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1 2016 5, 23(3): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J 马骞, 冯文荣, 柳淑芳, 庄志猛, 马慧,, 摘要 : (Cynoglossus semilaevis) 4(BMP4) cdna,, BMP4 cdna 1680 bp, 419 bp 5 (5 UTR) bp (ORF) 49 bp 3 (3 UTR), (Cichlidae), BMP4 BMP4 mrna 13,,, BMP4 ( ), ; ( 3 4 ),, BMP4 mrna ; BMP4 ; BMP4, : ; BMP4; ; ; ; 中图分类号 : S917 文献标志码 : A 文章编号 : (2016) (bone morphogenetic proteins, BMPs) β(transforming growth factor-β, TGF-β), 20, [1] BMP, [2 3] BMP4 BMP, BMP4,, [4 5], BMP4,, [6] [7] BMP4 mrna,, BMP4,, BMP4 [8], BMP4 [9], BMP4, BMP4 1997, Hwang [10] (Danio rerio) DNA 14.9 kb BMP4, 收稿日期 : ; 修订日期 : 基金项目 : ( ); ( ). 作者简介 : (1983 ),,,. maqian@ysfri.ac.cn 通信作者 :,,. zhuangzm@ysfri.ac.cn

2 3 : , BMP4 73%, BMP4 (Salmo salar) [11] 鳉 (Oryzias latipes) [12] (Paralichthys olivaceus) [13] BMP4, BMP4 mrna, 7 [14], BMP4, [13] BMP4 mrna, BMP4 mrna ; BMP4, BMP4,, [15 16], BMP4,, (Cynoglossus semilaevis),,, BMP4, cdna BMP4, BMP4 mrna ( ), BMP , 3 12 ( 6 ), (583.0± 61.3) mm (1479.2±280.8) g; (333.3± 14.3) mm (170.3±16.9) g 13,, 80, 3, [17] [18], PCR(qRT-PCR) ( 1), PCR RNA ; RNA 4% (PFA) 4 12~16 h, 30% 50% 70%, 70%, 20 item 表 1 早期发育阶段半滑舌鳎样品信息 Tab. 1 Information of Cynoglossus semilaevis samples in the early developmental stages cleavage stage embryo stage gastrula stage embryonic stage hatching stage larva stage earlystage larvae late- stage larvae juvenile stage fingerling stage sampling time 3 hpf 13 hpf 22 hpf 32 hpf 1, 2 dph 3, 4, 5, 10, 15 dph 20, 30, 40, 50 dph 90 dph qrt-pcr samples for qrt-pcr : hpf, dph. Note: hpf represents hours post fertilization; dph represents days post hatching.

