A clip domain serine protease (cSP) from the Chinese mitten crab Eriocheir sinensis: cDNA characterization and mRNA expression

Clip domain serine protease (cSP), characterized by conserved clip domains, is a new serine protease family identified mainly in arthropod, and plays important roles in development and immunity. In the present study, the full-length cDNA of a cSP (designated EscSP) was cloned from Chinese mitten crab Eriocheir sinensis by expressed sequence tags (ESTs) and PCR techniques. The 1380 bp EscSP cDNA contained a 1152 bp open reading frame (ORF) encoding a putative cSP of 383 amino acids, a 5'-untranslated region (UTR) of 54 bp, and a 3'-UTR of 174 bp. Multiple sequence alignment presented twelve conserved cysteine residues and a canonical catalytic triad (His(185), Asp(235) and Ser(332)) critical for the fundamental structure and function of EscSP. Two types of cSP domains, the clip domain and tryp_spc domain, were identified in the deduced amino acids sequence of EscSP. The conservation characteristics and similarities with previously known cSPs indicated that EscSP was a member of the large cSP family. The mRNA expression of EscSP in different tissues and the temporal expression in haemocytes challenged by Listonella anguillarum were measured by real-time RT-PCR. EscSP mRNA transcripts could be detected in all examined tissues, and were higher expressed in muscle than that in hepatopancreas. gill, gonad, haemocytes and heart. The EscSP mRNA expression in haemocytes was up-regulated after L anguillarum challenge and peaked at 2 h (4.96 fold, P < 0.05) and 12 h (9.90 fold, P < 0.05). Its expression pattern was similar to prophenoloxidase (EsproPO), one of the components of crab proPO system found in our previous report. These results implied that EscSP was involved in the processes of host-pathogen interaction probably as one of the proPO system members. (C) 2009 Elsevier Ltd. All rights reserved.

Sequence analysis of the ITS region and 5.8S rDNA of Porphyra haitanensis

The sequences of the ITS (internal transcribed spacer) and 5.8S rDNA of three cultivated strains of Porphyra haitanensis thalli (NB, PT and ST) were amplified, sequenced and analyzed. In addition, the phylogenic relationships of the sequences identified in this study with those of other Porphyra retrieved from GenBank were evaluated. The results are as follows: the sequences of the ITS and 5.8S rDNA were essentially identical among the three strains. The sequences of ITS l were 331 by to 334 bp, while those of the 5.8S rDNA were 158 by and the sequences of ITS2 ranged from 673 by to 681 bp. The sequences of the ITS had a high level of homology (up to 99.5%) with that of P. haitanensis (DQ662228) retrieved from GenBank, but were only approximately 50% homologous with those of other species of Porphyra. The results obtained when a phylogenetic tree was constructed coincided with the results of the homology analysis. These results suggest that the three cultivated strains of P. haitanensis evolved conservatively and that the ITS showed evolutionary consistency. However, the sequences of the ITS and 5.8S rDNA of different Porphyra species showed great variations. Therefore, the relationship of Porphyra interspecies phyletic evolution could be judged, which provides the proof for Porphyra identification study. However, proper classifications of the subspecies and the populations of Porphyra should be determined through the use of other molecular techniques to determine the genetic variability and rational phylogenetic relationships.

Effects of infection of EGH-expressing Escherichia coli on haemocytes in Ciona intestinalis

The effects of infection of EGFP-expressing Escherichia coli on the haemocytes of the ascidian Ciona intestinalis were investigated. The results showed that THC of the infected individuals changed significantly. Hyaline amoebocytes phagocytosed E. coli in 5 min and excreted lysosome particles that attached to the surface of the bacteria. Granular amoebocytes released lots of particles for Immoral immunity while stem-cell-like haemocytes remained intact. With the increase in THC, the stem-cell-like haemocytes showed division and proliferation. A small portion of hyaline amoebocytes was at early apoptosis stage I h after infection and typical apoptosis bodies emerged in granular amoebocytes. A few of the infected haemocytes showed DNA damage using SCGE assay. Flow cytometry analysis revealed an obvious apoptosis peak in infected haemocytes. In conclusion, apoptosis was found to be an important immune response of ascidian haemocytes response to bacterial infection. To our best knowledge, this is the first report of the occurrence of apoptosis of haemocytes in ascidians. (c) 2005 Elsevier B.V. All rights reserved.

