热休克诱导斑马鱼异源三倍体的研究

于１９９７年５月在广东省珠海市集斑马鱼和豹纹斑马鱼,采用热休克方法研究阻止第三极体排放诱导异源三倍体斑马鱼的适宜条件。按正交实验方案组合诱导参数,结果表明：在卵受精后２ｍｉｎ,采用３９℃持续处理２ｍｉｎ,三倍化率可达５３．８％,原肠期相对存活率为９１．０％,尾芽期相对存活率为９１％,孵化期相对存活率为９１．１％。本研究分析了热休克处理的参数与三倍体出现率和胚胎相对存活率的关系,并得出诱导参数中起始     

长牡蛎二倍体与三倍体壳型性状对体质量的影响分析

为了研究长牡蛎(Crassostrea gigas)二倍体和三倍体壳体性状对体质量的影响,随机选取养殖20月龄的二倍体和三倍体个体各75个,分别测量它们的壳体性状(壳高、壳长和壳宽)和体质量(全湿重和软体重),并进行多元回归和通径分析。结果显示,二倍体和三倍体的软体重变异系数都最高,绝大多数性状间的相关性达到了显著水平(P<0.05),全湿重和软体重的相关性较高分别为0.866和0.675;多元回归分析结果显示,壳长、壳高和壳宽对二倍体长牡蛎全湿重的影响相当,其中壳高最大,直接作用系数为0.474,对软体重影响最大的是全湿重,直接作用系数高达0.780;壳宽对三倍体长牡蛎全湿重的影响最大,直接作用系数为0.471,壳高和湿重对软体重的直接作用系数分别为0.348和0.367;对长牡蛎二倍体和三倍体全湿重和软体重分别进行回归,结果显示其r²值均小于0.85,说明存在对质量性状影响较大的其他因素。本研究为不同倍性长牡蛎的比较研究提供了数据支持。     

一株嗜碱兼性好氧反硝化菌Marinobacter sp.B3的分离鉴定及脱氮性能研究

为获得反硝化脱氮效率较好的菌株,实验从海水螺旋藻培养体系中分离获得一株嗜碱兼性好氧反硝化菌, 通过观察细菌形态以及16S rRNA基因序列的同源性分析, 鉴定该菌株为海杆菌属, 命名为Marinobacter sp. B3。为明确该海杆菌的反硝化性能及氮转化途径, 研究开展了溶解氧(DO), 碳氮摩尔比(C/N), pH和温度等不同单因素对反硝化性能影响实验和氮平衡实验。单因素影响实验结果表明, 当硝酸钾(KNO₃)作为唯一氮源, NO₃^--N的初始浓度为100 mg/L, 盐度32, 振荡速度为150 r/min (初始DO质量浓度是5.6 mg/L), C/N=10, pH=8.0±0.2, 温度为35 °C时, 可获得最大脱氮效果。氮平衡实验结果得出, 在好氧环境下, 有20.11%的NO₃^--N转化为胞内氮, 5.58 mg/L的NO₃^--N转化为其他形态(NO₂^--N、NO₄⁺-N和有机氮), 74.72%转化为N₂释放; 厌氧环境下, 有26.65%的NO₃^--N转化为胞内氮, 72.86%的NO₃^--N转化为气态产物释放。最终实验结果表明, Marinobactersp. B3在好氧和厌氧条件下, 48 h对NO₃^--N的去除率分别为99.89%和93.80%, 具有较好的反硝化脱氮能力, 且在好氧条件下NO₃^--N去除效率更高, 在海水工厂化循环水养殖尾水处理方面具有良好的应用前景。     

6—DMAP抑制九孔鲍受精卵第二极体的释放诱导三倍体

当50％九孔鲍受精卵出现第一极体后，用6－DMAP不同的浓度进行不同的持续时间诱导三倍体。结果表明，6－DMAP的诱导浓度为300μmol/dm^3、诱导持续时间为10min时，其诱导的三倍体率为90％以上，并且胚胎的孵化率高达85％，幼体的畸形率较低，为50％－55％。用该方法多次进行了九孔鲍三倍体生产诱导和苗种培育试验。本文还对6－DMAP的作用机理以及九孔鲍三倍体诱导中的诱导因子等问题进行了讨论。     