3 GenBank BMP4 cdna,, Primer Premier 5.0 BMP4-F BMP4-R( 2), BMP4, BMP4 cdna 3 5 RACE (BMP4-3 - OUTER/INNER BMP4-5 -OUTER/INNER), BMP4 cdna PCR (BMP4- RT-F/R), (BMP4-HY-F/R) BMP4 RNA Tab. 2 表 2 本研究所用的引物序列 Oligonucleotide primers used in this study primer (5 3 ) primer sequence (5 3 ) amplification target BMP4-F BMP4-R BMP4-5 -OUTER BMP4-5 -INNER BMP4-3 -OUTER BMP4-3 -INNER BMP4-RT-F BMP4-RT-R BMP4-HY-F BMP4-HY-R EF1A-F EF1A-R 18S-F 18S-R CCTGGTAAYCGAATGCAATGCTGATGGT GNGCCACDATCCARTCRTTCCA TGGTGTAAGGAACGGCTGATGCGGAC GGATGCTGCTGAGGTTGAACAGGAAA TCCTGTTCAACCTCAGCAGCATCCCA CGTCCGCATCAGCCGTTCCTTACACCA CGCATCAGCCGTTCCTT CATTCCAGCCTACGTCACTAAA ATGATTCCTGGTAATCGAATGC CGATTGCGTTTACGACCCCT GACAAACTGAAGGCHGAGCG CAGCCTGAGAGGTTCCAGTGAT CCTGAGAAACGGCTACCACATCC CCAATTACAGGGCCTCGAAAG BMP4 cdna fragment of partial BMP4 5 RACE 5 RACE amplification 3 RACE 3 RACE amplification BMP4 PCR expression of BMP4 BMP4 in situ hybridization of BMP4 mrna expression of EF1A mrna expression of 18S rrna 1.3 RNA cdna, Trizol (Invitrogen) RNA, RNA, PrimeScript RTase (TaKaRa) cdna 1.4 BMP4 cdna, BMP4-F/R, cdna, BMP4 cdna PCR 25 μl, : 94 3 min; s, s, s, 35 ; min RACE SMARTer RACE cdna (Clontech), BMP4 cdna PCR PCR, 1.5 BMP4 mrna PCR, BMP4-RT-F/R ( 2), cdna, BMP4 mrna 2, (elongation factor 1-alpha, EF1A) 18S DNA(18S ribosomal RNA, 18S) PCR [19] SYBR Premix Ex Taq TM (TaKaRa), ABI 7500 PCR (E> 90%; R 2 >0.990), PCR, RNA BMP4 (903 bp) (pgem-t, Promega) RNA, Noc I (NEB Biolabs), UTP(DIG-UTP) SP6 (DIG RNA Labeling Mix, Roche) RNA 10 μl,

4 3 : DNA 4 μg, 1 μl 10 buffer, 1 μl DIG-UTP Mixture, 1 μl, 1 μl RNA Holland [20],, K, 4% PFA, ; 60 3 h, RNA ( 1 ng/μl)60 16 h;, (blocking reagent, Roche) 2 h, ( )4 ;, NBT/BCIP( / ), ; 80%, SMZ DNA EditSeq DNAMAN ; NCBI BLAST DNASTAR, BMP4 ; BMP4 ClustalX BMP4 ; MEGA5.1 Neighbor-Joining (NJ),, 1000 [21] PCR C t, 2 - CT BMP4 ± ( x ±SD), SPSS 16.0 One-way ANOVA BMP4 mrna BMP4 cdna 1, BMP4 cdna 1680 bp(genbank : KC422339), BMP4 cdna 419 bp 5 (5 UTR) 1212 bp (ORF) 49 bp 3 (3 UTR), 403 BMP , kd, (pi) 7.96 BMP4 BLAST, Transforming growth factor-beta (TGF-beta), β, BMP4 C BMP 7 1 BMP RISR ( 1) 2.2 BMP4 BMP4 ( 2), BMP4 C, BMP4 7 3 N 1 (RXSR) MEGA5.1 BMP4,, BMP4, 0.043~0.051( 3) BMP4, (Danio rerio), 0.214,, 18 BMP4, (Xiphophorus maculatus) BMP4 ( 3) 2.3 BMP4 mrna PCR BMP4 mrna, BMP4 mrna 4, BMP4,,, BMP4 mrna (P<0.05) 5, BMP4 ( ) ; (22 hpf), ; ( 3 4 ) 10,

5 BMP4 5 UTR 3 UTR,,, RISR,. Fig. 1 Nucleotide and deduced amino acid sequences of Cynoglossus semilaevis BMP4 gene The lowercases indicate the 5 UTR and 3 UTR. The signal peptide is underlined; Cys residues are boxed; RISR sequence is double underlined. The stop codon is marked by an asterisk.

6 3 : BMP4,, RISR, N. Fig. 2 Alignment of BMP4 amino acid sequences of Cynoglossus semilaevis and other vertebrates The signal peptides are boxed by shaded rectangles; Cys residues are boxed; RISR sequences are double underlined; Conserved N-glycosylation sites are underlined.