Propagators and ridge jumps in a back-arc basin, the West Philippine Basin

We explore the tectono-magmatic processes in the western West Philippine Basin, Philippine Sea Plate, using bathymetric data acquired in 2003 and 2004. The northwestern part of the basin formed through a series of northwestward propagating rifts. We identify at least five sequences of propagating rifts, probably triggered by mantle flow away from the mantle thermal anomaly that is responsible for the origin of the Benham and Urdenata plateaus. Gravitational forces caused by along-axis topographic gradient and a similar to 30 degrees ridge reorientation appear to also be driving the rift propagations. The along-axis mantle flow appears to be reduced and deflected along the Luzon-Okinawa fracture zone, because the spreading system remained stable west of this major fault zone. North-east of the Benham plateau, a left-lateral fracture zone has turned into a NE-SW-trending spreading axis. As a result, a microplate developed at the triple junction.

Talc-bearing serpentinized peridotites from the southern Mariana forearc: implications for aseismic character within subduction zones

The serpentinized peridotites overlying the subducted zones in the Izu-Bonin-Mariana (IBM) arc system have been interpret as the cause of the low-velocity layer identified beneath the IBM froearc, in turn few earthquakes occurred along the plate boundary. Chrysotile, which is a low temperature and highly hydrated phase of serpentine with low frictional strength, has been suggested as the low velocity material in the serpentinized peridotites, besides, brucite is inferred to be likely conducive to stable sliding. However, such idea encounters challenging in our serpentinized peridotites from the southern Mariana forearc, which absent both the above minerals. The presence of talc, which characterized by its weak, low-friction and inherently stable sliding behavior, provides new clue. Here we report the occurrence of talc in serpentinized peridotites collected from the landward trench slope of the southern Mariana forearc. We infer that talc is mainly forming as a result of the reaction of serpentine minerals with silica-saturated fluids released from the subducting slab, and talc also occurs as talc veins sometimes. Due to its unique physical properties, talc may therefore play a significant role in aseismic slip in the IBM subduction zone.

Spatial and temporal variations of sound speed at the PN section

Gridded sound speed data were calculated using Del Grosso's formulation from the temperature and salinity data at the PN section in the East China Sea covering 92 cruises between February 1978 and October 2000. The vertical gradients of sound speed are mainly related to the seasonal variations, and the strong horizontal gradients are mainly related to the Kuroshio and the upwelling. The standard deviations show that great variations of sound speed exist in the upper layer and in the slope zone. Empirical orthogonal function analysis shows that contributions of surface heating and the Kuroshio to sound speed variance are almost equivalent.

EAWM-related air-sea-land interaction and the Asian summer monsoon circulation

Based on the data analysis, this study further explores the characteristics of East Asian winter monsoon (hereafter, EAWM, for brevity) as well as the related air-sea-land system, and illustrates how and to what degree anomalous signals of the subsequent Asian summer monsoon are rooted in the preceding EAWM activity. We identified an important air-sea coupled mode, i.e., the EAWM mode illustrated in Section 3. In cold seasons, strong EAWM-related air-sea two-way interaction is responsible for the development and persistence of the SSTA pattern of EAWM mode. As a consequence, the key regions, i.e., the western Pacific and South China Sea (hereafter, SCS, for brevity), are dominated by such an SSTA pattern from the winter to the following summer. In the strong EAWM years, the deficient snow cover dominates eastern Tibetan Plateau in winter, and in spring, this anomaly pattern is further strengthened and extended to the northwestern side of Tibetan Plateau. Thus, the combined effect of strong EAWM-related SSTA and Tibetan snow cover constitutes an important factor in modulating the Asian monsoon circulation. The active role of the EAWM activity as well as the related air-sea-land interaction would, in the subsequent seasons, lead to: 1) the enhancement of SCS monsoon and related stronger rainfall; 2) the northward displacement of subtropical high during Meiyu period and the related deficient rainfall over Meiyu rainband; 3) above-normal precipitation over the regions from northern Japan to northeastern China in summer; 4) more rainfall over the Arabian Sea and Northeast India, while less rainfall over southwest India and the Bay of Bengal. The strong EAWM-related air-sea interaction shows, to some degree, precursory signals to the following Asian summer monsoon. However, the mechanism for the variability of Indian summer monsoon subsequent to the strong EAWM years remains uncertain.