杂交与三倍体诱导对海湾扇贝(Argopecten irradians)幼虫的复合作用

基于2×2交叉实验设计,通过杂交和6-DMAP等诱导处理,构建了二倍体自交组、二倍体杂交组、三倍体诱导自交组和三倍体诱导杂交组等四个实验组家系,探讨了海湾扇贝(Argopecten irradians)幼虫期生长及存活的杂种优势和6-DMAP诱导处理效应的复合作用。分别计算各家系的孵化率,测定了各家系幼虫期第1、5、10天幼虫个体的壳长以及第5天和第10天各家系幼虫的存活率。利用一般线性模型(general linear model (GLM))计算表型变量的原因方差组分,评估了杂交和6-DMAP处理对扇贝幼虫生长和存活的影响。结果显示:6-DMAP处理组孵化率要显著低于非处理实验组(P0.05)。三倍体诱导自交组第5天幼虫存活率显著低于其他组(P0.05),到第10天三倍体自交组全部死亡。三倍体诱导杂交组第10天存活率低于二倍体组但不显著(P0.05)。在第1天幼虫生长中,三倍体诱导杂交组幼虫壳长显著小于其他组(P0.05),第5天幼虫生长中,三倍体诱导杂交组壳长则显著大于其他组,而三倍体诱导自交组则显著小于其他组(P0.05)。在第10天幼虫生长中,二倍体自交组显著小于二倍体杂交组和三倍体诱导杂交组组(P0.05)。上述结果显示扇贝幼虫生长期的杂种优势和三倍体诱导效应间存在一定相互作用。     

Cd~(2+)对青蛤(Cyclina sinensis)的毒性及蓄积过程研究

利用水生动物急性毒性实验方法,在6个浓度下Cd2+对青蛤的急性毒性进行测定,经回归分析后,得到Cd2+对青蛤96h半致死浓度为20.09mg/L,安全浓度为0.201mg/L。分别对半致死浓度和安全浓度下Cd2+在青蛤体内不同组织的蓄积情况进行了分析。结果表明,在半致死浓度条件下,168h青蛤各组织Cd2+的含量依次为肌肉内脏团鳃;而在安全浓度下,其结果为内脏团鳃肌肉,内脏团对于Cd2+富集能力远高于肌肉。讨论了青蛤的养殖环境及产品检验等问题。     

Cu2+对青蛤的胁迫效应

为研究青蛤(Cyclina sinensis)对重金属Cu~(2+)的蓄积作用及免疫机能的影响,开展了Cu~(2+)对青蛤的急性毒性实验,观察青蛤在96 h Cu~(2+)半致死浓度和安全浓度胁迫下,不同组织的蓄积趋势,及血淋巴液中SOD、CAT和ACP活性的变化。结果显示:Cu~(2+)的半致死浓度为0.807 mg/L,安全浓度为0.00807 mg/L。鳃和内脏团组织中蓄积的Cu~(2+)浓度与处理时间呈正相关;在半致死和安全浓度胁迫下,鳃组织中Cu~(2+)蓄积速度均快于内脏团;在半致死浓度胁迫下血淋巴液中SOD、CAT和ACP活性呈现先诱导再抑制的趋势,安全浓度下无明显变化,但均高于对照组水平。     

