Charybdis japonica (ACN ); Callinectes sapidus (2008) (AAS ); Discoplax hirtipes (AEM ); Cancer CHH 12, productus (ABQ ); Grapsus tenuicrustatus (AER ) CHH,,, ( ) (5), C1, C2,, C2 C1 30, C2,,

6 MEGA4.0 CHH NJ Fig.3 NJ tree based on CHH amino acid sequences using MEGA4.0 : Penaeus monodon: (AY ); Metapenaeus ensis: (AF ); Gecarcinus lateralis: (DQ ); Macrobrachium rosenbergii: (AF ); Pachygrapsus marmoratus: (AY ); Callinectes sapidus: (AY ); Grapsus tenuicrustatus: (JN ); Charybdis japonica: 蟳 (FJ ); Discoplax hirtipes: (JF ); Cancer productus: (EF ); Scylla olivacea: (EF ); Portunus trituberculatus: (EU ); Homarus gammarus: (DQ ); Marsupenaeus japonicus: (AB ); Macrobrachium nipponense: (HQ ); Scylla paramamosain C1: (JQ421462); Scylla paramamosain C2: (JQ421463) 5 PCR C2 24h Fig.5 C2 gene expression level in the eyestalk detected by qrt-pcr after 24h s stress in salinity * (P<0.05), ** (P<0.01) (1996) (Cancer pagurus), CHH Lorenzon (2000) (Palaemon elegans), Hg Cd, CHH 2h Stentiford (2001) (Hematodinium sp.) (Nephrops norvegicus), CHH, CHH CHH, CHH cdna,, CHH, 4 PCR C1 C2 Fig.4 C1 and C2 genes expression level in different tissues detected by qrt-pcr * (P<0.05), ** (P<0.01), CHH Chang (1998) ELISA, CHH,, CHH Webster,,, , 30(2): ,,, cdna., 27(3): ,,, (Scylla)., 31(2): Chang E S, Keller R, Chang S A et al, Quantification of crustacean hyperglycemic hormone by ELISA in hemolymph of the lobster, Homarus americanus, following various stresses. Gen Comp Endocrinol, 111(03): De KIeijn D, Van Herp F, Molecular biology of neurohomone precursors in the eyestalk of Crustacea. Comp Biochem Physiol, 112(2):

7 4 : (Scylla paramamosain) cdna 701 Fanjul-Moles M L, Biochemical and functional aspects of crustacean hyperglycemic hormone in decapod crustaceans: review and update. Comp Biochem Physiol C: Toxicol Pharmacol, 142(3 4): Garefa U, Araehiga H, Regulation of crustacean neurosecretory cell activity. Cell Mol Neurobiol, 18: Hsu Y W, Weller J R, Molecular cloning of four cdnas encoding prepro-crustacean hyperglycemic hormone (CHH) from the eyestalk of the red rock crab Cancer products: identification of two genetically encoded CHH isoforms and two putative post-translationally derived CHH variants. Gen Comp Endocrinol, 155(03): Keller R, Crustacean neuropeptides: structure, functions and comparative aspects. Exprientia, 48: KUMAR S, TAMURA K, NEI M, MEGA3: an integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinformatics, 5(2): Lorenzon S, Francese M, Heavy metal toxicity and differential effects on the hyperglycemic stress response in the shrimp Palaemon elegans. Arch Environ Contam Toxicol, 39(02): Mettulio R, Giulianini P G, Functional analysis of crustacean hyperglycemic hormone by in vivo assay with wild-type and mutant recombinant proteins. Regul Pept, 119(3): Okumura T, Ohira T, Katayama H et al, In vivo effects of a recombinant molt-inhibiting hormone on molt interval and hemolymph ecdysteroid level in the Kuruma Prawn, Marsupenaeus japonicas. Zoological Science, 22: Sedlmeier, The crustacean hyperglycemic hormone releases amylase from crayfish midgut gland. Peptides, 20: Spaning-Pierrot C, Soyez D, Van Herp F et al, Involvement of crustacean hyperglycemic hormone in the control of gill of the green crab Pachygrapsus marmoratus. Gen Comp Endocrine, 119: Stentiford G D, Chang E S, Chang S A et al, Carbohydrate dynamics and the crustacean hyperglycemic hormone (CHH): effects of parasitic infection in Norway lobsters (Nephrops norvegicus). Gen Comp Endocrinol, 121: Thannumalaya P S, Barbara R, Heimrich D et al, On the isolation and characterization of a crustacean hyperglycaemic hormone in the eyetalk of the shrimp Penaeus indicus. Aquaculture, 162: Tsai Kuo-Wei, Su-Jung Chang, Hsin-Ju Wu et al, Molecular cloning and differential expression pattern of two structural variants of the crustacean hyperglycemic hormone family from the mud crab Scylla olivacea. General and Comparative Endocrinology, 159: Webster S, Measurement of crustacean hyperglycemic hormone levels in the edible crab Cancer pagurus during emersion stress. Exp Biol, 199(7): CLONING AND EXPRESSION OF TWO GENES ENCODING TYPE CRUSTACEAN HYPERGLYCEMIC HORMONE FAMILY FROM THE MUD CRAB SCYLLA PARAMAMOSAIN SHU Miao-An, ZHANG Long-Tao, ZHOU Yu-Fang, HU Hang-Jiao, XU Bin-Peng, GUO Xiao-Ling (College of Animal Science, Zhejiang University, Hangzhou, ) Abstract The cdnas of two crustacean hyperglycemic hormones, designated as C1 and C2, were isolated from the eyestalk of mud crab Scylla paramamosain through RT-PCR and RACE. Sequence analysis indicated that C1 had an open reading frame of 420bp encoding 139aa of kDa and pi at 5.540; C2 had an open reading frame of 426bp encoding 141aa of kDa and pi at The amino acid sequences of C1 and C2 possessed 92%, 80%, 78% identity with the CHHs of S. olivacea, Charybdis japonica, Portunus trituberculatus respectively. C1 and C2 protein firstly clustered with CHHs of S. olivacea and Charybdis japonica in the phylogenetic analysis. The expression of C1 and C2 in tissues were analyzed by Real-Time PCR, the result showed that C1 and C2 were expressed in eyestalk, hepatopancreas, intestinal, heart, gill and stomach, but not in muscle. After 24h s stress in salinity, the expression of C2 increased by a factor of thirty compared with C1. The expression of C2 in salinity 10 was not significant compared with salinity 5 (P>0.05), but significant with salinity 15 (P<0.05) and great significant with salinity 20, 25, 30 (P<0.01). The more the salinity changed, the more the C2 expressed. These results served further studies on functions and regulation mechanism of CHHs. Key words Scylla paramamosain, Crustacean hyperglycemic hormone (CHH), Gene cloning, expression analysis

标题

吉林农业大学学报２０１６３８１８７９１ＪｏｕｒｎａｌｏｆＪｉｌｉｎＡｇｒｉｃｕｌｔｕｒａｌＵｎｉｖｅｒｓｉｔｙｈｔｔｐｘｕｅｂａｏｊｌａｕｅｄｕｃｎＥｍａｉｌｊｌｎｄｘｂｖｉｐｓｉｎａｃｏｍ神经生长因子ｍｒｎａ在鹅等级前卵泡中的表达刘璐璐隋玉健朱海洋武惠岩杨辉孙永峰吉林农业大学动物科学技术学院长春１３０１１８