马氏珠母贝(Pinctada fucata martensii)PmHEX基因的功能研究

几丁质是软体动物贝壳有机框架的重要成分,其代谢在贝壳矿化中发挥重要作用。β-N-乙酰-己糖胺酶(HEX, EC3.2.1.52)是几丁质代谢的关键水解酶。为了探究马氏珠母贝β-N-乙酰-己糖胺酶(Pm HEX)(登录号:MF555152)在贝壳形成中的作用,本研究利用原位杂交(ISH)技术检测Pm HEX基因在外套膜的定位,结果显示Pm HEX的mRNA主要分布于外侧褶的外上皮细胞、中褶的内侧上皮细胞和内褶上皮细胞。利用RNAi技术抑制Pm HEX表达后,Pm HEX在边缘区和套膜区的表达量均显著下调;SEM观察发现实验组的棱柱层和珍珠层的微观结构都出现不同程度的紊乱。综上所述,Pm HEX可能通过影响几丁质代谢,参与马氏珠母贝贝壳棱柱层和珍珠层的矿化过程。     

马氏珠母贝PmCOLVIA6基因的克隆和表达分析

collagenⅥ(COLⅥ)是胶原蛋白家族的重要成员,广泛分布于动物体细胞外基质,在脊椎动物骨骼形成过程中发挥重要作用。为了探究COLⅥ在马氏珠母贝贝壳形成中的作用,本研究运用聚合酶链式反应(PCR)和c DNA末端快速扩增技术(RACE)获得了马氏珠母贝Pm COLVIA6基因的全长序列,克隆获得其启动子区域,并对其蛋白结构进行了分析。荧光定量PCR技术检测了该基因在马氏珠母贝各个组织的表达量分布,并利用原位杂交技术检测Pm COLVIA6基因在外套膜的表达定位。结果表明:Pm COLVIA6序列全长为2 100 bp,开放阅读框(ORF)长为1 875 bp,编码624个氨基酸,5'非翻译区(5'UTR)长56 bp,3'非翻译区(3'UTR)长169 bp,于5'调控区克隆得到长为1 826 bp的序列,可能存在4个启动子序列。Pm COLVIA6蛋白含有典型的v WA结构域,但三股螺旋结构缺失。荧光定量分析表明Pm COLVIA6基因在马氏珠母贝的各个组织均有表达,而在珍珠囊和外套膜中央区的表达量显著高于其它组织。原位杂交结果显示Pm COLVIA6的m RNA主要分布于外套膜中央区的外表皮细胞。综上所述,Pm COLVIA6可能参与马氏珠母贝珍珠层的形成。     

合浦珠母贝IGF2BP1基因的克隆与表达分析

胰岛素样生长因子2信使核糖核酸结合蛋白(insulin-like growth factor 2 mRNA binding protein,IGF2BP)在脊椎动物体内功能很多,但在贝类中研究很少。为研究IGF2BP1是否参与了贝类的生物矿化过程,本研究通过RACE技术克隆获得合浦珠母贝(Pinctada fucata)IGF2BP1基因cDNA序列,命名为PfIGF2BP1。该基因全长2 980 bp,其中开放阅读框长1 737 bp,预测编码579个氨基酸,有4个KH结构域和2个RRM结构域。多重序列分析发现各物种的IGF2BP1氨基酸序列非常保守。实时定量PCR结果显示PfIGF2BP1在肠等8个组织和壳顶期等5个胚胎发育时期中均有表达,表达量最高的组织为珍珠囊(p0.05),说明Pf IGF2BP1可能参与了珍珠形成过程。Pf IGF2BP1在眼点期表达量最高,其次为壳顶期,说明PfIGF2BP1可能参与次生壳的形成过程。原位杂交表明PfIGF2BP1在外套膜边缘的外褶中表达,推测其可能参与棱柱层的形成。本研究为以后探讨IGF2BP1在贝类生物矿化过程中的作用奠定了基础。     