三倍体虹鳟(Oncorhynchus mykiss)卵巢分化及滞育的调控机制研究

尽管与性别决定和分化的相关基因在二倍体虹鳟中已被鉴定出来,如Cyp19a1a、Foxl2、Dmrt1、Amh和Sox9,但关于三倍体雌性虹鳟独特性腺表型及其相关性别控制基因的表达规律仍未得到深入研究,尤其是早期卵巢发育阻滞导致的生殖细胞去分化特征。因此,本研究以三倍体雌性虹鳟性腺发育中起重要调控作用的特异候选基因作为重点研究对象,辅助性腺体细胞与生殖细胞分化方向的组织学证据和类固醇激素表达规律,验证了各基因间的级联调控关系。结果表明,虹鳟三倍体与二倍体卵巢的分化特征基本一致。虹鳟二倍体与三倍体性腺分别在84dpf和98dpf就已分化为卵巢。但三倍体虹鳟卵巢分化存在障碍,早期发育停滞,卵原细胞及其滤泡细胞数量有限。Foxl2和Cyp19a1a在二倍体虹鳟卵巢和精巢中均有表达,它们在卵巢中表达量均呈逐渐升高趋势,但在精巢中的表达量非常低;这两个基因在雌性三倍体虹鳟性腺中的表达呈先升高后降低的趋势,其表达量均在8月龄时期达到最大。Dmrt1、Amh和Sox9在二倍体精巢和雌性三倍体卵巢中的表达均呈不断上调趋势,这些基因上调可能与血清睾酮含量的增加相关,因为在10月龄时期这两种鱼的血清含量相对较高,而这三个基因在二倍体虹鳟卵巢中的表达却呈下调趋势。因此本研究认为,维持鱼类卵巢分化需要持续高水平的雌激素,这可能是脊椎动物进化过程中的一个保守特征。可以推断雌性三倍体虹鳟卵巢滞育将导致其体细胞去分化,这对于Cyp19a1a表达及雌二醇合成存在抑制作用。因为这些雌激素是用来维持它们分化和继续发育的,可能产生一个负反馈圈,之后去分化会增强。     

香港牡蛎二倍体及三倍体幼虫生物学特征比较

为了评估香港牡蛎二倍体及三倍体幼虫生物学特征,于2013~2015年,在湛江和北海两地比较了二倍体及三倍体幼虫生长、存活及变态差异,计算了三倍体优势率,评估了倍性与环境效应互作对其表型性状的影响。结果表明:三倍体D形幼虫显著大于二倍体幼虫,之后,伴随着个体增长,生长优势逐渐增大,15日龄时的三倍体优势率为8.9%~9.1%。三倍体幼虫存活力与二倍体相当,未出现大量致死现象。三倍体变态规格显著大于二倍体,其优势率为5.1%~5.3%;三倍体变态时间显著低于二倍体,但其变态率与二倍体相当,不存在显著差异(p>0.05)。经过双因子分析模型的方差分析检测,发现倍性和环境效应都会对香港牡蛎幼虫生长产生显著影响(p<0.001),环境是影响幼虫存活率及变态的主要因子(p<0.05)。     

大菱鲆四倍体的人工诱导研究

大菱鲆(Scophthalmus maximus)为我国北方沿海主要的海水经济品种,其四倍体的获得对种质改良和三倍体的规模化生产具有重要的价值。到目前为止,有关大菱鲆四倍体的人工诱导尚未见报道。本文采用静水压抑制受精卵卵裂的方法,进行了四倍体诱导条件的摸索。结果显示,在14.8—15.5?C培育时,在卵裂前15min用67.5MPa处理6min可获得较好的诱导效果,其孵化率最高可达72%,染色体制片计数法计算其原肠胚期诱导率可达70%,初孵仔鱼流仪检测诱导率最高为73%。诱导组胚胎发育与对照组在形态学上基本一致,仅发育孵化时间比对照组略有滞后。     

中国对虾6种组织cDNA文库的构建

以中国对虾血液、眼柄、卵巢、雌虾头胸部、雄虾头胸部和三倍体对虾头胸部组织为材料,采用异硫氰酸胍-酸酚法提取总RNA;用磁珠法纯化mRNA;用Uni ZAP cDNA合成试剂盒合成双链cDNA并进行修饰,将cDNA定向连接在UniZAP载体上;经Gigapack Gold Package包装试剂盒包装成为噬菌体颗粒,转染宿主XL1 Blue MRF’菌株细胞,形成初级文库.初级文库经转染进一步扩增,形成稳定的cDNA文库.6个文库的库容在0.2×106~1.3×106之间,重组率都超过90%.从各文库随机取出6~10个清晰的噬菌斑进行PCR扩增,用琼脂糖凝胶电泳检测,其插入片段长度为500~2500bp.由初步功能基因克隆获得阳性结果.多项指标表明,所构建的对虾cDNA文库质量较高,为进一步筛选目的基因、EST测序和制作基因芯片提供了有效的工具.     

