48 4 Vol.48, No.4 2017 7 OCEANOLOGIA ET LIMNOLOGIA SINICA Jul., 2017 缩骨鲫 (Carassius auratus sogu var.)mc4r 克隆 * 组织分布以及与生长相关 SNPs 的筛选 1 1 11 2 1 2 (1. 510006; 2. 512006) -4 (Melanocortin reporter-4, MC4R) G, (Carassius auratus sogu var.)mc4r,, Real-time PCR mrna, MC4R SNPs MC4R 2854bp,, 981bp, 326, MC4R, MC4R MC4R 99.3%, MC3R MC5R MCRs MC4R mrna,,,, MC4R CDs 6 SNPs: (A199G A881C), 4 (A123G, C231T, A444G C480T) -4 (MC4R); ; ; ; Q953; S917.4 doi: 10.11693/hyhz20170100025-4 (melanocortin reporter-4, MC4R) (melanocortin reporters), G, 7, (Jangprai et al, 2011) (POMC) (Ellacott et al, 2004) MC4R,, (Ellacott et al, 2004) (Bertile et al, 2006) (Van der Ploeg et al, 2002) (Vrinten et al, 2000) MC4R, α-msh,,, ; (agouti), (AGRP) Y (NPY),,, (, 2011), MC4R (Vaisse et al, 1998), 4% MC4R (Froguel et al, 2001), (Huszar et al, 1997) MC4R,, MC4R MC4R, MC4R Mountjoy (1994) MC4R, MC4R * ( ), 201303048 ;, 2015A0303135092 ;, 2014A020208145, E-mail: 214453598@qq.com :,,, E-mail: shuhu001@126.com : 2017-01-29, : 2017-03-04
4 : (Carassius auratus sogu var.)mc4r SNPs 831 (2011) MC4R,,, MC4R (Schizothorax prenati, Wei et al, 2013), (C. auratus var., Cerdá-Reverter et al, 2003), (Oncorhynchus mykiss, Haitina et al, 2004) (Snakeskin gourami, Jangprai et al, 2011), (single nucleotide polymorphisms, SNPs) DNA (Lander, 1996) SNPs, 1000bp SNPs (Zhao et al, 2003) SNP mrna,, (Carroll et al, 2005) MC4R SNPs,, (Oreochromis niloticus) 鲀 (Takifugu rubripes) MC4R SNPs (, 2009;, 2012) (C. auratus sogu var.),, (, 2016), 1.6,, (, 1986),, MC4R,,, SNPs, 1 1.1 14, (106±8.7)g,, 1.2 Ezup DNA SanPrep DNA ( ) ; Taq RNA keeper Vazyme Biotech PMD19-T DH5α RNAiso PrimeScript TM RT reagent Kit with gdna Eraser (Perfect Real Time) SYBR Premix Ex Taq II (Tli RNaseH Plus), ROX plus TaKaRa 1.3 MC4R CDs (MC4R1F, MC4R1R, MC4R2F MC4R2R) 3 (MC4R3F MC4R3R), MC4R, MC4R4 MC4R5 MC4R6 Real-time PCR ( 1) 25mg, DNA DNA DNA,,, Seqman 表 1 用于缩骨鲫 MC4R 基因克隆 表达分析和 SNPs 筛选的引物 Tab.1 Primers used for cloning and expression analysis of MC4R in C. auratus sogu var. MC4R1F MC4R1R MC4R2F MC4R2R MC4R3F MC4R3R MC4R4 MC4R5 MC4R6 QMC4RF QMC4RR β-actinf β-actinr (5 3 ) ATGAACACCTCACATCATCATG TGGTGAAGAACATGCTGATAAG CTACATCACAATCTTCTACGCT CTATACACACAGAGAGGTCAAT ATGTGCAACTCGGTCATAGACC GGGATAAATGAGAGTTTATTGGAGG GGGTCATGATGTTGTGGTAGCG GAAGATCGTGATGTAGCGGTCC CAAATGGAGGCCAACAGTGAGC AGCTGTAGCAGACTTGTTGGTC TGTTCTTGATGATGCTCTCGCG CAGCCATCCTTCCTAGGTATG TCTGCATACGGTCAGCAATGC 1.4 MC4R MC4R DNAstar 7.1 Seqman, MC4R, NCBI KX988002 ExPASy-ProtParam tool MC4R TMHMM Server v. 2.0 MC4R Mega 5.0 MC4R MCR ( 2), NJ, Bootstrap 1000 1.5 MC4R 3 9 :