马氏珠母贝SRF基因的分子特征及组织特异性表达

血清反应因子(SRF)是一个高度保守的转录因子,在细胞增殖、细胞凋亡以及免疫反应中发挥重要作用。为了探究血清反应因子在马氏珠母贝中的生物学功能,本研究运用c DNA末端快速扩增(RACE)技术克隆得到马氏珠母贝SRF基因(Pm SRF)c DNA的全长序列,并且应用实时荧光定量PCR技术对Pm SRF基因在马氏珠母贝不同组织中的表达进行检测。结果显示,Pm SRF基因序列全长1 758 bp,其中开放阅读框(ORF)为1 440 bp,编码479个氨基酸,5'UTR为65 bp,3'UTR为253 bp,包含29 bp的poly A。预测其相对分子量为50 534.6 Da,理论等电点为7.71。多序列比对结果发现物种间SRF具有较高的保守性。SMART软件分析显示Pm SRF具有典型的MADS结构域。荧光定量PCR数据分析表明,Pm SRF基因在马氏珠母贝闭壳肌、肝胰腺、血细胞、外套膜、性腺、鳃六种组织中均有表达,其中在鳃中表达量最高。本研究可为进一步探究Pm SRF在贝类中的生物学功能提供重要的理论基础和参考价值。     

马氏珠母贝STA RDL3基因的克隆与表达性分析

采用RACE技术克隆获得马氏珠母贝STARDL3基因c DNA全长序列(Pm-STARDL3);利用荧光定量技术检测Pm-STARDL3基因在各个组织中的表达量。结果表明,Pm-STARDL3基因c DNA序列全长3 655 bp,开放阅读框(ORF)长1 491 bp,编码496个氨基酸,5'非翻译区(5'UTR)长110 bp,3'UTR长2 054 bp。PmSTARDL3氨基酸序列同源比对分析显示与华贵类栉孔扇贝(Mimachlamys nobilis)STARDL3的序列的相似度最高。SMART软件分析得出,Pm-STARDL3有STARD类蛋白特有的结构域。荧光定量PCR检测结果表明Pm-STARDL3在肝胰腺中表达量最高,其后依次是外套膜、鳃与闭壳肌,各组织的表达量差异具统计学意义(p0.05)。     

马氏珠母贝galectin-4基因cDNA的克隆及表达分析

半乳糖凝集素(Galectins)属于一种多功能凝集素家族,在机体抵抗微生物的感染中起重要作用。本研究利用cDNA末端快速扩增(rapid-amplification of cDNA ends,RACE)技术克隆获得马氏珠母贝galectin-4(PmGal-4)基因cDNA的全长,并对其序列进行分析。PmGal-4基因cDNA全长1 071 bp,5′非翻译区(5′UTR)长75 bp,3′非翻译区(3′UTR)长72 bp;其开放阅读框(open reading frame,ORF)长度为924 bp,编码307个氨基酸组成的前体肽,理论分子量约为34.6 kD,理论等电点为8.80。多序列比对结果显示各物种间galectin-4具有高保守性。序列分析结果显示PmGal-4的氨基酸序列具有典型富含半胱氨酸(cysteine-rich domain,CRD)的结构域。实时荧光定量PCR分析表明,PmGal-4基因在马氏珠母贝所检测的组织中呈组成型表达特征,但在闭壳肌和外套膜中表达量最高。上述结果表明PmGal-4基因可能参与了马氏珠母贝多种生理功能,特别是在免疫防御反应中具有重要的作用。     

马氏珠母贝组蛋白H3基因的克隆与表达特性分析

组蛋白H3是组成核小体的基本组分之一。研究表明,组蛋白不仅参与染色质的组装,也可以通过调节基因表达参与调控细胞分裂、凋亡、DNA修复等细胞生命过程。为了探究马氏珠母贝组蛋白H3(Pinctada martensii histone H3,Pm H3)的序列及表达特征,本文运用c DNA末端快速扩增(RACE)技术克隆得到Pm H3的c DNA全长序列,并且应用实时荧光定量PCR技术对Pm H3基因在马氏珠母贝不同组织中的表达进行检测。结果显示,Pm H3基因序列全长627 bp,其中开放阅读框(ORF)为411 bp,编码136个氨基酸,5'UTR为42 bp,3'UTR为174 bp,包含26 bp的poly A。预测其相对分子量为15 388.0 Da,理论等电点为11.27,不稳定系数为47.19,疏水性水平-0.604。多序列比对结果表明H3基因极为保守,在各物种间的相似度达到99%~100%。SMART软件分析发现Pm H3具有一个典型的H3结构域。荧光定量PCR数据结果显示,Pm H3基因在马氏珠母贝外套膜边缘区、外套膜中央区、鳃、血细胞、闭壳肌、足、性腺、肝胰腺八种组织中均有表达,其中在性腺中表达量最高。本研究可为进一步探讨Pm H3基因在贝类中的生物学特性以及组蛋白修饰提供理论依据。     

