合浦珠母贝3个养殖群体遗传多样性的微卫星分析

采用微卫星分子标记对我国广东大亚湾、广西北海、海南三亚的合浦珠母贝养殖群体进行了遗传多样性分析.从49对引物中筛选出31对有效扩增引物,种群扩增获得9个多态位点,多态位点比例为29.03%.它们在3个群体共96个个体中产生了44个等位基因,平均每个位点产生4.889个.3个群体的平均期望杂合度分别为0.590、0.600、0.615;平均观察杂合度分别是0.393、0.425、0.325;平均多态信息含量PIC值分别是0.530、0.551、0.556.表明3个群体的遗传多样性处于较高水平.平均遗传偏离指数分别为-0.324、-0.336、-0.304;哈代-温伯格平衡检测发现,3个群体27个位点中21个位点偏离平衡状态.遗传分化和遗传距离分析表明,海南群体与广东群体之间的亲缘关系较近,而与广西群体的亲缘关系较远.     

Molecular Cloning and Single Nucleotide Polymorphisms Identification of Myostatin cDNA in the Pearl Oyster,Pinctada fucata

Myostatin (MSTN or growth differentiation factor‐8) is considered a negative regulator of muscle growth and development. In this study, we cloned and characterized the full‐length MSTN cDNA from Pinctada fucata, and named it as the Pf‐MSTN cDNA. The single nucleotide polymorphisms (SNPs) in Pf‐MSTN cDNA were then screened and genotyped. The full‐length Pf‐MSTN cDNA was 2644 bp, including an open reading frame of 1248 bp encoding 415 amino acids which contained typical structural characteristics shared by all members of the transforming growth factor‐β (TGF‐β) superfamily including an N‐terminal signal peptide, a propeptide domain, and a TGF‐β superfamily bioactive domain. The Pf‐MSTN mRNA was detected in all tested tissues, with the highest mRNA levels observed in the adductor muscle, indicating that Pf‐MSTN may play a major role in this tissue. Furthermore, by sequencing and alignment, 32 SNP loci were identified in Pf‐MSTN cDNA. Genotyping 50 individuals from a common breeding stock revealed that 21 of these 32 loci were polymorphic. The minor allele frequency was in the range of 0.0400–0.4800, and the polymorphism information content value varied from 0.0739 to 0.3750. The observed and expected heterozygosity ranged from 0.0200 to 1.0000 and from 0.0776 to 0.5051, respectively. These SNPs identified in Pf‐MSTN will be useful for future studies investigating their utility in marker‐assisted selection for P. fucata breeding.     

Seasonal Fouling Stress on the Farmed Pearl Oyster,Pinctada fucata,from Southeastern Arabian Sea

Biofouling on the periostracum of pearl oysters and on the cages has been considered as a stress factor causing mortality of the farmed stock, reducing growth rates and also affecting pearl quality. In farming experiments using the pearl oyster, Pinctada fucata, at Kollam Bay (India), biofouling was found to be a problem. This study was conducted to understand the effect of fouling on the mortality of pearl oysters kept in suspended culture, to identify the main foulers, the seasonal variation in biofouling and species successions in the community with reference to abiotic factors. The average monthly mortality rate (MR) was estimated as 0.117 ± 0.002 and the monthly variations were significantly different (P < 0.01). The total fouling (0.163 ± 0.002 g/g oyster) and biofouling weights (0.166 ± 0.007 g/g oyster) were high in December when the fouling community was composed of several species and dominated by the ascidian, Didemnum sp. December was also the period when the MRs peaked indicating that this organism was the main cause of mortality in pearl farms in Kollam Bay. A clear seasonality in the fouling community (25 species belonging to nine phyla) has been observed in the present study. On the basis of this study, monthly cleaning of oysters is advocated except during December, January, and March when the cleaning should be fortnightly.     

马氏珠母贝激光育苗机理的探讨

马氏珠母贝激光育苗，其产量比常规育苗增加１０倍以上，激光能增强精子的活力，提高精液品质，可影响卵子的通透性，使卵子新陈代谢中的某些反应发生良性变化。激光能抑制中致病菌及霉菌的生长，使受精卵发育成健壮的胚胎、幼虫、幼苗。所以，激光育苗是一种投产少，效益极高的育苗方法。     

Purification and Characterization of an Antioxidant Protein from Pearl Oyster (Pinctada fucata martensii)