牙鲆生长相关性状遗传力估计

遗传力是数量遗传学的重要参数，对鱼类良种选育具有重要意义。可靠的遗传力估计值可以为合理制定育种计划提供宝贵信息，同时可以预测选择反应。本文按照10×3因子设计方法，10尾雄鱼与3尾雌鱼两两授精，产生10个父系半同胞、3个母系半同胞及30个全同胞家系，以微卫星分子标记为家系鉴定手段，对40日龄牙鲆生长相关性状遗传力进行了估计；同时，初步探讨了因子交配设计及人工控制条件下，亲本对子代遗传贡献率差异及有效群体大小。主要结论包括：1.初步筛选的14个微卫星位点中，有9个（Po91、Po1、Po56、Po20、Poli23、Po89、Poli121、Po42、Po13）在亲本中呈现中、高度多态性：平均等位基因9.4个；平均亲本特异性等位基因4个。用5个位点（Po91、Po1、Po56、Po20、Poli23）为346个子代中的227个个体找到所属家系；继续用另外4个位点（Po89、Poli121、Po42、Po13）分型，成功鉴定72个个体。鉴定率约86%，其中，亲本特异性等位基因的存在使鉴定效率大大提高。以上9个位点可为该群体良种选育工作提供技术支持。2. 发生降解的DNA与完整的DNA，在相同引物、相同PCR体系、相同模板浓度下扩增的带型一致。该结果证实了微卫星分型对降解的DNA同样稳定。3. 亲本对子代的遗传贡献率存在差异。雄亲的贡献率为5.8—14.3%，除3号、9号子代数较少，5号、6号子代数较多外，其它父本基本一致；母本对子代的贡献率差异较大（18.5%—50.6%），这与人工授精前雌亲发育状况、卵子质量检测结果基本一致，在一定程度上说明，母本尤其是卵子质量对鱼类早期存活具有较大影响。4. 家系内子代数目的不平衡导致实际有效群体大小下降。Ne=7.44，比理论有效群体大小（9.23）下降约19%。但与自然交配相比（有效群体下降可达75%），人工控制下的交配在一定程度上可有效限制遗传多样性的下降。5. 基于父本方差组分，40日龄生长相关性状遗传力估计值（h2s±S.E.）为（0.157±0.052）－（0.440±0.137）。加性遗传方差在表型方差中所占的比例，在一定程度上表明该牙鲆群体生长性状具有一定的选择力度。