合浦珠母贝外套膜细胞engrailed、BMP2和Smad3的mRNA表达水平的相关性

软体动物engrailed蛋白和骨形成相关蛋白对胚胎贝壳区域边界形成可能具有重要作用,engrailed还被推测为调节基质蛋白在外套膜组织区域化表达的重要调控因子．因此,弄清调控engrailed在软体动物中特征表达的分子机制有着重要的研究意义．但是,由于贝类基因组测序尚不完整,目前也没有建立获得贝类细胞系,以致于许多预测可能参与调控的基因需要通过克隆来鉴定,而且经典的研究细胞信号通路的方法也很难得到应用．目前,在中国南海广泛养殖的合浦珠母贝中,已获知其BMP2和Smad3的cDNA全长,以该贝的基因组为模板,PCR扩增获得了一段engrailed编码区片段．经软件分析,该片段含有EH4结构域,且与其他物种engrailed蛋白具有很高的同源性．研究的贝中,特别是外套膜组织中,engrailed、BMP2和Smad3三者表达之间的相关性,将有助于我们理解贝壳形成的分子机制．贝壳缺刻后半定量PCR试验结果表明,三者均参与贝壳修复,且在贝壳缺刻后的修复过程中,engrailed和Smad3的mRNA表达变化规律非常相似,提示它们之间可能存在相互影响的联系．用地塞米松(DXM)和过氧化氢(H₂O₂)分别处理原代培养的贝外套膜组织迁出细胞,实时相对定量PCR检测engrailed、BMP2和Smad3的mRNA表达水平,统计分析结果表明,三者具有显著的相关性．上述所有结果为进一步研究贝类生物矿化的发育和信号转导机制提供了新的思路和基础．     

马氏珠母贝(Pinctada fucata martensii)PmTYRL-1基因的分子特征与表达分析

酪氨酸酶(Tyrosinase)是Ⅲ-型铜蛋白超家族的一员,在色素沉着、伤口愈合、先天免疫和角质层形成等过程发挥重要作用。为了探究酪氨酸酶样蛋白1基因在马氏珠母贝贝壳形成中的作用,本研究运用RACE技术获得马氏珠母贝酪氨酸酶样蛋白1基因(Pm TYRL-1)的cDNA序列全长,并检测Pm TYRL-1在各个组织的表达模式。结果表明:PmTYRL-1序列全长2 406 bp,其中5'UTR为169 bp,3'UTR为116 bp,开放阅读框(ORF)为2 121 bp,编码706个氨基酸。预测其分子量(MW)为81 112.60 Da,理论等电点(PI)为8.67。SMART软件分析PmTYRL-1氨基酸序列具有典型的酪氨酸酶结构域。多序列比对发现PmTYRL-1与其它物种的同源性较低。qPCR结果表明,PmTYRL-1在边缘区显著高表达,其次是套膜区和肝胰腺。综上所述,PmTYRL-1可能参与马氏珠母贝的贝壳形成,可为进一步探究PmTYRL-1在马氏珠母贝生物矿化中的作用提供理论基础。     