Protein from pearl oyster (Pinctada fucata martensii) was purified and characterized. Results showed that the pearl muscle protein (PMP) was composed of a single polypeptide with a molecular weight of approximate 11.6 kDa and contained 95.1% of the protein and 4.92% of the carbohydrate of the oyster. The polypeptide appears to be a glycoprotein, since the Fourier transform infrared (FTIR) spectroscopy of the PMP shows the typical characteristics of protein and polysaccharides. The denaturation temperature of the PMP was 81.3°C by differential scanning calorimetry. Circular dichroism spectroscopy showed that the PMP has a highly ordered structure. The PMP (200 μg/mL) showed a high oxygen radical absorbance activity (ORAC) of 6.57 μmol Trolox/μmol protein. Its antioxidant activity was stable at temperatures from 30–80°C and pH 2–10. The antioxidant activity was significantly inhibited by Zn²⁺, Ca²⁺, and EDTA and enhanced by Mn²⁺. The results suggest that the PMP could be used as a potential natural antioxidant.     

马氏珠母贝肉软罐头的研制

研究了以马氏珠母贝肉为主要原料，配以各种调味料，经调味、渗透、干燥、加工生产色泽、质地和风味良好的马氏珠母贝肉软罐头的工艺技术。     

两个马氏珠母贝养殖群体遗传多样性微卫星分析

以两个马氏珠母贝(Pinctada martensii Dunker)养殖群体为对象,采用微卫星分子标记技术对两个群体的遗传结构进行了研究,分析了各群体内的遗传多样性和两个群体间的遗传分化.结果表明,在广东养殖贝和三亚养殖贝群体中,分别获得232和230条扩增片段,两群体的平均观测杂合度分别为0.28和0.29,平均多态信息含量分别为0.44和0.46,平均有效等位基因数分别为2.25和2.13,显示两个群体的群体遗传多样性水平相差较小(p<0.05),两群体间遗传变异性小,其遗传多样性处于中等水平.两个养殖群体的基因分化系数(GST)为0.0448,群体之间属于轻度偏中度分化水平.     

马氏珠母贝术前处理的研究

实验结果表明:经术前处理后的插核贝休养期存活率为95 2%,育珠期存活率达85 5%,每贝只插1个核,休养期留核率为0 86,育珠期留核率达0 71。珍珠的质量明显提高,优质珍珠的生成率高达34 6%,比对照组高出27 5%。     

Identification,Characterization, and Evaluation of Polymorphic Microsatellite Markers for Parentage Assignment in Pinctada fucata

Parentage analysis is a useful tool for establishing pedigree relationships in selective breeding. In this study, 31 microsatellite markers were tested, and 13 loci were subsequently used to identify parenthood in Pinctada fucata. According to simulation analysis, the power of nine loci to exclude false parents was 99.94%, and that of 10 loci was 99.95%. Moreover, using 10 loci and known parental and filial information, the cumulative assignment success rate to one true parental pair was as high as 99%. Of the 120 progeny tested, 93.3% were exclusively assigned to their parental pairs in mixed pedigrees. These results suggested that these microsatellite markers can be used for rapid and effective parentage assignment and contribute to the selective breeding of P. fucata.     

马氏珠母贝蛋白的分离及分子量分布研究

试验结果表明,马氏珠母贝全脏器各分离蛋白组分占总蛋白含量分别为:非蛋白氮1.48%,肌浆蛋白33.20%,肌原纤维蛋白13.14%,碱溶性蛋白21.54%,基质蛋白30.65%;贝肌肉各分离蛋白组分占总蛋白含量分别为:非蛋白氮0.83%,肌浆蛋白26.56%,肌原纤维蛋白18.48%,碱溶性蛋白30.34%,基质蛋白23.79%;马氏珠母贝蛋白氨基酸组成均衡,呈味氨基酸及支链氨基酸含量高,但在各蛋白组分中的含量存在一定差异性.利用聚丙烯酰胺凝胶电泳(SDS-PAGE)测定的分子量分布结果显示,2种原料蛋白的肌浆蛋白分子量分布范围较宽,集中在200～19 kDa,肌原纤维蛋白分子量分布集中在200～45 kDa,碱溶性蛋白集中分布在41～3 kDa.     