L-DOPA对西施舌眼点幼虫附着变态诱导的研究

采用试验水体中添加化学诱导物的方法研究L-DOPA(L-多巴)对西施舌(Coelomctraantiquata)眼点幼虫附着变态的诱导作用。结果表明,L-DOPA能诱导西施舌眼点幼虫变态,但对其附着的诱导效果不明显。用直径小于1 mm的细沙为附着基质,1×10-6mol/L的L-DOPA处理西施舌眼点幼虫12 h,变态率为73.3%,对照组为62.6%。浓度为1×10-7mol/L的L-DOPA处理西施舌幼虫12 h,生长速率为25.6%,对照组为14.7%,表明适当浓度的L-DOPA能促进西施舌幼虫的生长。在无沙附着的条件下,西施舌幼虫可正常附着变态,但生长速率较低且变态后死亡率高。     

单体牡蛎诱导变态的研究

为了得到壳型规则、大小均一的单体牡蛎,作者以葡萄牙牡蛎(Crassostrea angulata)和长牡蛎(Crassostrea gigas)眼点幼虫为材料,从肾上腺素(EPI)浓度梯度、处理时间梯度和眼点幼虫密度梯度3个因素诱导产生不固着变态的单体牡蛎。结果表明,葡萄牙牡蛎用EPI处理24 h的最适浓度为5×10–4 mol/L,不固着变态率为72.8%,在该最适浓度下,最佳处理时间为12 h,不固着变态率为82.7%。长牡蛎用EPI处理6 h的最适浓度为5×10–5 mol/L,不固着变态率为53.2%,在该最适浓度下,最佳处理时间为8h,不固着变态率为56.8%,眼点幼虫密度在80个/m L以下EPI处理效果没有显著性差异。对长牡蛎幼虫进行后续生长测定,结果显示EPI处理组幼虫的壳长、壳高和存活率要高于对照组,表明EPI可能促进牡蛎幼虫变态长出次生壳并提高其生存能力。     

兼养小球藻在不同浓度Fe3+培养下的蛋白质组学研究

为研究小球藻(Chlorella vulgaris)对不同浓度Fe~(3+)的响应,测定了自养和乙酸兼养小球藻在不同浓度Fe~(3+)条件下的生长速率和油脂含量,比较分析了兼养小球藻在不同浓度Fe~(3+)下的蛋白质组表达差异。结果表明,兼养小球藻比自养小球藻生长速率快,积累的生物量多,在缺铁条件下中性脂含量高。蛋白质组分析显示:在缺铁条件下光合作用相关蛋白含量最低;在缺铁和高浓度铁条件下,热激蛋白、蛋白合成和糖代谢等过程相关蛋白表达都下调,表明缺铁和高浓度铁条件对小球藻的生长都是逆境条件;在缺铁条件下氨基酸代谢相关蛋白表达上调,这可能是由于NO_3~–同化下降,氨基酸合成减少,需要进行氮的回收利用。上述结果表明:在兼养条件下缺铁培养的小球藻可获得较高生物量和中性脂含量;而高浓度铁能促进小球藻生长,但不能提高中性脂含量,对藻细胞也有一定的胁迫效应。     

Remineralization and accumulation of organic carbon and nitrogen in marine sediments of eutrophic bays: the case of the Bay of Concepcion, Chile