832 48 RNA keeper, trizol RNA, 2% RNA PrimeScript TM RT reagent Kit with gdna Eraser (Perfect Real Time) PCR ABI7000, SYBR Premix Ex Taq TM II (Tli RNaseH Plus) Real-time PCR 9 mrna, PCR,, β-actin ( 1), QMC4RF QMC4RR MC4R 2 ΔΔct 表 2 用于构建系统发育树的 MC4R 以及 MCRs 氨基酸序列 Tab.2 GenBank accession numbers of MC4R and MCRs used in this study C. auratus sogu var. KX988002 MC4R C. auratus CAD58853.1 MC4R Cyprinus carpio CBX89936.1 MC4R Danio rerio NP_775385.1 MC4R S. prenanti AGF80338.1 MC4R Gasterosteus aculeatus ALF99996.1 MC4R Dasyatis akajei BAU98233.1 MC4R 鳉 Aphyosemion striatum SBP15058.1 MC4R 鳉 Nothobranchius rachovii SBR74851. 1 MC4R Xiphophorus maculatus AHC02878.1 MC4R Channa argus AMM02541.1 MC4R Scatophagus argus AOQ25859.1 MC4R Paralichthys olivaceus ADP09415.1 MC4R Dicentrarchus labrax CBN82190.1 MC4R Xenopus tropicalis XP_004915370.1 MC4R Gallus gallus NP_001026685.1 MC4R Anser anser ABF19809.1 MC4R Columba livia ACC60975.1 MC4R Mus musculus NP_058673.2 MC4R Homo sapiens NP_005903.2 MC4R Bos Taurus NP_776535.1 MC4R Ovis aries AFJ95892.1 MC4R C. auratus CAE11349.1 MC5R D. rerio AAI62747.1 MC3R D. rerio AAO24742.1 MC1R D. rerio AAO24743.1 MC2R 1.6 SNPs,, 5 5, SNPs,, DNA 10 DNA, MC4R1F MC4R1R MC4R2F MC4R2R CDs,, Chromas 2.33, SNPs, SNPs 2 2.1 MC4R MC4R CDs 3 1,, MC4R 2854bp,, 981bp, 326, MC4R 2 MC4R 36531.58, 8.45 (Leu) (12.6%), (Trp), 1.5%, 1.8% 10.7% 35.65,, (GRAVY) 0.713, 1 MC4R Fig.1 PCR result of MSTN of C. auratus sogu var. : 1 3 1 PCR, M DL2000 DNA Marker 2.2 MC4R MC4R TMHMM Server v. 2.0,,, G N-, C- 45 67, 79 101, 121 143, 163 185, 195 217, 243 265 280 302( 2)
4 : (Carassius auratus sogu var.)mc4r SNPs 833 2 MC4R Fig.2 MC4R nucleotide sequence and predicted amino acid sequence of C. auratus sogu var. :,,,,, SNPs, 2.3 MC4R GenBank MC4R MCRs,, MC4R 99.3%, 98.6%, 97.9% 97.5%, MC4R 83.4% 79.7%, 75.3%, 71.2% 68.9% MCRs, MC4R MC5R, 68.4%, MC3R 60.5%, MC1R 42.5%, MC2R 25.7%, MC4R, MCRs
834 48 3 MC4R MCRs Fig.3 Phylogenetic tree of MC4R of different species and MCRs family 2.4 MC4R Real-time PCR MC4R Real-time PCR, Real-time PCR, MC4R,,,, ( 4) 2.5 MC4R SNPs CDs, 6 SNPs ( 2), 1 1,, SNPs, A123G A199G C231T A444G C480T A881C( 5), A123G (CTA>CTG), ; A199G (ATC>GTC),,, ; C231T (TAC>TAT), ; A444G 4 MC4R mrna Fig.4 Relative expression of MC4R mrna in tissues of in C. auratus sogu var.
