合浦珠母贝的配子发生

本文研究了合浦珠母贝精子和卵子的发生过程,详细描述了原始生殖细胞,精原细胞,卵原细胞,精母细胞,卵母细胞以及精子和卵子的形态结构;另外还报道了合浦珠母贝中独特的精、卵退化现象,并讨论了它们对合浦珠母贝人工育苗生产的影响。     

合浦珠母贝珍珠的生物学性能初步检测

本研究报道了合浦珠母贝珍珠的生物学性能初步检测结果。珍珠作为—种天然的生物材料。在医药、化妆品工业等领域中的应用已很广泛。本研究对合浦珠母贝珍珠进行了体外细胞毒性试验、溶血试验和经静脉全身急性毒性试验的研究。实验结果表明,合浦珠母贝珍珠具有较好的细胞相容性,其浸提液对实验动物无明显毒性。在本研究中,合浦珠母贝珍珠的浸提液对兔血红细胞的溶血指数高于对照组。     

合浦珠母贝三个养殖群体微卫星遗传多样性

以广东徐闻金碧公司养殖场、广西北海营盘镇养殖场和南海水产研究所海南实验基地3个合浦珠母贝养殖群体为对象,利用8个微卫星位点M1、M2、M3、M4、M5、M6、M7、M8的引物进行了遗传多样性分析.结果表明:8个微卫星标记位点在3个养殖群体中共检测到58个等位基因,观测等位基因数为2～9个,平均有效等位基因数3.72～5.06,平均观察杂合度0.41～0.56,平均期望杂合度0.67～0.75,3个群体的平均多态信息含量PIC值为0.62～0.70,全部为高度多态(PIC≥0.5),表明这几个合浦珠母贝养殖群体目前仍具有较高的遗传多样性,遗传信息丰富,遗传变异大,可以作为良好的育种材料;在这3个养殖群体中,南海水产研究所海南实验基地的养殖群体的遗传多样性最高,广西北海营盘镇养殖群体遗传多样性最低,这一结果可以为今后选择育种、种质保护提供可资借鉴的资料.

Abstract：

By using eight mierosatellite loci (M1, M2, M3, M4, M5, M6, M7 and MS), the genetic diversity of three Pinctada fucata populations from the pearl farms in Xuwen of Guang-dong and Beihai of Guangxi, and from the experimental base of South China Sea Fisheries Re-search Institute in Hainan was studied. A total of fifty eight alleles of these eight microsatellite lo-ci were detected, among which, the observed allele number was 2-9, average effective allele number was 3.72-5.06, average observed population heterozygosity was 0. 41-0. 56, and aver-age observed expected heterozygosity was 0. 67-0. 75. All the three populations had a polymor-phie information content (PIC) of 0. 62-0.70, suggesting their high polymorphism (PIC > 0. 5). Among the three populations, the cultured population from the experimental base of South China Sea Fisheries Institute had the highest polymorphism, and that from Beihai of Guangxi had the lowest one. These results provided useful information for the selective breeding and germplast conservation of P. Fucata.     

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。     

合浦珠母贝幼虫变态中的形态、器官变化及运动与摄食观察

为揭示合浦珠母贝幼虫至稚贝生长发育过程中其外部形态变化及内部器官改变的内在规律, 掌握其形态和器官与运动和摄食行为之间的关联。在光学显微镜下对整个幼虫生长发育及变态过程中的外部形态、内部器官特征进行了系列观察和性状测量; 利用非线性回归参数拟合, 描述各形态性状生长特点及不同属性之间的联系; 观察不同发育阶段其运动与摄食过程。结果显示, 幼虫在正常生长过程中, 其壳长生长方式为加速正增长、壳高为减速正增长、绞合线长为加速负增长, 壳高相对于壳长的生长为快速生长、绞合线长相对于壳长为慢速生长。幼虫生长至壳长为(209.26±9.22) μm时, 内部器官发生改变, 面盘开始逐渐退化从而发育成鳃, 斧足逐渐形成; 壳长生长至(234.30±14.00) μm时, 次生壳开始长出, 外部形态逐渐向稚贝转变。稚贝阶段, 其鳃丝长、鳃丝间距和鳃丝数量相对于壳长的生长均表现为慢速生长。幼虫在水中的运动和摄食过程主要依靠面盘外周纤毛的摆动来完成, 俯视观幼虫绕不规则圆沿顺时针方向运动, 垂直观幼虫螺旋上升或下降。稚贝阶段, 依靠斧足的往复伸缩来完成爬行, 依靠鳃的过滤完成摄食。在幼虫变态过程中, 面盘退化至鳃具备滤食功能期间, 变态幼虫运动功能降低, 摄食能力丧失, 依靠自身能量储备来完成生长和器官发育, 这一过程是苗种培育中的重要关键点。     

