马氏珠母贝(Pinctada fucata martensii)细胞粘附分子3(Pm-CAM3)基因的克隆与表达分析

细胞粘附分子3 (cell adhesion molecule 3, CAM3)是免疫球蛋白家族(immunoglobulin family, IgSF)的一员,在细胞黏连、外源病原体的识别等方面具有重要作用。本实验利用末端快速克隆(RACE)技术,克隆获得马氏珠母贝(Pinctada facata martensii)细胞粘附分子3的全长序列(Pm-CAM3),并使用荧光定量PCR(Real-time PCR, q RT-PCR)技术检测了Pm-CAM3在马氏珠母贝不同组织中的表达模式。结果显示,Pm-CAM3基因全长2 245 bp。其中5'UTR 335 bp,3'UTR 166 bp,开放阅读框(ORF)长度为1 744 bp,共编码581个氨基酸;预测其相对分子量为63.21 kD,等电点为5.07,脂溶指数(aliphatic index)为67.21;总平均亲水性(grand averageofhydropathy, GRAVY)为-0.549,属于亲水性蛋白;Pm-CAM3具有跨膜结构域,其胞外区域具有一个Ig SF家族典型的Ig结构域以及一个Ig-like结构域。多序列比对结果显示,Pm-CAM3在物种间的保守性较低,其中Pm-CAM3与太平洋牡蛎的CAM3 (Crassostrea gigas, Cg-CAM3)的氨基酸序列相似性最高,但仅为37%。qRT-PCR分析表明Pm-CAM3在马氏珠母贝的9个组织中均有表达,其中在鳃中的表达量最高(p<0.05)。     

Population genetics of a marine bivalve, Pinctada maxima, throughout the Indo-Australian Archipelago shows differentiation and decreased diversity at range limits

Intraspecific genetic diversity governs the potential of species to prevail in the face of environmental or ecological challenges; therefore, its protection is critical. The Indo-Australian Archipelago (IAA) is a significant reservoir of the world's marine biodiversity and a region of high conservation priority. Yet, despite indications that the IAA may harbour greater intraspecific variation, multiple-locus genetic diversity data are limited. We investigated microsatellite DNA variation in Pinctada maxima populations from the IAA to elucidate potential factors influencing levels of genetic diversity in the region. Results indicate that genetic diversity decreases as the geographical distance away from central Indonesia increases, and that populations located towards the centre of P. maxima 's range are more genetically diverse than those located peripherally ( P <  0.01). Significant partitioning of genetic variation was identified ( F _ST = 0.027; R _ST = 0.023, P  < 0.001) and indicates that historical biogeographical episodes or oceanographic factors have shaped present population genetic structure. We propose that the genetic diversity peak in P. maxima populations may be due to (i) an abundance of suitable habitat within the IAA, meaning larger, more temporally stable populations can be maintained and are less likely to encounter genetic bottlenecks; and/or (ii) the close proximity of biogeographical barriers around central Indonesia results in increased genetic diversity in the region because of admixture of genetically divergent populations. We encourage further genetic diversity studies of IAA marine biota to confirm whether this region has a significant role in maintaining intraspecific diversity, which will greatly assist the planning and efficacy of future conservation efforts.     

A novel putative tyrosinase involved in periostracum formation from the pearl oyster (Pinctada fucata)

Tyrosinase (monophenol, L-DOPA: oxygen oxidoreductase, EC 1.14.18.1), a kind of copper-containing phenoloxidase, arouses great interests of scientists for its important role in periostracum formation. A cDNA clone encoding a putative tyrosinase, termed OT47 because of its estimated molecular mass of 47kDa, was isolated from the pearl oyster, Pinctada fucata. This novel tyrosinase shares similarity with the cephalopod tyrosinases and other type 3 copper proteins within two conserved copper-binding sites. RT-PCR analysis showed that OT47 mRNA was expressed only in the mantle edge. Further in situ hybridization analysis and tyrosinase activity staining revealed that OT47 was expressed at the outer epithelial cells of the middle fold, different from early histological results in Mercenaria mercenaria, suggesting a different model of periostracum secretion in P. fucata. Taken together, these results suggest that OT47 is most likely involved in periostracum formation. The identification and characterization of oyster tyrosinase also help to further understand the structural and functional properties of molluscan tyrosinase.     

