翡翠贻贝外套膜转录组及贝壳珍珠质层和肌棱柱层蛋白质组分析

贝类贝壳在生物材料学及仿生学研究中占据着重要地位。贝壳基质蛋白质是贝壳中的主要有机质成分,对贝壳的形成以及贝壳的力学性能至关重要。翡翠贻贝(Perna viridis)贝壳主要由肌棱柱层和珍珠质层两种微观结构组成,其结构层次较简单,是研究贝壳基质蛋白质及其与贝壳形成关系的极好材料。为深入研究翡翠贻贝贝壳基质蛋白质的分子组成以及分布特点,首先采用扫描电子显微镜,观察翡翠贻贝贝壳内表面珍珠质层和肌棱柱层的微观结构;采用刮取法获得贝壳内表面珍珠质层和肌棱柱层的粉末;对不同层次的贝壳粉末,利用酸溶法去除碳酸钙成分,所获得的有机质组分通过离心将其分为酸可溶性组分和酸不溶性组分。采用Illumina深度测序技术对翡翠贻贝外套膜组织进行大规模测序和序列组装,在此基础上,采用LC-MS/MS质谱技术结合外套膜转录组数据库搜索,对翡翠贻贝肌棱柱层和珍珠质层贝壳基质蛋白质开展组学分析。扫描电镜观察结果表明,翡翠贻贝贝壳有两种不同形貌结构的层次,其中珍珠质层为片状堆叠结构,而肌棱柱层为柱状结构。翡翠贻贝外套膜转录组测序共计获得 69 859 条Unigene。蛋白质组学鉴定结果表明,翡翠贻贝贝壳中总计鉴定到蛋白质54种,其中38种为肌棱柱层所特有蛋白质,3种珍珠质层特有蛋白质,另有13种在珍珠质层和肌棱柱层均被鉴定到。肌棱柱层特有蛋白质的分子多样性明显强于珍珠质层。上述研究为进一步探讨贝壳不同微观层次的形成机制,以及贝壳基质蛋白质对贝壳不同结构层次的调控作用机制奠定了基础。     

椭圆背角无齿蚌贝壳棱柱层的结构

用光学显微镜和扫描电子显微镜对椭圆背角无齿蚌Anadonta woodiana elliptica贝壳的横截面、纵截面和水平截面进行了观测。结果表明,边长为10～50μm、高度为35～47μm的多边棱柱体是贝壳棱柱层结构的基本单元。它们在贝壳的水平轴和横轴的二维空间方向逐步进行堆砌,以形成棱柱层。在堆砌时所形成的夹角,导致了贝壳在横轴和纵轴方向形成一定的弧度。在外套膜分泌形成贝壳的过程中,依次形成角质层、棱柱层、珍珠层。     

绢丝丽蚌年龄与生长的研究

绢丝丽蚌一年生长一个生长轮。年轮可肉眼观察贝壳外表面凹陷的生长轮来鉴定,用纵剖贝壳明暗相间层数与打磨后观察棱柱层和珍珠层上的生长轮来验证。绢丝丽蚌10龄以前生长较快,10龄以后生长逐渐减慢。10龄以前年龄(A)与壳长(L)呈直线相关,年龄与壳重(Ws)、体重(W)均呈幂函数相关,其10龄以前的方程式分别为：L=0．8980A 0．8600(r=0．9883),Ws=1．0175A^2.3399(r=0．9997),W=1．3188A^2.3333=0．9997)。10龄以后年龄与壳长、壳重和体重均呈直线相关,其回归方程式分别为：L=0．1817A 7．9085(r=0．9813),Ws=10．7720A 61．1930(r=0．9902),W=13．6960A 78．8690(r=0．9903)。壳长与壳重、体重之间均呈幂函数相关,其相关方程式分别为：Ws=0．6303L^2.4846(r=0．9999),W=0．8181L^2.4775(r=0．9999)。壳重与体重之间呈线性相关,其回归方程式为：W=0．3560 1．2744Ws(r=0．9999)。     