马氏珠母贝磺基转移酶PmCHST11基因的分子特征与表达分析

硫酸软骨素(CS)具有抗炎、抗病毒和软骨保护功能等免疫学功能。硫酸软骨素磺基转移酶CHST11(carbohydrate sulfotransferase 11,chondroitin 4)催化磺酸基从供体PAPS(3'-磷酸腺苷酰-5'-磷酸硫酸盐)转移到软骨素N-乙酰半乳糖胺的4位碳上,实现磺酸化反应,是合成硫酸软骨素的关键酶。本研究通过对马氏珠母贝磺基转移酶基因Pm CHST11(Pinctada martensii carbohydrate sulfotransferase 11)的全长克隆及组织表达分析,探究Pm CHST11在马氏珠母贝免疫调节中的作用。结果表明:Pm CHST11序列全长为1 418 bp,编码426个氨基酸;Pm CHST11序列含有信号肽结构区域,跨膜结构域和磺基转移酶-2结构域,为高尔基体膜偶联磺基转移酶;多序列比对结果显示,Pm CHST11与其它物种的CHST11同源性较低;实时荧光定量结果表明,Pm CHST11基因在多个组织中均有表达,其中在外套膜边缘区显著高表达(p0.05)。本研究为分析Pm CHST11在马氏珠母贝中的免疫调节中的作用积累基础资料。     

Expression of genes responsible for biomineralization of Pinctada fucata during development

This study compares the expression levels of nacrein, N16, MSI60, Prismalin-14, aspein and MSI31 genes during the ontogeny of Pinctada fucata. Several novel findings were obtained: 1) The early calcitic prismatic layer was distinguished as a thin membrane-like structure. 2) Initial formation of the nacreous layer started from the mantle pallial region at the age of 31 days. 3) 18S rRNA of P. fucata was determined to be more suitable as a real-time PCR reference gene compared with GAPDH and β-actin genes. 4) A relationship was recognized between the expression levels of the above six organic matrix genes and biomineralization of the larval shell. The lack of calcite in the shells of the veliger and pediveliger stages, when MSI31 and Prismalin-14 genes were expressed, makes a role of polymorph control by these genes less likely. The hypothetical involvement of N16 and MSI60 proteins in aragonitic nacreous layer formation was corroborated by the expression levels of N16 and MSI60 genes during ontogeny. Our results are important with respect to the control of CaCO₃ crystal polymorphism and shell microstructures in P. fucata.     

A novel matrix protein family participating in the prismatic layer framework formation of pearl oyster, Pinctada fucata

Understanding the molecular composition and the formation mechanism of shell matrix framework is of great interest for biomineralization in mollusk shell. The cDNAs encoding a novel matrix protein family (KRMP) were cloned from the mantle of pearl oyster, Pinctada fucata. Analysis of the deduced amino acid sequences revealed that KRMP have a high proportion of lysine, glycine, and tyrosine, and their predict isoelectric points are higher than any other identified shell matrix protein to our knowledge. The deduced amino acid sequences of KRMP can be divided into three regions, including an N-terminal signal peptide, a lysine-rich basic region interacting with acidic proteins or CO(3)(2-), and a Gly/Tyr-rich region involved in the protein cross-link via quinone-tanning process. RT-PCR and in situ hybridization demonstrated that KRMP mRNA was specifically expressed in the mantle edge, involved in the prismatic layer formation. Taken together, it seems that KRMP is a matrix protein family participating in the framework formation of prismatic layer.     

A novel matrix protein participating in the nacre framework formation of pearl oyster,Pinctada fucata

Understanding the molecular composition is of great interest for both nacre formation mechanism and biomineralization in mollusk shell. A cDNA clone encoding an MSI31 relative, termed MSI7 because of its estimated molecular mass of 7.3 kDa, was isolated from the pearl oyster, Pinctada fucata. This novel protein shares similarity with MSI31, a prismatic framework protein of P. fucata. It is peculiar that MSI7 is much shorter in size, harboring only the Gly-rich sequence that has been proposed to be critical for Ca(2+) binding. In situ hybridization result showed that MSI7 mRNA was expressed specifically at the folds and outer epithelia of the mantle, indicating that MSI7 participates in the framework formation of both the nacreous layer and prismatic layer. In vitro experiment on the function of MSI7 suggested that it accelerates the nucleation and precipitation of CaCO(3). Taken together, we have identified a novel matrix protein of the pearl oyster, which may play an important role in determining the texture of nacre.     