合浦珠母贝PU3基因的克隆及功能鉴定

目的:克隆获得合浦珠母贝PU3基因的序列,并研究其在生物矿化中的功能。方法:使用RACE获得PU3基因的全长;利用实时荧光定量PCR的方法检测PU3基因在不同组织中的表达分布;利用实时荧光定量PCR的方法检测贝壳损伤修复过程中PU3基因的表达量的变化;通过RNAi实验,抑制PU3基因的表达,之后用扫描电子显微镜观察合浦珠母贝贝壳表面的变化。结果:合浦珠母贝PU3基因的cDNA全长为2361bp,编码618个氨基酸。氨基酸序列的功能结构域分析表明其含有4个FN3结构域。该基因在外套膜中高表达,且在外套膜边缘区的表达量高于外套膜中心区。在贝壳损伤修复的过程中,该基因的表达水平呈现上升的趋势。利用RNAi技术抑制PU3基因的表达后,贝壳的棱柱层结构发生了变化,缝隙变宽,且出现空洞。结论:PU3基因所表达的蛋白作为正调控因子参与生物矿化的过程,并主要作用于贝壳的棱柱层,抑制其表达会影响棱柱层的框架结构。     

合浦珠母贝SOX9对Prismalin-14基因的调控研究

目的:研究合浦珠母贝转录因子SOX9对Prismalin-14的转录调控机制。方法:应用在线预测软件PROMO分析Prismalin-14的启动子序列,以预测Prismalin-14启动子上可能的转录因子与其结合位点;运用细胞共转染实验和双荧光素酶报告系统以检测SOX9对Prismalin-14启动子的激活作用;构建Prismalin-14启动子截短体的荧光素酶报告载体,并和SOX9的真核载体共转到HEK-293T细胞中,再进行双荧光素酶报告系统检测Prismalin-14启动子的活性;构建SOX9截短体的真核表达载体,并与Prismalin-14启动子的荧光素酶报告载体共转到HEK-293T细胞中,再进行双荧光素酶报告分析Prismalin-14启动子的活性。结果:SOX9能激活Prismalin-14的启动子的活性,并具有剂量效应;对Prismalin-14启动子进行截短后,不包含结合位点的Prismalin-14启动子的活性是野生型Prismalin-14启动子活性的49%,推测Prismalin-14启动子上的-415bp到-405bp区域是SOX9激活作用的关键区域;对SOX9的SRY-related HMG结构域进行截短后,其对Prismalin-14启动子的激活作用显著减少,因此SOX9结构的完整对Prismalin-14启动子活性的激活作用是必须的。结论:Prismalin-14的转录可能受SOX9调控,为进一步研究合浦珠母贝的转录调控机制提供基础,将有助于从分子水平上理解贝壳形成的上游调控机理。     

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．     

射肋珠母贝生殖腺变化的观察

本文报道了射肋珠母贝生殖腺周年变化情况。观察发现该贝生殖腺变化经历五个时期,其中滤泡期持续时间极短。     

合浦珍珠母贝的养殖和研究新进展

北部湾是我国养殖海水珍珠的海域之一,民间曾流传着“东珠不如西珠,西珠不如南珠”的说法,广西北海市合浦县营盘乡则是著名的“南珠之乡”.据清康熙年间成书的〈粤闽巡视纪略〉就有7个古珠池的记载：“相传有七：曰青莺、曰断望、曰杨梅、曰白沙、曰平江、曰海渚、曰乌泥,俱在冠头岭外大海中,上下相去约一百八十三里.”     