4 : (Carassius auratus sogu var.)mc4r SNPs 835 5 MC4R SNPs Fig.5 The SNPs in CDs of C. auratus sogu var. : A F A123G, A199G, C231T, A444G, C480T A881C (ACG>ACA), ; C480T (ACT>ACC),, A881C (CAC>CAA),, 3 MC4R GenBank MC4R MCRs, 99.3%, (2005) RAPD :, MC4R, 97.5% 99.3%, MC4R MC4R MCR 25.7% 68.4%,, MC4R MC3R MC5R MCRs, 68.4% 60.5%, (Shukla et al, 2013), MC4R MC5R MC3R MCR, Klovins (2004) (Squalus acanthias), MC4R MC3R MC5R MCRs,, MC4R, MC4R 326 327aa, MC4R 332aa, MC4R MC4R mrna,,,,,,,,, (Jangprai et al, 2011);, MC4R, (Cerdá-Reverter et al, 2003), MC4R,,,
836 48 MC4R, (Ellacott et al, 2004); MC4R, MC4R (Lh et al, 2002); MC4R, POMC (Bertile et al, 2006) MC4R, (, 2012), MC4R 6 SNPs,,,,,, (Kimchi-Sarfaty et al, 2007), (, 2008;, 2016) Komar(2007), A199G (ATC>GTC),,,, A881C (CAC>CAA),,,,,, SNPs 4 MC4R, MC4R 2854bp,, 981bp, 326, MC4R,, MC4R mrna,,, 6 SNP,,, 2011. MC4R., 20(1): 29 34,, 2011. MC4R., 31(5): 45 50,,, 2016. DGAT2 7., 24(5): 689 696,,, 2012. MC5R., 22(17): 12 16,, 粦, 2005. RAPD., 35(2): 13 16,,, 2009. MC4R SNPs., 16(6): 816 823,,, 2008. Leptin SNP., 27(2): 251 257,,, 2012. 鲀 (Takifugu rubripes)mc4r., (7): 97 102,, 1986.., (3): 21,,, 2016.., (4): 13 17 Bertile F, Raclot T, 2006. The melanocortin system during fasting. Peptides, 27(2): 291 300 Carroll L, Voisey J, van Daal A, 2005. Gene polymorphisms and their effects in the melanocortin system. Peptides, 26(10): 1871 1885 Cerdá-Reverter J M, Ringholm A, Schiöth H B et al, 2003. Molecular cloning, pharmacological characterization, and brain mapping of the melanocortin 4 receptor in the goldfish: involvement in the control of food intake. Endocrinology, 144(6): 2336 2349 Ellacott K L, Cone R D, 2004. The central melanocortin system and the integration of short- and long-term regulators of energy homeostasis. Recent Progress in Hormone Research, 59: 395 408 Froguel P, Boutin P, 2001. Genetics of pathways regulating body weight in the development of obesity in humans. Experimental Biology and Medicine, 226(11): 991 996 Haitina T, Klovins J, Andersson J et al, 2004. Cloning, tissue distribution, pharmacology and three-dimensional modelling of melanocortin receptors 4 and 5 in rainbow trout suggest close evolutionary relationship of these subtypes. Biochemical Journal, 380(2): 475 486 Huszar D, Lynch C A, Fairchild-Huntress V et al, 1997. Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell, 88(1): 131 141 Jangprai A, Boonanuntanasarn S, Yoshizaki G, 2011. Characterization of melanocortin 4 receptor in Snakeskin gourami and its expression in relation to daily feed intake and short-term fasting. General and Comparative Endocrinology, 173(1): 27 37 Kimchi-Sarfaty C, Oh J M, Kim I W et al, 2007. A silent polymorphism in the MDR1 gene changes substrate specificity. Science, 315(5811): 525 528 Klovins J, Haitina T, Ringholm A et al, 2004. Cloning of two