7 表 3 已报道的脊椎动物 BMP4 基因核苷酸编码区序列来源及其与半滑舌鳎 BMP4 的同源性比较 Tab. 3 Nucleotide sequences identity of Cynoglossus semilaevis BMP4 gene with corresponding published sequences species GenBank GenBank access no pairwise distance Cynoglossus semilaevis KC Tanganicodus irsacae AB Ophthalmotilapia nasuta AB Haplotaxodon microlepis AB Gnathochromis permaxillaris AB Boulengerochromis microlepis AB Steatocranus casuarius AB Julidochromis transcriptus AB Tropheus duboisi AB Cyprichromis leptosoma AB Ctenochromis horei AB Neolamprologus brichardi XM_ Maylandia zebra XM_ Tilapia rendalli AB Haplochromis burtoni AB Astatoreochromis alluaudi AB Labidochromis caeruleus AB Haplochromis chilotes AB Pundamilia nyererei AB Salmo salar NM_ Xiphophorus maculatus XM_ Salvelinus alpinus JQ 鲀 Takifugu rubripes XM_ 鳉 Oryzias latipes XM_ Astyanax mexicanus DQ Lepisosteus oculatus XM_ Danio rerio DRU Rattus norvegicus AY Papio anubis NM_ Homo sapiens NM_ Xenopus laevis AF BMP4 mrna, BMP4 mrna ( ) 7 ; 3~4 BMP4 mrna (1~90 ), BMP4 2, BMP4 mrna, BMP4 mrna 6, BMP4 mrna 1, ( 6 A); 2 ( 6B); 3 ( 6C) 5 ( 6D F)

8 3 : BMP4 (NJ ) Bootstrap 1000, Fig. 3 Phylogenetic tree of BMP4 amino acid sequences based on Neighbor-Joining (NJ) method The tree is based on a 1000 bootstrap procedure; the scale bar 0.02 in terms of genetic distance is indicated below the tree. 4 BMP4 mrna B: ; C: ; D: ; Gi: ; Go: ; H: ; I: ; K: ; L: ; M: ; Sp: ; Sc: ; St:. BMP4 mrna (P<0.05). Fig. 4 Expression of BMP4 mrna in various tissues of Cynoglossus semilaevis B: brain; C: cartilage; D: dorsal fin; Gi: gill; Go: gonad; H: heart; I: intestine; K: kidney; L: liver; M: muscle; Sp: spleen; Sc: spinal cord; St: stomach. Different letters indicate significant differences among BMP4 mrna levels in various tissues (P<0.05). 5 BMP4 mrna hpf, dph. BMP4 mrna (P<0.05). Fig. 5 BMP4 mrna expression levels in early developmental stages of Cynoglossus semilaevis hpf represents hours post fertilization; dph represents days post hatching. Different letters indicate significant differences among BMP4 mrna levels at different developmental stages (P<0.05).

508 中国水产科学第 23 卷的 BMP4 基因在冠状幼鳍上下颌及胸鳍表达明分析结果表明, 半滑舌鳎与已报道的其他脊椎动显, 而在背鳍腹鳍处尚无表达; BMP4 mrna 在物 BMP4 氨基酸序列在 C 端高度保守(长度约为 10 日龄仔鱼冠状幼鳍上下颌鳃盖骨及背鳍 110~130 aa), 具备较为保守的 7 个半胱氨酸残基, 腹鳍臀鳍处均有表达 (图 6G);