The Bay of Concepcion (36°40′S; 73°02′W) is a semi-enclosed and shallow embayment in which biogeochemical processes are seasonally coupled to coastal upwelling during the austral spring and summer. The nutrient cycle in the bay is complex due to the combined effects of a pronounced O₂ minimum layer and high nutrient concentrations both originating from subsurface equatorial water during coastal upwelling and a rapid rate of sediment nutrient recycling. The sediments are characterized by a high content of organic matter mainly due to the extremely high rates of phytoplankton production and deposition. During the upwelling period, a black flocculent layer frequently covers the sediment–water interface in the inner part of the bay where an extensive mat of Beggiatoa spp. develops. Three approaches are used to analyse the extent to which the benthic system recycles or retains nutrients at two stations, located at the centre (station C, St. C) and mouth (station B, St. B) of the bay for a 1-year period (March 1996–1997): (1) estimation of C and N remineralization rates based on SO₄²⁻ reduction measurements, (2) calculation of C and N turnover rates using a diagenetic model applied to total organic carbon and total nitrogen vertical distributions and, (3) construction of C and N budgets from direct measurements of sedimentation (from a sediment trap) and estimates of the C and N burial rates. Depth-integrated SO₄²⁻ reduction rates varied between 3.4 (winter) and 25.5 (summer) mmol m⁻² d⁻¹. Estimated C and N oxidation rates ranged between 7.9 and 87.8 mol C m⁻² yr⁻¹ and between 0.9 and 6.9 mol N m⁻² yr⁻¹, respectively. Each approach yielded minor differences in the C and N remineralization rates (and also minor differences between both studied stations), except when the kinetic model was applied to C and N distribution without including the presence of the flocculent layer. The rates of carbon oxidation and sulphate reduction were considerably higher than in other coastal sediments with similar depositional regime. The C and N burial rates were 2.23 and 0.21 (St. C) and 1.30 and 0.09 (St. B) mol m⁻² yr⁻¹, respectively. The C/N ratio of the buried fraction was ca. 10.6 at St. C and 14.4 at St. B. Because the observed differences in burial rates could not be ascribed to distinctive depositional (both stations have similar sediment accumulation rates) and oceanographic (similar O₂ concentration and hydrography) conditions, differences may be due to in part spatial heterogeneity in the supply of organic matter. The degree of preservation of organic matter as plankton detritus and nitrogen accumulating bacterial biomass associated with Beggiatoa spp. at St. C may also be involved.     

Ammonium uptake kinetics and interactions between nitrate and ammonium uptake inChattonella antiqua

Ammonium uptake kinetics and interactions between nitrate and ammonium uptake were examined inChattonella antiqua. After the addition of ammonium to the culture ofC. antiqua, the ammonium concentration decreased linearly with time. The ammonium uptake rate as a function of ammonium concentration followed the Michaelis-Menten equation; the maximal uptake rate was 2.0 pmol cell^–1hr^–1 and the half saturation constant, 2.2M. Although the ammonium uptake was not affected by nitrate, uptake of nitrate was rapidly (15min) suppressed by ammonium and a 50% reduction in nitrate uptake was observed at an ammonium concentration ofca. 2M.     

Effects of cold eddy on phytoplankton production and assemblages in Luzon strait bordering the South China Sea

The biochemical effects of a cold-core eddy that was shed from the Kuroshio Current at the Luzon Strait bordering the South China Sea (SCS) were studied in late spring, a relatively unproductive season in the SCS. The extent of the eddy was determined by time-series images of SeaWiFS ocean color, AVHRR sea surface temperature, and TOPEX/Jason-1 sea surface height anomaly. Nutrient budgets, nitrate-based new production, primary production, and phytoplankton assemblages were compared between the eddy and its surrounding Kuroshio and SCS waters. The enhanced productivity in the eddy was comparable to wintertime productivity in the SCS basin, which is supported by upwelled subsurface nitrate under the prevailing Northeastern Monsoon. There were more Synechococcus, pico-eucaryotes, and diatoms, but less Trichodesmium in the surface water inside the eddy than outside. Prochlorococcus and Richelia intracellularis showed no spatial differences. Water column-integrated primary production (IPP) inside the eddy was 2–3 times that outside the eddy in the SCS (1.09 vs. 0.59 g C m⁻²d⁻¹), as was nitrate-based new production (INP) (0.67 vs. 0.25 g C m⁻²d⁻¹). INP in the eddy was 6 times that in the Kuroshio (0.12 g C m⁻²d⁻¹). IPP and INP in the eddy were higher than the maximum production values ever measured in the SCS basin. Surface chlorophyll a concentration (0.40 mg m⁻³) in the eddy equaled the maximum concentration registered for the SCS basin and was higher than the wintertime average (0.29 ± 0.04 mg m⁻³). INP was 3.5 times as great and IPP was doubled in the eddy compared to the wintertime SCS basin. As cold core eddies form intermittently all year round as the Kuroshio invades the SCS, their effects on phytoplankton productivity and assemblages are likely to have important influences on the biogeochemical cycle of the region.     