The structure-function relationship of MSI7, a matrix protein from pearl oyster Pinctada fucata

We previously identified a matrix protein, MSI7, from pearl oyster Pinctada fucata. According to the structural analysis, the DGD site in the N-terminal of MSI7 is crucial for its role in the shell formation. In this study, we expressed a series of recombinant MSI7 proteins, including the wild-type and several mutants directed at the DGD site, using an Escherichia coli expression system to reveal the structure-function relationship of MSI7. Furthermore, in vitro crystallization, crystallization speed assay, and circular dichroism spectrometry were carried out. Results indicated that wild-type MSI7 could induce the nucleation of aragonite and inhibit the crystallization of calcite. However, none of the mutants could induce the nucleation of aragonite, but all of them could inhibit the crystallization of calcite to some extent. And all the proteins accelerated the crystallization process. Taken together, the results indicated that MSI7 could contribute to aragonite crystallization by inducing the nucleation of aragonite and inhibiting the crystallization of calcite, which agrees with our prediction about its role in the nacreous layer formation of the shell. The DGD site was critical for the induction of the nucleation of aragonite.     

马氏珠母贝雌雄同体和自体受精的研究

对马氏珠母贝(Pinctada martensiiDunker)成体的性腺进行外观和切片观察,可把马氏珠母贝雌雄同体的个体分为2种类型,其中A类型可以从性腺外观特征上辨别,而B类型需性腺穿刺或组织切片来确认。对试验群体的性腺的类型进行的初步统计表明,雌雄同体个体比例A类型为4.6%,B类型为3.4%,其中雌雄生殖细胞均接近成熟的个体分别约为1.8%和2.1%。通过解剖、0.07‰的氨海水处理,对雌雄同体个体的自体授精及其早期胚胎发育进行了研究,发现自体授精的受精率低于异体授精,同时在发育过程中有较多的卵裂球解体,但仍有部分胚胎可以发育成D型幼虫。     

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.     

Mantle histology and histochemistry of three pearl oysters: Pinctada persica,Pinctada radiata and Pteria penguin

Shells in pearl oysters are produced by the mantle which is also used as a graft in pearl operations. Here, we investigate the mantle structure of a new pearl oyster species of the Persian Gulf, Pinctada persica, and compare it to two other pearl-producing species, Pinctada radiata and Pteria penguin. The anterior, ventral and posterior segments of the mantle edge of each valve were fixed, and tissue sections were stained with haematoxylin and eosin. A new pentachrome method and PAS-alcian blue staining were used to characterise the different mucosubstances. The mantle edges were found to have an outer, middle and inner fold, which have different morphology in each species. The mantle edge is lined by cuboidal and columnar epithelia, and interspersed among these epithelial cells we found mucous cells and cells that contained brown granules. The outer and middle folds of the two Pinctada species show different shapes to that of Pteria penguin. Most of the mucous cells in the mantle contain acidic mucosubstances and small amounts of mixed acidic-neutral mucosubstances were observed in the middle and inner fold of Pinctada persica. This study reveals that the mantle edges of the three species possess similar cellular structure, but vary in the shape of the folds.     

Isolation and characterization of eight microsatellite loci from silver‐lipped pearl oyster Pinctada maxima

We have developed di‐ and tetra‐nucleotide microsatellite markers for the silver‐lipped pearl oyster Pinctada maxima. Screening of approximately 50 000 clones resulted in the identification of 74 microsatellites. Fluorescent PCR was optimized for eight polymorphic loci. In a sample of 1637 individuals, these eight loci possessed between 14 and 68 alleles (average 29.8), with observed and expected heterozygosity ranging from 0.479 to 0.891 and 0.872 to 0.972, respectively.