9 508 中国水产科学第 23 卷的 BMP4 基因在冠状幼鳍上下颌及胸鳍表达明分析结果表明, 半滑舌鳎与已报道的其他脊椎动显, 而在背鳍腹鳍处尚无表达; BMP4 mrna 在物 BMP4 氨基酸序列在 C 端高度保守(长度约为 10 日龄仔鱼冠状幼鳍上下颌鳃盖骨及背鳍 110~130 aa), 具备较为保守的 7 个半胱氨酸残基, 腹鳍臀鳍处均有表达 (图 6G); BMP4 基因在符合 TGF-β 超家族成员在氨基酸序列的共同特 15~20 日龄稚鱼上下颌鳃盖骨及背鳍腹鳍征 [2] 此外, 半滑舌鳎 BMP4 基因也具备脊椎动臀鳍处均有表达(图 6H, 图 6I), 此阶段冠状幼鳍物 BMP 家族共有的 RXXR 蛋白水解位点, 已报道逐渐萎缩直至消失的脊椎动物 BMP4 酶切位点序列为 R(I/V)SR, 并 3 且该位点的位置较为保守 [22] 讨论已有研究表明, BMP4 基因在脊椎动物中具有本研究克隆得到半滑舌鳎 BMP4 cdna 全长广泛的组织表达分布例如, 褐牙鲆 BMP4 mrna 序列, 并推导出其编码的氨基酸序列序列比对在除肝之外的组织中均有表达, 在肠道肾和皮图6 BMP4 基因在半滑舌鳎早期发育阶段的空间表达分布 A.1 日龄, B. 2 日龄, C. 3 日龄, D-F. 5 日龄, G.10 日龄, H. 15 日龄, I. 20 日龄, J. 对照组(10 日龄). BMP4 表达的部位为淡蓝色或深蓝色. Fig. 6 Spatial expression pattern of BMP4 gene in early developmental stages of Cynoglossus semilaevis by whole mount in situ hybridization A. 1 dph; B. 2 dph; C. 3 dph; D-F. 5 dph; G. 10 dph; H. 15 dph; I. 20 dph; J. Negative control (10 dph). Hybridization signals were reflected as blue color.

10 3 : [13] BMP4 mrna 13, BMP4,, BMP4 mrna, [14] BMP4, [3], BMP4 mrna,, BMP4 mrna, BMP4 mrna [23], BMP [24] BMP4,, PCR, BMP4 ( ),, BMP4, 16 h 7 [14], (Senegalese sole)bmp4 2 mrna [23],, [25],, BMP4 [8, 26 27], BMP4,,, BMP4 [25, 28], BMP4, 1~2 BMP4 mrna : 1, ( ) ; 2,, [18],, 3~4 BMP4 mrna, ; 5, BMP4 mrna,, BMP4, BMP4, BMP4 mrna, 3, BMP4,, ( ) ;,,, [18], BMP4 ( 6G I),, BMP4,, [29], [30], 1 ( ), [18],,