我国蚌科一新纪录

查氏拟齿蚌Pseudodon chaperi (Morgan, 1885) 见壳近似长方形,略膨胀。前部短圆,后背缘呈截状与后缘相交处有一不明显的角。壳顶部低平,略低于后背缘,位于贝壳的前部,距壳前端1/3处。腹缘略直。壳面呈黄褐色或棕褐色,常被腐蚀,生长     

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。     

我国蛾螺科新纪录

海伦闭螺Clea(Anentome)helena(Phillippi) 本种为蛾螺科中较小型的种类。壳高约20毫米,壳宽7毫米,个体大的壳高可达30毫米。有6—8个螺层,螺层缓慢均匀增长;壳坚固,外形呈长圆锥形。壳面呈黄褐色或绿褐色,各螺层具有1—3条红褐色色带及粗的纵肋,体螺层上约有12条纵肋。壳口具有短而宽的前沟。     

三种不同因子对三角帆蚌外套膜细胞Ca~(2+)流动性的影响

<正>三角帆蚌(Hyriopsis cumingii)是我国主要的育珠母蚌,其所培育出的珍珠色泽鲜艳、细腻光滑,因而成为目前淡水育珠生产中首选的育珠母蚌之一。蚌的外套膜是贝壳和珍珠形成的重要组织器官,有内外两层表皮细胞及其间的结缔组织构成。它对钙具有高度通透性,其上皮细胞具有通过细胞膜主动吸收Ca~(2+)和贮存Ca~(2+)的功能,并通过胞吐作用排出钙至外套膜外腔中,这些钙就是形成珍珠的基础。维     

蚌科钩介幼虫比较形态学研究──Ⅰ.四个种幼虫的形态

本文报道圆顶珠蚌、鱼尾楔蚌、中国尖嵴蚌、卵形尖嵴蚌育儿囊的特点和钩介幼虫的形态。应用光镜和扫描电镜对四种蚌的钩介幼虫形态进行了观察和比较。结果表明,四种蚌的育儿囊均为外鳃类的同生型,钩介幼虫为有钩型,幼虫的大小、形状、壳表面、壳钩、棘刺、幼虫丝、感觉毛等在不同种之间存在着差异。文中对这些特征在分类上的意义进行了讨论。     

三角帆蚌贝壳珍珠层颜色遗传规律的初步研究

为了探明三角帆蚌(Hyriopsis cumingii)贝壳珍珠层颜色的遗传规律,为珍珠层颜色的选择育种提供理论指导,利用三角帆蚌紫色和白色选育品系进行自交和正反杂交,建立了白色♂×白色♀、白色♂×紫色♀、紫色♂×白色♀和紫色♂×紫色♀4个交配组合,统计分析了每个交配组合子代的珍珠层颜色分离情况.结果显示,白色自交组合的子代贝壳珍珠层颜色全部表现为白色,没有发生颜色分离;杂交组合的子代珍珠层颜色出现两种情况,一是全部表现为紫色,二是颜色发生分离,且紫色和白色个体比例符合1∶1的比例关系;紫色自交组合的子代珍珠层颜色也出现两种情况,一是全部表现为紫色,二是颜色分离出紫色和白色,且比例符合3∶1的比例关系.结果表明,三角帆蚌贝壳珍珠层颜色受遗传基因控制,可以稳定遗传,属质量性状.珍珠层紫色性状对白色性状为显性,两种颜色性状均不存在母性遗传.白色个体为隐性纯合体,选育纯化较为容易,而紫色个体既有显性纯合体又有杂合体,选育纯化相对较困难.     