刺参遗传连锁图谱构建及卵母细胞成熟过程研究

第一部分 刺参遗传连锁图谱的构建 利用AFLP和微卫星标记以一个远缘地理杂交家系为作图群体，以拟测交理论为作图策略，构建了刺参Apostichopus japonicus的雌性和雄性遗传连锁图谱。利用62对AFLP引物组合，对亲本和93个个体进行了分离分析，共得到3572个标记，其中具有多态性的标记为点数为1133个，多态性比例为31.5%。平均每个引物对产生18.3个多态片段。1133个多态性位点中，294个标记在父母本中都出现并在后代中分离，839个标记在父本或母本中出现并在子代分离，其中母本443(52.8%)个分离标记，父本396(47.2%)个分离标记。卡方检验显示，556个标记符合孟德尔1:1分离。筛选微卫星标记中30个在家系中具有作图信息，其中19个可用于母本作图，19个可用于父本作图。 利用Mapmaker 3.0/EXP软件对分离标记进行连锁分析。358个分子标记用于雌性遗传连锁分析，其中包括19个微卫星标记，199个AFLP标记和11个微卫星标记定位在雌性框架图谱中。雌性框架图谱共有25个连锁群，总长度为3148.3 cM。标记之间最大间隔为37.5 cM，平均间隔为17.0 cM，连锁群的分子标记数最大为21个最小为4个。用于雄性连锁分析的分子标记共有332个，其中包含19个微卫星标记。207个标记定位于23个连锁群上，其中AFLP标记数为196个，微卫星标记数为11个。23个连锁群总长度为3059.8 cM，标记间最大间隔38.6 cM，平均间隔16.6 cM。每个连锁群的长度范围在40 cM到271.4 cM之间，标记数在18到4个之间，包括二、三连体在内的雄性连锁图谱共覆盖3227 cM。雌雄基因组的预测长度分别为4053.7 cM和3816.3 cM，因此，所构建的雌雄连锁图谱覆盖率分别为84.0%和84.5%。通过共有微卫星标记，有3个连锁群在两个图谱中对应。分子标记在两个图谱中呈均匀分布，没有成簇现象。 分子标记筛选、遗传图谱的构建为刺参分子标记辅助选择育种，经济性状QTL定位和比较基因作图打下基础，并且最终将促进中国刺参遗传改良和新品种的培育工作。 第二部分 卵母细胞成熟过程研究 对刺参卵母细胞的体外诱导成熟方法进行探索，并且通过透射电子显微镜、扫描电子显微镜和激光共聚焦显微镜结合超薄切片技术及间接免疫荧光染色技术研究卵母细胞成熟的生物学过程。 分别利用在海参中有诱导作用的海星神经提取液和二硫苏糖醇(DTT)对从成熟卵巢解剖出的刺参卵母细胞进行诱导成熟试验，结果显示，刺参的卵母细胞在海水中不会发生自发成熟，海星神经提取液以及神经提取液加滤泡悬浮液对卵母细胞没有诱导成熟的作用，而二硫苏糖醇处理可以诱导卵母细胞生发泡破裂，当DTT溶液的浓度处于10-1 mol/L到10-3 mol/L之间时，对刺参卵母细胞的促熟作用较为明显，在10-2 mol/L左右时，卵母细胞的诱导成熟率可以达到90%以上。未成熟的卵母细胞处于第一次减数分裂前期，不具备受精能力。经DTT诱导后，生发泡发生移动并破裂，染色体排列到赤道板上，然后第一、二极体先后排出，诱导成熟的卵母细胞排出极体的时间与自然受精的卵子一致。在卵母细胞成熟过程中有受精膜举起的现象，说明精子入卵不是受精膜举起的必要条件。卵母细胞只有在生发泡破裂之后才具有受精能力，从生发泡破裂到第二极体排出前的整个减数分裂过程中，卵母细胞都可以受精，但是只有在第一极体排出前受精的卵母细胞才能发生卵裂。人工促熟的卵母细胞受精后，形成的胚胎似乎并不能正常发育，试验过程中最多只得到了8细胞期的胚胎，之后细胞便产生畸形并停止分裂，最终裂解消失。 利用DTT诱导刺参卵母细胞成熟，综合运用光镜、电子显微镜及激光共聚焦技术观察卵母细胞成熟过程，分析了卵母细胞表面动物极突起在卵母细胞脱滤泡中的作用，以及卵母细胞动物极突起和微管组织在生发泡移动过程中所起的作用。卵母细胞通过动物极突起连接到滤泡上，在脱滤泡作用开始时，卵母细胞动物极突起刺入并穿过滤泡膜，使得滤泡膜表面形成一个缺口，然后整个卵母细胞开始从缺口处挤出。脱滤泡的作用力来源有两种：胶膜层的水合作用以及滤泡的收缩。通过激光共聚焦显微镜观察，未成熟的卵母细胞中存在5种类型的微管，并且有两个微管组织中心锚定在卵母细胞突起下方。减数分裂重新启动时，生发泡沿微管向动物极移动，至细胞膜下时破裂。微管解聚对照实验证明驱动生发泡移动的细胞结构为微管束。生发泡破裂后，两个微管组织中心组织形成减数分裂纺锤体，然后第一极体以“收缩-排出”的独特方式排出。 本研究填补了刺参繁殖生物学研究中的缺失环节，为刺参人工繁育技术提供理论基础和指导。

塞隆骨提取物对CFA诱导活化的小鼠腹腔巨噬细胞功能的影响

目的：研究塞隆骨提取物对弗氏完全佐剂诱导活化的腹腔巨噬细胞的免疫抑制作用。方法：小鼠腹腔注射弗氏完全佐剂诱导腹腔巨噬细胞活化并回收，体外加入LPS及IFN-γ刺激培养巨噬细胞，利用酶联免疫法测定培养上清中细胞因子（IL-1β、IL-6、IL-12p40、TNF-α水平。结果：塞隆骨水提物及90％醇沉部分体内给药均能够能显著抑制弗氏完全佐剂诱导的巨噬细胞向腹腔的浸润，能抑制巨噬细胞在LPS及IFN-γ刺激下生成细胞因子（IL-12p40、IL-1β、IL-6、TNF-α。结论：塞隆骨提取物对巨噬细胞功能有一定程度的影响，能够抑制其产生炎症因子，可能是其治疗类风湿性关节炎的作用机理之一。

