长牡蛎 dpp 同源基因的克隆及其在贝壳发生中的功能研究

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1 长牡蛎 dpp 同源基因的克隆及其在贝壳发生中的功能研究 1, 刘刚 2, 郇聘 1 1, 刘保忠 (1., ; 2., ) 摘要 : dpp 是转化生长因子 β 家族的重要成员, 在胚胎发育中作为形态发生子参与体轴形成及附肢发育等过程 为了研究牡蛎 dpp 同源基因在贝壳发生中的作用, 揭示贝壳发生的分子机理, 作者克隆了一种长牡蛎 (Crassostrea gigas) 的 dpp 基因, 命名为 cgdpp 序列和进化分析显示, cgdpp 分子中不同位点的氨基酸残基变异程度不同 作者用整装原位杂交的手段研究了 dpp 同源基因在长牡蛎早期发育中的表达情况 结果表明, dpp 同源基因参与了从贝壳形成区开始分化到早期贝壳形成的全过程 在担轮幼虫 (Trochophore) 中, dpp 同源基因似乎调控了贝壳的形状与扩张速度 ; 在早期 D 形幼虫中, dpp 同源基因表达量突然下降至痕量水平, 并与贝壳发育区无明显的关联, 提示 dpp 同源基因可能仅仅参与了贝壳的发生, 不参与其进一步的发育过程 关键词 : 长牡蛎 (Crassostrea gigas); 发育 ; 整装原位杂交 ; 转化生长因子中图分类号 : Q344 文献标识码 : A 文章编号 : (2014) doi: /hykx ,, [1-5] [6-7],,,,, ;,, D,,, Ⅰ, dpp dpp β (TGF-β) (BMP), BMP2/4 [8], dpp ( ) [9-12], (Crassostrea gigas) dpp, ;, dpp 1 材料与方法 1.1 实验动物与幼虫培养,, 25,, 4%, 长牡蛎 dpp 基因的克隆 EST dpp HS HS cdna 3, dpp 3 cdna 5 3 EST (HS HS140609) cgdpp-f1(tacacacgggattatttact) cgdpp- R1(CGCAAATAAATAATATCACA), cdna dpp cdna, cgdpp : ; : : ( ); 973 (2010CB126403) : (1987-),,,,, liugang10@mails.ucas.ac.cn;,, : , bzliu@qdio.ac.cn Marine Sciences / Vol. 38, No. 9 /

2 1.3 序列特征和进化分析 cgdpp : Expasy (http: //web.expasy.org/compute_pi/) ; SignalP ( NCBI CDD cgdpp (http: // cgdpp dpp, Neighbor-Joining [13] 1.4 整装原位杂交 ORF, cgdpp-f2(ttctctgggagagagcgtca) cgdpp-r2(ctaccggcaaccacagccttc) PCR, pgem-t (Promega), (DH5α), T7 SP6 RNA RNA; DIG RNA Labeling Mixture (Roche) RNA RNA,, D cgdpp Maures [14] 2 结果 2.1 cgdpp 基因 cgdpp cdna 1448bp, 131bp 5 UTR, 1257bp (ORF) 60bp 3 UTR, 418, kda, 9.59 GenBank(Accession No. KC ) 1~20 (SP), -, C 106~ R(K)-X-X-R(K), ( 1) [15] C 105 7, BMP2 Cgdpp, 1 1 Cgdpp Fig.1 A scheme of Cgdpp 3, N (SP), (Pro-domain),,, (mature domain) Cgdpp (PS), R(K)-X-X-R(K) (R K ) 7 (C) Cgdpp is comprised of three domains. The signal peptide locates at the N terminal, followed by the Pro-domain and mature domain. The molecule is cleaved at a consus site R(K)-X-X-R(K) between the two domains and release mature domain. There are multiple putative proteolytical sites (PSs) in Cgdpp. Seven conserved cysteine residuals in the mature domain of Cgdpp 2.2 进化分析 cgdpp, ( 2) cgdpp dpp, dpp ; (Homo sapiens) (Rattus norvegicus) (Danio rerio) BMP2 BMP4 ; (Drosophila melanogaster) ; 2.3 cgdpp 在长牡蛎早期发育中的时空表达分析 14 h(14 hours post fertilization, 14 hpf) cgdpp ( 3) 4 (2 hpf) cgdpp 4 hpf, cgdpp, (8 hpf); 8 / 2014 / 38 / 9