13种双壳类贝壳的扫描电镜观察

采用扫描电子显微镜(SEM)对湛江地区的丽文蛤(Meretrix lusoria)、琴文蛤(M.lyrata)、文蛤(M.meretrix)、波纹巴非蛤(Paphia undulata)、菲律宾蛤仔(Ruditapes philippinarum)、杂色蛤仔(R.variegata)、伊萨伯雪蛤(Clausinella isobellina)、格粗饰蚶(Anadara clathrata)、泥蚶(Tegillarca granosa)、栉孔扇贝(Chlamys farreri)、尖紫蛤(Sanguinolaria aauta)、锈色朽叶蛤(Coecella turgida)和栉江珧(Atrina pectinata)这6科13种双壳类的贝壳形态微观结构特征进行观察。结果表明,不同种的贝壳表面和横切面微观结构有一定差异。这些差异主要表现在晶体的组成和排列方式两个方面。贝壳角质层根据表面形态特征分为5种类型:光滑平整、颗粒状、不规则多边形、蜂窝状和沟壑状。贝壳棱柱层的晶体形状有棱柱状、短柱状、片状和不规则形状。不同贝壳的晶体有两种排列方向,垂直于横切面和平行于横切面。13种贝壳珍珠层的晶体有颗粒状、砖块状、圆形、块状和不规则的多边形。不同种的贝壳角质层、棱柱层和珍珠层的厚度也不同。研究贝壳微观结构之间的差异,可以为分类提供基本资料。     

淡水贝类贝壳多层构造形成研究

对几种淡水贝（包括蚌、螺）进行形态及组织学观察，并通过实验方法重现贝壳三种物质，即：角质、棱柱质、珍珠质的生成过程，结果表明：外套膜外表皮细胞是由相同类型细胞组成，这些相同细胞在不同的作用条件下形成贝壳多层构造。     

Differential community development of fouling species on the pearl oysters Pinctada fucata, Pteria penguin and Pteria chinensis (Bivalvia, Pteriidae)

A field experiment documented the development of fouling communities on two shell regions, the lip and hinge, of the pearl oyster species Pinctada fucata, Pteria penguin and Pteria chinensis. Fouling communities on the three species were not distinct throughout the experiment. However, when each species was analysed separately, fouling communities on the lip and hinge of P. penguin and P. chinensis were significantly different during the whole sampling period and after 12 weeks, respectively, whereas no significant differences could be detected for P. fucata. There was no significant difference in total fouling cover between shell regions of P. fucata and P. chinensis after 16 weeks; however, the hinge of P. penguin was significantly more fouled than the lip. The most common fouling species (the hydroid Obelia bidentata, the bryozoan Parasmittina parsevalii, the bivalve Saccostrea glomerata and the ascidian Didemnum sp.) showed species-specific fouling patterns with differential fouling between shell regions for each species. The role of the periostracum in determining the community development of fouling species was investigated by measuring the presence and structure of the periostracum at the lip and hinge of the three pearl oyster species. The periostracum was mainly present at the lip of the pearl oysters, while the periostracum at the hinge was absent and the underlying prismatic layer eroded. The periostracum of P. fucata lacked regular features, whereas the periostracum of P. penguin and P. chinensis consisted of a regular strand-like structure with mean amplitudes of 0.84 microm and 0.65 microm, respectively. Although the nature and distribution of fouling species on the pearl oysters was related to the presence of the periostracum, the periostracum does not offer a fouling-resistant surface for these pearl oyster species.     

Differential community development of fouling species on the pearl oysters Pinctada fucata,Pteria penguin and Pteria chinensis (Bivalvia,Pteriidae)

Abstract

A field experiment documented the development of fouling communities on two shell regions, the lip and hinge, of the pearl oyster species Pinctada fucata, Pteria penguin and Pteria chinensis. Fouling communities on the three species were not distinct throughout the experiment. However, when each species was analysed separately, fouling communities on the lip and hinge of P. penguin and P. chinensis were significantly different during the whole sampling period and after 12 weeks, respectively, whereas no significant differences could be detected for P. fucata. There was no significant difference in total fouling cover between shell regions of P. fucata and P. chinensis after 16 weeks; however, the hinge of P. penguin was significantly more fouled than the lip. The most common fouling species (the hydroid Obelia bidentata, the bryozoan Parasmittina parsevalii, the bivalve Saccostrea glomerata and the ascidian Didemnum sp.) showed species-specific fouling patterns with differential fouling between shell regions for each species. The role of the periostracum in determining the community development of fouling species was investigated by measuring the presence and structure of the periostracum at the lip and hinge of the three pearl oyster species. The periostracum was mainly present at the lip of the pearl oysters, while the periostracum at the hinge was absent and the underlying prismatic layer eroded. The periostracum of P. fucata lacked regular features, whereas the periostracum of P. penguin and P. chinensis consisted of a regular strand-like structure with mean amplitudes of 0.84 μm and 0.65 μm, respectively. Although the nature and distribution of fouling species on the pearl oysters was related to the presence of the periostracum, the periostracum does not offer a fouling-resistant surface for these pearl oyster species.     