白云石珠核对马氏珠母贝外套膜表皮细胞作用的研究

利用光镜和透射电镜技术对白云石珠核"平板珍珠"生成过程中马氏珠母贝外套膜表皮细胞的显微与超微结构变化进行观察。观察结果表明,在"平板珍珠"生成过程中,白云石珠核与贝壳珠核结果一致。外套膜受到异缘物的刺激,发生一系列的表皮细胞形态和细胞器的变化。(1)细胞形态变化:外表皮细胞高度发生变化,先增高再变矮,其中插片后6、12d变化较明显,而12d最高,且胞质中的空泡化最明显。(2)黏液细胞量变化:插片后Ⅱ型黏液细胞数量增多,而插片后6、12、19d外表皮中则很少黏液细胞分布。(3)细胞器变化:插片后22h,柱状细胞内质网上的核糖体消失,高尔基体减少,线粒体也多转变为分泌泡;39h,原来具成群线粒体和分泌泡的细胞内的线粒体很少,分泌泡则非常多;6d,粗面内质网增多;12、19d,有较多含有成群的线粒体的细胞,分泌泡则很少。     

脊尾白虾(Exopalaemon carinicauda)回交家系遗传变异的微卫星分析

为解析脊尾白虾(Exopalaemon carinicauda)回交家系的遗传变异规律,本研究利用自行开发的25个微卫星标记对本实验室构建的A、B、C3个回交家系的遗传结构特征进行了分析.结果显示,回交家系A、B、C的平均等位基因数(Na)分别为2.21、2.18、2.09,观测杂合度(Ho)分别为0.3805、0.3703、0.2489,期望杂合度(He)分别为0.3629、0.3998、0.2503,多态信息含量(PIC)分别为0.3257、0.3391、0.2585,相较于野生群体与杂交家系已有了大幅度的降低,属中度或低度多态.回交家系A、B、C在25个位点共得到62个等位基因,其中,A家系56个,B家系54个,C家系51个,3个回交家系共同存在的等位基因为39个,6个位点在回交家系中已经纯合(其中,ECL10在A、B、C中都已纯合),计算得出3个回交家系的纯合率分别达到了53.86％、56.53％、63.33％.由此可见,回交家系的基因纯化速率较快,适用于脊尾白虾重要经济性状的遗传解析和优良种质的选育.     

Imidazole‐dicarboxylic Acid Nickel Complex as a Novel Grafting Agent for Pearl Oyster,Pinctada martensii

In the pearl cultivation industry, the implantation of the nucleus is a critical step in the production process. The surgical insertion of the nucleus and the mantle results in a wound and stress that affect not only the survival chances of the pearl oysters but also the quality of the pearls produced. This study proposes a synthesized imidazole‐dicarboxylic acid nickel complex that may hold potential in assisting the wound healing and nacre formation processes. The acute toxicity of the complex to pearl oysters, Pinctada martensii, was examined in a dose–response study over a 90‐h exposure. The safe concentrations of the nickel complex determined using probit analysis were 0.916 mg/L and 0.471 mg/L for 72‐h and 96‐h treatments, respectively. The reliability of the regression was verified using the chi‐square test. The alkaline phosphatase (ALP) activity in different regions of the treated and control oysters, along with relevant blood biochemical indices, were measured over a 90‐d period to predict the potential effects of the nickel complex on wound healing and nacre formation. A significant increase in ALP activity in the mantle, together with elevated blood levels of Mg (II) and globulin, and reduced blood levels of Zn (II) and blood urea nitrogen were observed. These results indicate that the prepared nickel complex may hold promise in helping wound healing post mantle implantation, promote the secretion of nacre, and enhance the immunity of the oysters, thus ultimately improving the yield and quality of the pearls.     

大珠母贝（Pinctada maxima）金属硫蛋白cDNA克隆与序列特征分析

金属硫蛋白（Metallothionein,MT）是一类普遍存在于生物体内、分子量低、半胱氨酸含量丰富、能够与二价重金属离子结合的蛋白质。MT在清除自由基、解除重金属毒性、参与体内微量元素代谢、防止细胞癌变等方面具有重要作用。本研究通过构建大珠母贝外套膜全长cDNA文库,首次克隆得到大珠母贝金属硫蛋白（PmMT）全长cDNA序列。该序列全长599bp,5’UTR（Untranslated Region）为75bp,3’UTR为296bp,开放阅读框（Open Reading Frame,ORF）为228bp,编码75个氨基酸。编码的氨基酸中半胱氨酸含量丰富,达到29．3％;赖氨酸和甘氨酸含量也较高,均为9．3％;不含苯丙氨酸、组氨酸、色氨酸、酪氨酸等芳香族氨基酸;含有软体动物等无脊椎动物金属硫蛋白的特征序列。序列特征分析表明,该序列具备金属硫蛋白的典型特征,是金属硫蛋白家族成员。     