4 : (Carassius auratus sogu var.)mc4r SNPs 837 melanocortin (MC) receptors in spiny dogfish: MC3 receptor in cartilaginous fish shows high affinity to ACTH-derived peptides while it has lower preference to γ-msh. European Journal of Biochemistry, 271(21): 4320 4331 Komar A A, 2007. Silent SNPs: impact on gene function and phenotype. Pharmacogenomics, 8(8): 1075 1080 Lander E S, 1996. The new genomics: global views of biology. Science, 274(5287): 536 539 Lh V D P, Martin W J, Howard A D et al, 2002. A role for the melanocortin 4 receptor in sexual function. Proceedings of the National Academy of Sciences of the United States of America, 99(17): 11381 11386 Mountjoy K G, Mortrud M T, Low M J et al, 1994. Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain. Molecular Endocrinology, 8(10): 1298 1308 Shukla C, Koch L G, Britton S L et al, 2013. Differential contribution of brain-region-specific melanocortin 5 receptor to physical activity levels in lean vs. obesity-prone rats. The FASEB Journal, 27: 935.1 Vaisse C, Clement K, Guy-Grand B et al, 1998. A frameshift mutation in human MC4R is associated with a dominant form of obesity. Nature Genetics, 20(2): 113 114 Van der Ploeg L H T, Martin W J, Howard A D et al, 2002. A role for the melanocortin 4 receptor in sexual function. Proceedings of the National Academy of Sciences of the United States of America, 99(17): 11381 11386 Vrinten D H, Gispen W H, Groen G J et al, 2000. Antagonism of the melanocortin system reduces cold and mechanical allodynia in mononeuropathic rats. The Journal of Neuroscience, 20(21): 8131 8137 Wei R B, Yuan D Y, Zhou C W et al, 2013. Cloning, distribution and effects of fasting status of melanocortin 4 receptor (MC4R) in Schizothorax prenanti. Gene, 532(1): 100 107 Zhao Z M, Fu Y X, Hewett-Emmett D et al, 2003. Investigating single nucleotide polymorphism (SNP) density in the human genome and its implications for molecular evolution. Gene, 312: 207 213 CLONE, EXPRESSION, AND SCREENING OF GROWTH ASSOCIATED SNPS OF CARASSIUS AURATUS SOGU VAR. YANG Yang 1, ZHOU Hui-Qiang 1, SHU Hu 1, LAN Zhao-Jun 2, LI Qiang 1, GU Ping-Hua 2 (1. School of Life Science, Guangzhou University, Guangzhou 510006, China; 2. Shaoguan Fisheries Research Institute, Shaoguan 512006, China) Abstract Melanocortin reporter-4 (MC4R) belongs to G-protein-coupled receptors family; it plays an important role in energy regulation. By homologous cloning and chromosome walking, MC4R gene of Carassius auratus sogu var. was cloned, and the expression of MC4R gene were studied based on real-time PCR. Meanwhile, SNPs (single nucleotide polymorphisms) of the MC4R in CDs region were detected by Sanger sequences. The result show that the length of MC4R genome gene was 2854bp that contain one 981-bp exon, the CDs code 326 amino acid. The MC4R contained 7 transmembrane structure and was a stable hydrophobin. Through sequence alignment of MC4Rs from different species and other members of MCRs, we found that the C. auratus is the closest species to C. auratus sogu var. The homology ratio is 99.3%, the MC4R was far from other MCRs members. The real-time PCR result shows that MC4R mrna was most expressed in brain, and then in eyes; and less expressed in muscle, ovary, and kidney. Weak MC4R mrna was expressed in gill, intestine, heart and hepatopancreas. In the codon sequence, six SNPs were detected: two missense mutations, and four silent mutations. The results shall be insightful to understand the growth of C. auratus sogu var., and offer fundamental data for molecular markers of C. auratus sogu var. Key words melanocortin reporter-4 (MC4R); Carassius auratus sogu var.; clone; tissue expression; single nucleotide polymorphisms (SNPs)