Low genetic differentiation among widely separated populations of the pearl oyster Pinctada fucata as revealed by AFLP

Genetic variation within and among five populations of the pearl oyster Pinctada fucata, from China (Daya Bay, Sanya Bay and Beibu Bay), Japan (Mie Prefecture) and Australia (Port Stephens) was studied using AFLP. Three primer pairs generated 184 loci among which 91.8-97.3% is polymorphic. An overall genetic diversity of 0.38 among populations and an average of 0.37 within populations (ranging from 0.35 in Japanese population to 0.39 in Beibu Bay population) were observed. Genetic differentiation among the five populations is low but significant as indicated by pairwise G_ST (0.0079-0.0404). AMOVA further shows that differentiation is significant among the five populations but is not significant at a broader geographical scale, among the three groups of Chinese, Japanese and Australian populations or among the two groups of Australian and north Pacific populations. The low level of genetic differentiation indicated that P. fucata populations in the west Pacific are genetically linked. Among the five populations, the Australian one is more differentiated from the others, based on both pairwise AMOVA and G_ST analyses, and is genetically isolated by distance as indicated by Mantel test. However, genetic differences among the three Chinese populations are not correlated with the geographic distances, suggesting that Hainan Island and Leizhou Peninsula may act as barriers blocking gene flow.     

Haemocyte morphology and function in the Akoya Pearl Oyster, Pinctada imbricata

The morphology and cytochemistry of Pinctada imbricata haemocytes were studied in vitro. Three distinct blood cell types were identified; hyalinocytes, granulocytes, and serous cells. Haemocytes were classified based on the presence/absence of granules, and nucleus to cytoplasm ratio. Granulocytes were the most common cell type (62 ± 2.81%), followed by hyalinocytes (36 ± 2.35%), and serous cells (2 ± 0.90%). Granulocytes, and hyalinocytes were found to be immunologically active, with the ability to phagocytose Congo red stained yeast. Of the cells involved in phagocytosis, granulocytes were the most active with 88.8 ± 3.9% of these haemocytes engulfing yeast. Cytochemical stains (phenoloxidase, peroxidase, superoxide, melanin, neutral red) showed that enzymes associated with phagocytic activity were localised in granules within granulocytes. Based on their affinities for Giemsa/May-Grünwald stain, haemocytes were also defined as either acidic, basic or neutral. Hyalinocytes and serous cells were found to be eosinophilic, whilst granulocytes were either basophilic (large granulocytes), eosinophilic (small granulocytes) or a combination of the two (combination granulocytes). Light, differential interference contrast and epi-fluorescence microscopy identified three sub-populations of granulocytes based on size and granularity; small (4.00-5.00 μm in diameter, with small granules (0.05-0.5 μm in diameter), large (5.00-9.00 μm in diameter, with large granules (0.50-2.50 μm in diameter) and combination (5.00-9.00 μm in diameter, with both large and small granules). These observations demonstrate that P. imbricata have a variety of morphologically and functionally specialized haemocytes, many of which maybe associated with immunological functions.     

Tissue inhibitor of metalloproteinase gene from pearl oyster Pinctada martensii participates in nacre formation

Tissue inhibitors of metalloproteinases (TIMPs) are nature inhibitors of matrix metalloproteinases and play a vital role in the regulation of extracellular matrix turnover, tissue remodeling and bone formation. In this study, the molecular characterization of TIMP and its potential function in nacre formation was described in pearl oyster Pinctada martensii. The cDNA of TIMP gene in P. martensii (Pm-TIMP) was 901 bp long, containing a 5′ untranslated region (UTR) of 51 bp, a 3′ UTR of 169 bp, and an open reading fragment (ORF) of 681 bp encoding 226 amino acids with an estimated molecular mass of 23.37 kDa and a theoretical isoelectric point of 5.42; The predicted amino acid sequence had a signal peptide, 13 cysteine residues, a N-terminal domain and a C-terminal domain, similar to that from other species. Amino acid multiple alignment showed Pm-TIMP had the highest (41%) identity to that from Crassostrea gigas. Tissue expression analysis indicated Pm-TIMP was highly expressed in nacre formation related-tissues, including mantle and pearl sac. After decreasing Pm-TIMP gene expression by RNA interference (RNAi) technology in the mantle pallium, the inner nacreous layer of the shells showed a disordered growth. These results indicated that the obtained Pm-TIMP in this study participated in nacre formation.     