3 2 cgdpp, 1000 bootstrap Fig. 2 A phylogenetic tree of dpp-homologs constructed through the Neighbor-Joining method. The bootstrap values were calculated from 1000 replicates, 6 hpf, cgdpp (11 hpf), cgdpp, ( ) D (14 hpf), cgdpp,, cgdpp, 4 (8hpf) (11hpf) cgdpp 3 讨论与结论 cdna dpp cgdpp, TGF-β,,, 7, BMP2 cgdpp,, ( ), C 105,, cgdpp dpp,, cgdpp,, ( 2), bootstrap ( 45),, cgdpp, cgdpp D, cgdpp, cgdpp, cgdpp, Kin [16] (Saccostrea kegaki) (4hpf 8hpf),,, cgdpp Ninov [17], dpp, cgdpp,, dpp, [18], dpp [8, 12], cgdpp dpp,, Marine Sciences / Vol. 38, No. 9 /

4 cgdpp, ( 3 4), cgdpp Nederbragt [2] (Patella vulgata) Nederbragt dpp engrailed P. vulgata,, dpp engrailed 3 cgdpp Fig. 3 Expression pattern of cgdpp mrna during early development of C. gigas 14hpf ( ), Sf., =50 μm Non-specific staining occurred around the shell plates at 14 hpf (white stars), Bar = 50 μm 4 cgdpp (8 hpf) (11 hpf) Fig. 4 Schemes of the cgdpp expression pattern in the gastrula (8 hpf) and trochophore (11 hpf) of C. gigas cgdpp, (sf),, Gray regions indicate the distributions of cgdpp mrna, dpp engrailed D, cgdpp, cgdpp,,,, ( Dpp ) Kin [16] dpp,, D, Kin cgdpp,, cgdpp,, cgdpp mrna,,,, cgdpp, cgdpp, : [1] Jackson D J, Wörheide G, Degnan B M. Dynamic expression of ancient and novel molluscan shell genes during ecological transitions[j]. BMC Evolutionary Biology, 2007, 7 (1): 160. [2] Nederbragt A J, Van Loon A E, Dictus W J A G. Expression of Patella vulgata orthologs of engrailed and dpp-bmp2/4 in adjacent domains during molluscan shell development suggests a conserved compartment boundary mechanism[j]. Developmental Biology, 2002, 246 (2): [3] Shen X, Belcher A M, Hansma P K, et al. Molecular cloning and characterization of lustrin A, a matrix protein from shell and pearl nacre of Haliotis rufescens 10 / 2014 / 38 / 9

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6 Clone of dpp gene and its functions in larval shell formation of the Pacific oyster Crassostrea gigas LIU Gang 1, 2, HUAN Pin 1, LIU Bao-zhong 1 (1. Institute of Oceanology, Chinese Academy of Sciences, Qingdao , China; 2. University of Chinese Academy of Sciences, Beijing , China) Received: Feb., 5, 2013 Key words: Crassostrea gigas; development; whole mount in situ hybridization; transforming growth factor Abstract: Dpp is an important member of the transforming growth factor (TGF) β family, which acts as a morphogen in many animals. It was revealed that dpp gene was involved in the shell formation of mollusks. The Pacific oyster (Crassostrea gigas) is a world-wide economic bivalve species, and is a potential model organism of bivalves. In this work, we cloned a dpp-homolog from C. gigas (cgdpp), which exhibited typical characteristics of TGF-β members. The expression pattern of cgdpp was investigated in the early development of C. gigas using whole mount in situ hybridization. The results indicated that cgdpp might participate in cell motility and differentiation during development of the shell field. In trochophore, the cgdpp might regulate the shape and extension of the shell. Only trace mount of cgdpp was detected in the early D-veliger, suggesting that the cgdpp is not involved in the later development of shells since D-veliger. In addition, different from previous report, our results did not support the involvement of cgdpp in the formation of the shell hinge. Our studies demonstrated the roles of cgdpp in the shell formation of C. gigas, which would help to reveal the mechanism of shell formation. ( 本文编辑 : 谭雪静 ) 12 / 2014 / 38 / 9