青海地区唐古特大黄药材HPLC指纹图谱研究

目的建立了青海地区唐古特大黄药材的HPLC指纹图谱.方法采用反相高效液相色谱法,乙腈-水（0.04%的磷酸）为流动相,梯度洗脱,体积流量1.0 mL/min,检测波长为280 nm,柱温为40 C.结果精密度、重现性、稳定性试验中共有峰面积和保留时间的RSD均小于5%.青海不同采集地唐古特大黄的平均相似度为0.925.结论该方法简便、实用、可靠,可用于以青海果洛地区为主产地不同海拔唐古特大黄药材质量标准的分析检测,也为栽培大黄代替野生大黄提供理论基础.

青藏高原高寒灌丛CO2通量日和月变化特征

采用涡度相关法对青藏高原高寒灌丛CO2通量进行连续观测的结果表明,青藏高原高寒灌丛CO2通量呈明显的日和月变化特征.就日变化而言,暖季(7月)CO2通量峰值出现在12:00左右(-1.19 g CO2/(m2•h)-1),08:00～19:00时CO2净吸收,而20:00～07:00为CO2净排放; 冷季(1月)CO2通量变化振幅极小,除11:00～17:00时少量的CO2净排放以外(0.11 g CO2/(m2•h)-1左右),其余时段CO2通量接近于零.从月变化来看,6～9月为CO2净吸收阶段,8月CO2净吸收最大,6～9月CO2净吸收的总量达673 g CO2/m2; 1～5月及10～12月为CO2净排放,共排放446 g CO2/m2,4月CO2净排放最大.全年CO2通量核算表明,无放牧条件下青藏高原高寒灌丛是显著的CO2汇,全年CO2净吸收量达227 g CO2/m2.

不同类型生态系统水热碳通量的监测与研究

亚太地区环境革新战略项目(APEIS)在中国5种主要生态系统类型区(草地:海北、耕地:禹城、稻田:桃源、林地:千烟洲、荒漠:阜康)建立了一个以连续观测能量、水分和碳素通量为中心,包括气象、水文、土壤、植被等各项生态要素的监测网络系统,被称之为APEIS-FLUX系统.作者首先对APEIS-FLUX系统的观测数据进行了初步分析,表明该系统稳定可靠,它可以实时地提供高质量、高精度、长期而连续的通量及生态要素的观测数据.对数据的比较清楚地反映出了不同生态系统类型区的水热碳通量的差异性.其次,利用APEIS-FLUX数据对美国航空航天局(NASA)的MODIS数据产品进行比较验证后发现,除部分产品如地表面温度(MOD11)等与观测数据较吻合以外,大部分数据产品如土地覆盖(MOD12),叶面积指数(MOD15)和光合速率与净第一性生产力(MOD17)等都与观测数据相差深远,有必要对其处理程序和模式进行修正.为此,我们利用APEIS-FLUX的数据作为MOD15和MOD17的生成模型(BIOME-BGC)的输入数据,并对该模型的有关参数进行了修订.结果表明,该模式在通过修正后,可以很好地模拟植被的生长过程及其相应的水热碳循环过程.

高寒草甸藏嵩草种群繁殖对策的研究

对高寒草甸藏嵩草 (Kobresiatibetica)种群的繁殖对策进行了初步研究 .结果表明 :藏嵩草属寒冷中生密丛短根茎地下芽植物 ,在高寒生境中采用了以营养繁殖为主、有性繁殖为辅的繁殖策略 ,具体体现在以下几个方面 :藏嵩草种子产量nA=2 0 0 .1m-2 ,但种子萌发率较低 ,室内和野外萌发率分别仅有 4%和 2 % ,经氢氧化钠溶液和赤霉素溶液处理后的种子萌发率分别为 1%和 6 .7% ,而剥去种皮后种子萌发率达 47.3% ,所以种皮坚硬是造成种子萌发率低的主要原因 ;进入种子库、保留至返青期且具有活性的种子仅占种子总数的 31.49% ;单位面积上理论实生苗数仅为1.2 6m-2 ,与此相反 ,藏嵩草营养繁殖所形成的新个体数为 10 1.32m2 ,远远多于种子萌发所形成的实生苗数 .此外 ,藏嵩草营养繁殖效力也远高于有性繁殖效力，营养繁殖效力占总繁殖效力的83.46%（167）。