珠江流域9个大鳞副泥鳅群体遗传多样性的微卫星分析

为探索我国珠江流域大鳞副泥鳅(Paramisgurnus dabryanus)群体的遗传分化及亲缘关系,本研究采用8个微卫星分子标记,对我国珠江流域9个大鳞副泥鳅群体(佛山、高要、封开、肇庆、乳源、乐昌、韶关、河源和惠州)进行了遗传多样性分析。结果显示,8个微卫星位点共检测到 69个等位基因,平均等位基因(Na)和有效等位基因(Ne)分别为8.6和4.0个,平均观测杂合度(Ho)和期望杂合度(He)分别为0.4426和0.7030。9个大鳞副泥鳅群体间的遗传分化系数(Fst)和基因流(Nm)分别为0.2452和0.7697,表明群体间遗传分化水平较高,遗传交流水平低。采用UPGMA法,对 9个群体基于遗传距离进行聚类,可分为两大支,韶关、佛山和乳源群体聚为一支;另一支包含了其余的6个大鳞副泥鳅群体,分为3个小分支,分别为乐昌与肇庆群体、河源与惠州群体及高要与封开群体。研究表明,珠江流域的9个大鳞副泥鳅群体具有较高的遗传多样性,且群体间存在着一定的遗传分化,具有进一步选育的价值。     

Experimental Collection of Pearl Oyster, Pinctada mazatlanica and Pteria sterna, Spat on a Filament Substrate

Juveniles of Pinctada mazatlanica and Pteria sterna were collected with artificial collectors from Falsa Bay, Baja California Sur, Mexico during a 12 month experimental period. Filament substrate artificial collectors contained in 2 mm plastic mesh bags (25 ± 25 cm) were deployed from the surface to 17 m of depth, and replaced monthly. Pinctada mazatlanica had the greatest recruitment (12 juveniles/cm²) in late summer and P. sterna (5 juveniles/cm²) in winter. Maximum spat collection from September 1986 to April 1987 occurred at 10 m depth for P. mazatlanica and at 4–7 m depth for P. sterna . However, peak spatfall under 11m depth occurred from late July to August 1987. The mean prodissoconch shell height was 254 μ m (SE = 0.003; N = 50) for P. mazatlanica and 313 μm (SE = 0.004; N = 50) for P. sterna . The growth equations for the first five weeks of both species, based on the prodissoconch shell height and the maximum spat height (measured from the umbo to distal edge) are presented.     

马氏珠母贝选育群体4个世代的遗传变异

采用9个多态性微卫星位点分析马氏珠母贝(Pinctada fucata)壳高选育品系(JCS)4个世代的遗传多样性与遗传结构的变化。结果表明, 随着选育的进行,  4个世代每个位点的平均有效等位基因数(N_e)从2.096下降至1.435, 平均等位基因丰度(R_s)从3.556下降至 2.556, 平均观测杂合度从0.492渐次下降至0.269, 遗传多样性呈明显的下降趋势。随着选育世代的增加, 从等位基因频率的变化来看, 部分呈上升趋势, 部分呈下降趋势, 部分基本保持稳定, 其中, 一些低频基因甚至会趋于消失。在所有等位基因中, 大部分的频率呈随机变化趋势, 并在不同的世代间上下波动; HHM20- 158 bp的频率呈有规律的变化, 由中高频逐步上升至高频, 呈富集趋势。结论认为, 经过4代的选育, 马氏珠母贝选育群体遗传多样性呈降低趋势, 遗传结构趋于稳定。本研究旨在为后续的马氏珠母贝选育策略提供理论指导。

     

Fertilization,Hatching, Survival,and Growth of Third-Generation Colored Pearl Oyster (Pinctada martensii) Stocks

In April of 2009, third-generation black (BS₃), red (RS₃), yellow (YS₃), and white (WS₃) colored stocks of pearl oyster Pinctada martensii were compared in terms of fertilization, hatching, survival, and growth. Among stocks, there were no significant differences in fertilization rate (P > 0.05; values ranging from 94.8%–98.9%). Hatching rates of WS₃ were significantly lower (P < 0.05) at 61.3 ± 6.1% than other stocks (74.3 ± 5.4%, 75.9 ± 5.2%, 73.9 ± 4.9%, for BS₃, RS_3, and YS_3, respectively). Larval survival rates of the BS₃ stock (65.8 ± 9.4%), RS₃ (61.5 ± 8.7%), and YS₃ (58.2 ± 9.2 %) were significantly (P < 0.05) higher than that of the WS₃ stock (44.8 ± 11.2%). On day 8, there were no significant differences in mean shell length among the four colored stocks (P > 0.05). At days 15, 23, 90, 150, 210, and 325, however, significant differences in mean shell length among the four colored stocks were observed (P < 0.05). The BS₃ stock had consistently larger mean shell length than the other colored stocks.