Molecular and histological identification of Marteilioides infection in Suminoe Oyster Crassostrea ariakensis, Manila Clam Ruditapes philippinarum and Pacific Oyster Crassostrea gigas on the south coast of Korea

The oyster ovarian parasite Marteilioides chungmuensis has been reported from Korea and Japan, damaging the oyster industries. Recently, Marteilioides-like organisms have been identified in other commercially important marine bivalves. In this study, we surveyed Marteilioides infection in the Manila clam Ruditapes philippinarum, Suminoe oyster Crassostrea ariakensis, and Pacific oyster Crassostrea gigas, using histology and Marteilioides-specific small subunit (SSU) rDNA PCR. The SSU rDNA sequence of M. chungmuensis (1716 bp) isolated from C. gigas in Tongyoung bay was 99.9% similar to that of M. chungmuensis reported in Japan. Inclusions of multi-nucleated bodies in the oocytes, typical of Marteilioides infection, were identified for the first time in Suminoe oysters. The SSU rDNA sequence of a Marteilioides-like organism isolated from Suminoe oysters was 99.9% similar to that of M. chungmuensis. Marteilioides sp. was also observed from 7 Manila clams of 1840 individuals examined, and the DNA sequences of which were 98.2% similar to the known sequence of M. chungmuensis. Unlike Marteilioides infection of Pacific oysters, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected Suminoe oysters or Manila clams. Distribution of the infected Manila clams, Suminoe oysters and Pacific oysters was limited to small bays on the south coast, suggesting that the southern coast is the enzootic area of Marteilioides infection.     

Molecular Cloning and Expression of a Pearl Oyster (<Emphasis Type="Italic">Pinctada fucata</Emphasis>) Homologue of Mammalian Putative Tumor Suppressor QM

The QM gene was originally identified as a putative tumor suppressor gene from a Wilms tumor cell line by subtractive hybridization assay. Later studies showed that the QM protein is multifunctional, involved in cell growth and differentiation, energy metabolism, respiration, and cytoskeletal function. In this report a full-length complementary DNA encoding a QM counterpart in pearl oyster (Pinctada fucata) was isolated. Phylogenetic analysis shows that oyster QM is more closely related to its insect homologues than to the mammalian homologues. Analysis of the tissue expression pattern of the oyster QM gene showed that oyster QM messenger RNA is expressed in all tissues tested, with highest levels in the digestive gland and mantle. Furthermore, we expressed the QM protein in Escherichia coli; Western blotting showed that the antibody of human QM is immunoreactive to the expressed oyster QM protein. Incubation of the oyster QM with Zn²⁺ resulted in the reduction of intrinsic emission fluorescence and a red-shift in the _max emission, indicating the occurrence of Zn²⁺-induced conformational changes. This evidence presents a possible mechanism for the critical function of zinc ion in the interaction of QM with Jun.     

Cloning and characterization of an mRNA encoding a novel G protein α-subunit abundant in mantle and gill of pearl oyster Pinctada fucata

Nacre formation is an ideal model to study biomineralization processes. Although much has been done about biomineralization mechanism of nacre, little is known as to how cellular signaling regulates this process. We are interested in whether G protein signaling plays a role in mineralization. Degenerate primers against conserved amino acid regions of G proteins were employed to amplify cDNA from the pearl oyster Pinctada fucata. As a result, the cDNA encoding a novel G_sα (pfG_sα) from the pearl oyster was isolated. The G_sα cDNA encodes a polypeptide of 377 amino acid residues, which shares high similarity to the octopus (Octopus vulgaris) G_sα. The well-conserved A, C, G (switch I), switch II functional domains and the carboxyl terminus that is a critical site for interaction with receptors are completely identical to those from other mollusks. However, pfG_sα has a unique amino acid sequence, which encodes switch III and interaction sites of adenylyl cyclase respectively. In situ hybridization and Northern blotting analysis revealed that the oyster G_sα mRNA is widely expressed in a variety of tissues, with highest levels in the outer fold of mantle and epithelia of gill, the regions essential for biomineralization. We also show that overexpression of the pfG_sα in mammalian MC3T3-E1 cells resulted in increased cAMP levels. Mutant pfG_sα that has impaired CTX substrate diminished its ability to induce cAMP production. Furthermore, the alkaline phosphatase (ALP) activity, an indicator for mineralization, is induced by the G_sα in MC3T3-E1 cells. These results indicated that G_sα may be involved in regulation of physiological function, particularly in biological biomineralization.     