cDNA Microarray Analysis Revealing Candidate Biomineralization Genes of the Pearl Oyster, <Emphasis Type="Italic">Pinctada fucata martensii</Emphasis>

Biomineralization is a common biological phenomenon resulting in strong tissue, such as bone, tooth, and shell. Pinctada fucata martensii is an ideal animal for the study of biomineralization. Here, microarray technique was used to identify biomineralization gene in mantle edge (ME), mantle center (MC), and both ME and MC (ME-MC) for this pearl oyster. Results revealed that 804, 306, and 1127 contigs expressed at least three times higher in ME, MC, and ME-MC as those in other tissues. Blast against non-redundant database showed that 130 contigs (16.17 %), 53 contigs (17.32 %), and 248 contigs (22.01 %) hit reference genes (E?≤??10), among which 91 contigs, 48 contigs, and 168 contigs could be assigned to 32, 26, and 63 biomineralization genes in tissue of ME, MC, and ME-MC at a threshold of 3 times upregulated expression level. The ratios of biomineralization contigs to homologous contigs were similar at 3 times, 10 times, and 100 times of upregulated expression level in either ME, MC, or ME-MC. Moreover, the ratio of biomineralization contigs was highest in MC. Although mRNA distribution characters were similar to those in other studies for eight biomineralization genes of PFMG3, Pif, nacrein, MSI7, mantle gene 6, Pfty1, prismin, and the shematrin, most biomineralization genes presented different expression profiles from existing reports. These results provided massive fundamental information for further study of biomineralization gene function, and it may be helpful for revealing gene nets of biomineralization and the molecular mechanisms underlining formation of shell and pearl for the oyster.     

Tyrosinase localization in mollusc shells

In molluscan shellfish, pigmentation is frequently observed in the calcified shell, but the molecular basis of this process is not understood. Here, we report two tyrosinase proteins (Pfty1 and Pfty2) found in the prismatic shell layer of the pearl oyster Pinctada fucata; this layer is recognized as the pigmented region in P. fucata. The protein sequences were deduced from the corresponding cDNAs and confirmed by MALDI-TOF/TOF analysis. The sequences suggest that both tyrosinases have two copper-binding sites in similar N-terminal domains that are homologous to tyrosinases of cephalopods and hemocyanins of gastropods. In turn, this suggests that bivalve tyrosinases are evolved from a common ancestral copper-binding protein in the mollusc. Pfty1 and Pfty2 were specifically expressed in the mantle, and their expression in the mantle is different from each other, suggesting that these tyrosinases have distinctive roles in melanogenesis in shells.     

Identification of genes directly involved in shell formation and their functions in pearl oyster, Pinctada fucata

Mollusk shell formation is a fascinating aspect of biomineralization research. Shell matrix proteins play crucial roles in the control of calcium carbonate crystallization during shell formation in the pearl oyster, Pinctada fucata. Characterization of biomineralization-related genes during larval development could enhance our understanding of shell formation. Genes involved in shell biomineralization were isolated by constructing three suppression subtractive hybridization (SSH) libraries that represented genes expressed at key points during larval shell formation. A total of 2,923 ESTs from these libraries were sequenced and gave 990 unigenes. Unigenes coding for secreted proteins and proteins with tandem-arranged repeat units were screened in the three SSH libraries. A set of sequences coding for genes involved in shell formation was obtained. RT-PCR and in situ hybridization assays were carried out on five genes to investigate their spatial expression in several tissues, especially the mantle tissue. They all showed a different expression pattern from known biomineralization-related genes. Inhibition of the five genes by RNA interference resulted in different defects of the nacreous layer, indicating that they all were involved in aragonite crystallization. Intriguingly, one gene (UD_Cluster94.seq.Singlet1) was restricted to the 'aragonitic line'. The current data has yielded for the first time, to our knowledge, a suite of biomineralization-related genes active during the developmental stages of P. fucata, five of which were responsible for nacreous layer formation. This provides a useful starting point for isolating new genes involved in shell formation. The effects of genes on the formation of the 'aragonitic line', and other areas of the nacreous layer, suggests a different control mechanism for aragonite crystallization initiation from that of mature aragonite growth.