Aragonitic dendritic prismatic shell microstructure in Thracia (Bivalvia,Anomalodesmata)

The shells of most anomalodesmatan bivalves are composed of an outer aragonitic layer of either granular or columnar prismatic microstructure, and an inner layer of nacre. The Thraciidae is one of the few anomalodesmatan families whose members lack nacreous layers. In particular, shells of members of the genus Thracia are exceptional in their possession of a very distinctive but previously unreported microstructure, which we term herein “dendritic prisms.” Dendritic prisms consist of slender fibers of aragonite which radiate perpendicular to, and which stack along, the axis of the prism. Here we used scanning and transmission electron microscopical investigation of the periostracum, mantle, and shells of three species of Thracia to reconstruct the mode of shell calcification and to unravel the crystallography of the dendritic units. The periostracum is composed of an outer dark layer and an inner translucent layer. During the free periostracum phase the dark layer grows at the expense of the translucent layer, but at the position of the shell edge, the translucent layer mineralizes with the units typical of the dendritic prismatic layer. Within each unit, the c‐axis is oriented along the prismatic axis, whereas the a‐axis of aragonite runs parallel to the long axis of the fibers. The six‐rayed alignment of the latter implies that prisms are formed by {110} polycyclically twinned crystals. We conclude that, despite its distinctive appearance, the dendritic prismatic layer of the shell of Thracia spp. is homologous to the outer granular prismatic or prismatic layer of other anomalodesmatans, while the nacreous layer present in most anomalodesmatans has been suppressed.     

A new model for periostracum and shell formation in Unionidae (Bivalvia, Mollusca)

The periostracum in Unionidae consists of two layers. The outer one is secreted within the periostracal groove, while the inner layer is secreted by the epithelium of the outer mantle fold. The periostracum reaches its maximum thickness at the shell edge, where it reflects onto the shell surface. Biomineralization begins within the inner periostracum as fibrous spheruliths, which grow towards the shell interior, coalesce and compete mutually, originating the aragonitic outer prismatic shell layer. Prisms are fibrous polycrystalline aggregates. Internal growth lines indicate that their growth front is limited by the mantle surface. Transition to nacre is gradual. The first nacreous tablets grow by epitaxy onto the distal ends of prism fibres. Later growth proceeds onto previously deposited tablets. Our model involves two alternative stages. During active shell secretion, the mantle edge extends to fill the extrapallial space and the periostracal conveyor belt switches on, with the consequential secretion of periostracum and shell. During periods of inactivity, only the outer periostracum is secreted; this forms folds at the exit of the periostracal groove, leaving high-rank growth lines. Layers of inner periostracum are added occasionally to the shell interior during prolonged periods of inactivity in which the mantle is retracted.     

Die Mikro- und Ultrastruktur abgedeckter Hohlelemente und die Conellen des Ammoniten-Gehäuses

Hollow floored spines in the shell ofKosmoceras (Kosmoceras) spinosum (Sow.) and the hollow floored keel ofEleganticeras elegantulum (Young & Bird) have been studied with the scanning electron microscope. In both cases the shell wall is complete in so far as it consists of the outer prismatic layer, the nacreous layer and at least the distal zones of the inner prismatic layer. Both types of hollow shell elements are separated from the lumen of the whorl by a floor which is made up by the proximal zones of the inner prismatic layer. This explains why conellae occur, with preference, along the floors of hollow spines and keels. The origin of primary aragonitic conellae and of secondary calcitic conellae is discussed as well as their dependence on structural properties of the corresponding shell layer, which is the inner prismatic layer. An attempt is made to reconstruct the way of formation of the floored hollow spines and the floored hollow keels by the mantle epithelium.     