11 [29, 31 32], ; 28, [33] BMP4, BMP4 mrna,,, BMP4 cdna,, BMP4, 参考文献 : [1] Chen D, Zhao M, Mundy G R. Bone morphogenetic proteins[j]. Growth Fact, 2004, 22(4): [2] Bragdon B, Moseychuk O, Saldanha S, et al. Bone morphogenetic proteins: A critical review[j]. Cell Signal, 2011, 23(4): [3] Nishimura R, Hata K, Matsubara T, et al. Regulation of bone and cartilage development by network between BMP signalling and transcription factors[j]. J Biochem, 2012, 151(3): [4] Hongwei C, Wei J, Frank M, et al. Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs)[J]. J Bone Joint Surg, 2003, 85(8): [5] Canalis E. Growth factor control of bone mass[j]. J Cell Biochem, 2009, 108(4): [6] Sato M, Ochi T, Nakase T, et al. Mechanical tension stress induces expression of bone morphogenetic protein (BMP) 2 and BMP 4, but Not BMP 6, BMP 7, and GDF 5 mrna, during distraction osteogenesis[j]. J Bone Mineral Res, 1999, 14(7): [7] Luo Y W, Li S, Shen L N. The expression of bone morphogenetic protein-4 in the Meckel s Cartilage[J]. Academic Journal of Kunming Medical College, 2004, 25(3): 9 12.[,,. BMP4 mrna Meckel s [J]., 2004, 25(3): 9 12.] [8] Chang H, Brown C W, Matzuk M M. Genetic analysis of the mammalian transforming growth factor-β superfamily[j]. Endocr Soc, 2013, 23(6): [9] Little S C, Mullins M C. Extracellular modulation of BMP activity in patterning the dorsoventral axis[j]. Birth Defects Res C, 2006, 78(3): [10] Hwang S P L, Tsou M F, Lin Y C, et al. The zebrafish BMP4 gene: sequence analysis and expression pattern during embryonic development[j]. DNA Cell Biol, 1997, 16(8): [11] Leong J S, Jantzen S G, Schalburg K R V, et al. Salmo salar and Esox lucius full-length cdna sequences reveal changes in evolutionary pressures on a post-tetraploidization genome[j]. BMC Genom, 2010, 11: 279. [12] Wise S B, Stock D W. Conservation and divergence of Bmp2a, Bmp2b, and Bmp4 expression patterns within and between dentitions of teleost fishes[j]. Evol Dev, 2006, 8(6): [13] Zeng X, Shi Z Y, Chen X W, et al. Skeleton development of Japanese flounder (Paralichthys olivaceus) and expression analysis of related genes (Sox9, Bmp4 and Bmp2) during metamorphosis[j]. Marine Fisheries, 2009, 31(4): [,,,. Sox9, Bmp4 Bmp2 [J]., 2009, 31(4): ] [14] Marques C L, Fernandez I, Viegas M N, et al. Comparative analysis of zebrafish bone morphogenetic proteins 2, 4 and 16: molecular and evolutionary perspectives[j]. Cell Mol Life Sci, 2016, 73(4): [15] Blaxter J H S, Ehrlich K F. Change in behaviour during starvation of herring and plaice larvae[m]//the Early Life History of Fish. Heidelberg: Springer Verlag, 1974: [16] Yin M C. Advances and studies on early life history of fish[j]. Journal of Fisheries of China, 1991, 15(4): [. [J]., 1991, 15(4): ] [17] Chen D G. Fishery Ecology of the Yellow Sea and Bohai Sea[M]. Beijing: China Ocean Press, 1991: [. [M]. :, 1991: ] [18] Wan R J, Jiang Y W, Zhuang Z M. Morphological and developmental characters at the early stages of the tongue fish Cynoglossus semilaevis[j]. Acta Zoologica Sinica, 2004, 50(1): [,,. [J]., 2004, 50(1): ] [19] Ma Q, Zhuang Z M, Feng W R, et al. Evaluation of reference genes for quantitative realtime PCR analysis of gene expression during early development processes of the tongue sole (Cynoglossus semilaevis)[j]. Acta Oceanologica Sinica,