Differential effects of thermal and chemical stressors on tissue balls from scleractinian corals

Coral cell aggregates (tissue balls) from four species (Acropora muricata, Fungia repanda, Pavona cactus and Pocillopora damicornis) were used as an indicator to investigate the effects on the corals of thermal stress and of chemical extracts from three sponges (Adocia sp., Haliclona sp. and Lissodendoryx sp.) and one ascidian (Didemnum molle). The formation and disintegration of tissue balls were studied through exposure to a temperature range of 23–30 °C at time intervals of 0–90 min, and to sponge and ascidian crude extracts at concentrations of 50–200 µg ml^?1 at temperatures of 23 and 30 °C and at time intervals of 10, 60 and 120 min. The negative effect of temperature on overall tissue ball density (number per cm² of coral surface) was greatest at higher temperatures (28 and 30 °C) but varied among coral species. Tissue balls of P. damicornis were the most robust whereas those of A. muricata were the most sensitive. High concentrations of extracts of Adocia sp., Haliclona sp. and Lissodendoryx sp. generally inhibited the formation of tissue balls or caused their disintegration, or both, most markedly at 30 °C. Adocia sp. induced the least negative effects and Haliclona sp. the most. No tissue balls were formed in the presence of D. molle extracts (50 and 100 µg ml^?1), indicating a high level of interference with tissue ball formation. The differential susceptibility to thermal and chemical stressors exhibited by the corals under study have possible implications for the interactions of the corals with other sedentary reef organisms under climate change-driven ocean warming.     

三种化学诱导剂诱导太平洋牡蛎三倍体的比较研究

从三倍体率、D幼孵化率、幼虫生长速度、生产成本等方面比较了细胞松驰素 B、二甲基氨基嘌呤、咖啡因三种化学诱导剂诱导太平洋牡蛎产生三倍体的效果。 CB的三倍诱导率最高 ,为91.5% ,但 D幼孵化率最低 ,仅为 5.8% ,且成本最高 ,为 6 - DMAP和咖啡因的 4倍和 50倍。 6 -DMAP的三倍体诱导率略低于 CB,为 88.0 % ,但 D幼孵化率最高 ,为 6 0 .5% ,其成本仅为 CB的1/ 4。咖啡因的三倍体诱导率最低 ,为 82 .4 % ,幼虫孵化率为 30 .2 % ,但其成本最低 ,仅为 CB的 2 %和 6 - DMAP的 8%。三种诱导剂诱导的三倍体群幼虫在生长速度及成活率方面无显著差别。     

胶州湾微微型浮游植物丰度及其与环境因子的相关性分析

利用流式细胞仪对胶州湾微微型浮游植物4个季节的丰度分布进行了研究,并分析了微微型浮游植物与环境因子的相关性。结果表明,聚球藻的丰度在2.17×102—2.329×104个/ml之间,高值区主要分布在湾内西部和湾口海域;仅夏季、冬季丰度之间有显著性差异;夏季在垂直分布上差异显著,在B3、C4、D5连续站昼夜变化趋势基本一致,分别在13:00和3:00出现峰值。微微型真核浮游植物的丰度分布在1.028×103—8.651×104个/ml之间,主要活跃于湾内西部海域;四季丰度在垂直分布上差异不显著;春、夏季丰度明显高于秋、冬季;夏季连续站昼夜变化趋势与聚球藻基本一致。通过主成分分析表明,聚球藻和微微型真核浮游植物丰度在不同季节受不同环境因子的影响,在冬季与温度有关,温度升高,二者的丰度增高。在其它季节,二者丰度主要受营养盐等环境因子的影响。