Biphasic and Dually Coordinated Expression of the Genes Encoding Major Shell Matrix Proteins in the Pearl Oyster Pinctada fucata

Regional expression patterns of shell matrix protein genes of Pinctada fucata were investigated using real-time quantitative polymerase chain reaction (PCR) and in situ hybridization. Six shell matrix proteins examined in this study indicated a distinct biphasic pattern of expression, falling into one of the following three groups: (1) expressed only in the more dorsal region of the mantle (MSI60 and N16); (2) expressed only in the more ventral region (MSI31, Prismalin-14, and Aspein); and (3) expressed in both regions (nacrein). The ubiquity of the last protein probably reflects its general role as a carbonate-producing enzyme, while the other groups are interpreted as corresponding to the distinction between the two varieties of shell layers, the aragonitic nacreous layer and the calcitic prismatic layer. In addition, the constituent genes of each of these two groups indicated similar levels of relative expression among different sites even among different individuals, suggesting that the genes of each group share a single upstream regulatory factor, respectively, and that these genes are expressed in a dually coordinated fashion.     

中华白海豚雄性生殖系统组织学初步研究

采用组织学和形态学方法研究了中华白海豚(Sousa chinensis)的雄性生殖系统。以睾丸和附睾中是否存在精母细胞及精子细胞等作为判断雄性中华白海豚性成熟的标准,通过比较不同个体睾丸和附睾的组织结构特征,发现成熟个体的生精小管和附睾的组织学结构与未成熟个体间存在显著差异。通过测量样本睾丸的2个形态参数:生精小管直径和生精小管的相对面积,得到性成熟个体的生精小管直径为(118.3±12.8)μm,生精小管相对面积为0.52;未成熟个体的生精小管直径(47.4±3.5)～(60.3±6.0)μm,生精小管相对面积为0.27～0.40。     

孕育对褶纹冠蚌滤食率的影响及鳃微结构变化

对比了褶纹冠蚌(Cristaria plicata)孕育蚌、未孕雌蚌和雄蚌的滤食率,并运用组织学、扫描电镜和透射电镜对实验蚌的鳃结构进行比较观察,以此探讨鳃结构变化对滤食功能的影响。滤食实验结果表明:孕育事件显著降低了雌蚌的滤食率,而未孕雌蚌与雄蚌的滤食率无显著差异。孕育雌蚌内外鳃均由两鳃小瓣愈合而成,每一鳃小瓣由成排的鳃丝组成,在中介区鳃丝之间通过丝间隔连接,在内部侧区则通过瓣间隔相连。雌蚌内鳃的鳃间隔为外鳃的2～3倍,而雄蚌的内外鳃无差异。孕育雌蚌外鳃在初级水管、瓣间隔等出现明显的变化,并出现了二级水管结构,而内鳃未发现显著变化。扫描电镜显示:在褶纹冠蚌鳃丝表面存在3种类型的纤毛(前纤毛、前侧纤毛和侧纤毛),其形态结构和分布各具特点,长径58～85μm椭圆形的鳃小孔成排相间分布于鳃丝之间,而3组实验蚌的内外鳃丝之间无明显差异。透射电镜观察发现:孕育雌蚌鳃丝表皮细胞表面形成突起,显著增加了表面微绒毛的数量,可能有利于雌蚌在孕育期间由于初级水管转化成育儿囊后对呼吸、滤食等功能的补偿,与其他蚌科物种报道类似。综合实验表明,孕育雌蚌外鳃结构的变化,尤其是初级水管的结构改变和二级水管、鳃丝表面褶皱的出现可能是影响孕育雌蚌滤食功能的主要原因。