12 3 : , 34(10): [20] Holland L Z, Holland P W H, Holland N D. Revealing homologies between body parts of distantly related animals by in situ hybridization to developmental genes: amphioxus versus vertebrates[m]//molecular Zoology: Advances, Strategies, and Protocols. New York: Wiley-Liss, 1996, : [21] Tamura K, Peterson D, Peterson N, et al. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[j]. Mol Biol Evol, 2011, 28(10): [22] Goldman D C, Hackenmiller R, Nakayama T, et al. Mutation of an upstream cleavage site in the BMP4 prodomain leads to tissue-specific loss of activity[j]. Development, 2006, 133(10): [23] Marques C L, Marques I, Rosa J, et al. Spatiotemporal expression and retinoic acid regulation of bone morphogenetic proteins 2, 4 and 16 in Senegalese sole[j]. J Appl Ichthyol, 2014, 30(4): [24] Kultz D. The combinatorial nature of osmosensing in fishes[j]. Physiology, 2012, 27(4): [25] Kimmel C B, Ballard W W, Kimmel S R, et al. Stages of embryonic development of the zebrafish[j]. Dev Dynam, 1995, 203: [26] Menshykau D, Kraemer C, Iber D. Branch mode selection during early lung development[j]. PLoS Comput Biol, 2012, 8(2): e [27] Mikawa S, Wang C, Sato K. Bone morphogenetic protein-4 expression in the adult rat brain[j]. J Comp Neurol, 2006, 499(4): [28] Chin A, Chen J N, Weinberg E S. Bone morphogenetic protein-4 expression characterizes inductive boundaries in organs of developing zebrafish[j]. Dev Genes Evol, 1997, 207(2): [29] Chen S Q, Gao T X, Wang C, et al. Study on developmental characters in early stage of spotted halibut Verasper variegatus[j]. Periodical of Ocean University of China, 2006, 36(2): [,,,. [J]. :, 2006, 36(2): ] [30] Du J, Xu G F, Han Y, et al. Development of embryos, larvae, fry and juveniles in lenok Brachymystax lenok[j]. Journal of Dalian Ocean University, 2010, 25(5): [,,,.,, [J]., 2010, 25(5): ] [31] Qin Z Q, Lin Y J, Zhang Y Z, et al. Study on the early development of southern flounder Paralichthys lethostigma[j]. Journal of Oceanography in Taiwan Strait, 2008, 27(4): [,,,.,, [J]., 2008, 27(4): ] [32] Liu X Z, Xu Y J, Liu X F, et al. Embryonic and postembryonic development of barfin flounder Verasper Moseri[J]. Oceanologia et Limnologia Sinica, 2009, 40(6): [,,,. (Verasper moseri) [J]., 2009, 40(6): ] [33] Tong X H. Studies on early development and related physiological traits in turbot[d]. Qingdao: Institute of Oceanology, Chinese Academy of Sciences, 2010: [. [D]. :, 2010: ]

13 Molecular cloning and expression analysis of BMP4 gene in tongue sole (Cynoglossus semilaevis) MA Qian, FENG Wenrong, LIU Shufang, ZHUANG Zhimeng, MA Hui Key Laboratory for Fishery Resources and Eco-environment, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao , China Abstract: The growth and development of the vertebrate skeleton is regulated by the bone morphogenetic proteins (BMPs), among which BMP4 is one of the most active osteoinduction factors. In this study, we cloned the BMP4 gene of the tongue sole(cynoglossus semilaevis) and described its expression profiling. The BMP4 cdna sequence was 1680 bp, and contained a 5 -untranslated region (5 -UTR) of 419 bp, an open reading frame (ORF) of 1212 bp encoding a protein of 403 amino acids, and a 3 -UTR of 49 bp. Homology and phylogenetic analyses revealed that the deduced amino acid sequence of the C. semilaevis BMP4 protein shares highest identity with those of Cichlidae species, and fish BMP4 forms a single lineage distinct from the BMP4 proteins of other vertebrates. The tissue expression distribution of the BMP4 gene was examined in 13 adult tissues. BMP4 mrna was predominantly expressed in the gill, and less in the dorsal fin, cartilage, etc. In the early developmental stages, BMP4 mrna was also detected at the egg, larval, juvenile, and fingerling stages, with the highest expression in the egg stage (cleavage and gastrula periods), and the second highest in the larval stage (3 4 days old). Whole-mount in situ hybridization showed that BMP4 mrna was firstly detected in head and anterior region in the early larval stage, and then appeared in the crown-like larval fin, jaw, and pectoral fin. BMP4 mrna was then predominantly expressed in all the fins and the operculum during the early juvenile stage. These results demonstrate the important role of BMP4 in the fin development of C. semilaevis, and provide a theoretical foundation for investigating the mechanisms regulating fish bone development. Key words: Cynoglossus semilaevis; BMP4; molecular cloning; gene expression; whole-mount in situ hybridization; skeletal development Corresponding author: ZHUANG Zhimeng. zhuangzm@ysfri.ac.cn

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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

Microsoft Word - 2——马 骞_news_.doc

Microsoft Word - 2——马骞_news_.doc