褶纹冠蚌外套膜组织培养的分泌物的偏光显微镜观察

以淡水育珠贝中珍珠形成较快的褶纹冠蚌为材料,用相差显微镜观察组织培养的外套膜的分泌物的形成和变化,用偏光显微镜观察分泌物的双折射现象,并与活体外套膜的分泌物、贝壳的角质层、棱柱层、珍珠层的双折射现象进行比较。结果表明;离体培养的外套膜细胞不仅能产生活体细胞相同的分泌物,而且分泌物还能在培养过程中形成结晶,并逐渐生长。发现外套膜的不同部位分区培养所形成的分泌物的性状与结晶性质和活体有一致性,表明组织培养的外套膜小片具有贝体原来的组织结构、分化特征和分泌功能。     

Identification of chitin in the prismatic layer of the shell and a chitin synthase gene from the Japanese pearl oyster, Pinctada fucata

The shell of the Japanese pearl oyster, Pinctada fucata, consists of two layers, the prismatic layer on the outside and the nacreous layer on the inside, both of which comprise calcium carbonate and organic matrices. Previous studies indicate that the nacreous organic matrix of the central layer of the framework surrounding the aragonite tablet is beta-chitin, but it remains unknown whether organic matrices in the prismatic layer contain chitin or not. In the present study, we identified chitin in the prismatic layer of the Japanese pearl oyster, Pinctada fucata, with a combination of Calcofluor White staining with IR and NMR spectral analyses. Furthermore, we cloned a cDNA encoding chitin synthase (PfCHS1) that produces chitin, contributing to the formation of the framework for calcification in the shell.     

Identification and Characterization of the Lysine-Rich Matrix Protein Family in <Emphasis Type="Italic">Pinctada fucata</Emphasis>: Indicative of Roles in Shell Formation

Mantle can secret matrix proteins playing key roles in regulating the process of shell formation. The genes encoding lysine-rich matrix proteins (KRMPs) are one of the most highly expressed matrix genes in pearl oysters. However, the expression pattern of KRMPs is limited and the functions of them still remain unknown. In this study, we isolated and identified six new members of lysine-rich matrix proteins, rich in lysine, glycine and tyrosine, and all of them are basic matrix proteins. Combined with four members of the KRMPs previously reported, all these proteins can be divided into three subclasses according to the results of phylogenetic analyses: KRMP1–3 belong to subclass KPI, KRMP4–5 belong to KPII, and KRMP6–10 belong to KPIII. Three subcategories of lysine-rich matrix proteins are highly expressed in the D-phase, the larvae and adult mantle. Lysine-rich matrix proteins are involved in the shell repairing process and associated with the formation of the shell and pearl. What’s more, they can cause abnormal shell growth after RNA interference. In detail, KPI subgroup was critical for the beginning formation of the prismatic layer; both KPII and KPIII subgroups participated in the formation of prismatic layer and nacreous layer. Compared with different temperatures and salinity stimulation treatments, the influence of changes in pH on KRMPs gene expression was the greatest. Recombinant KRMP7 significantly inhibited CaCO₃ precipitation, changed the morphology of calcite, and inhibited the growth of aragonite in vitro. Our results are beneficial to understand the functions of the KRMP genes during shell formation.     

Formation of the prismatic layer in the freshwater bivalve Hyriopsis cumingii: the feedback of crystal growth on organic matrix

The squeezing hypothesis and the organic frameworks preformation hypothesis propose two different mechanisms to explain the interaction between organic frameworks and crystals during biomineralization of the prismatic layer of the mollusk shell. In this study, we began to study Hyriopsis cumingii shell formation and discover that this species seemed to follow the squeezing hypothesis. During the formation of the aragonite prismatic layer in the freshwater bivalve H. cumingii, we found that crystal growth was involved in controlling initiation of formation of the interprismatic organic membranes. First, newly formed crystals were embedded in the periostracum. Next, the interprismatic organic membranes of the prismatic layer were produced via squeezing between neighboring crystals. The organic matrix secreted by the mantle continuously self‐assembled into the interprismatic organic membranes as the crystals grew. In the mature stage, the interprismatic organic membranes were shaped by crystal growth. These findings provide evidence to support the squeezing hypothesis and add to the existing knowledge about interactions that occur at the organic–inorganic interfaces during mollusk shell biomineralization.