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.

The influence of the cation of quaternized chitosans on antioxidant activity

Various quaternized chitosans (QCSs) were synthesized according to previous method. Their reducing power and antioxidant potency against hydroxyl radicals ((OH)-O-center dot) and hydrogen peroxide (H2O2) were explored by the established systems in vitro. The QCSs exhibited markedly antioxidant activity, especially TCEDMCS, whose IC50 on hydroxyl radicals was 0.235 mg/mL. They showed 65-80% scavenging effect on hydrogen peroxide at a dose of 0.5 mg/mL. Generally, the antioxidant activity decreased in the order TCEDMCS > TBEDMCS > EDMCS > PDMCS > IBDMCS > Chitosan. Furthermore, the order of their (OH)-O-center dot and H2O2 scavenging activity was consistent with the electronegativity of different substituted groups in the QCSs. The QCSs showed much stronger antioxidant activity than that of chitosan may be due to the positive charge density of the nitrogen atoms in QCSs strengthened by the substituted groups. (C) 2009 Elsevier Ltd. All rights reserved.

Isolation, antimicrobial activity, and metabolites of fungus Cladosporium sp associated with red alga Porphyra yezoensis

Cladosporium sp. isolate N5 was isolated as a dominant fungus from the healthy conchocelis of Porphyra yezoensis. In the re-infection test, it did not cause any pathogenic symptoms in the alga. Twenty-one cultural conditions were chosen to test its antimicrobial activity in order to obtain the best condition for large-scale fermentation. Phenylacetic acid, p-hydroxyphenylethyl alcohol, and L-beta-phenyllactic acid were isolated from the crude extract as strong antimicrobial compounds and they are the first reported secondary metabolites for the genus Cladosporium. In addition, the Cladosporium sp. produced the reported Porphyra yezoensis growth regulators phenylacetic acid and p-hydroxyphenylacetic acid. No cytotoxicity was found in the brine shrimp lethality test, which indicated that the environmental-friendly Cladosporium sp. could be used as a potential biocontrol agent to protect the alga from pathogens.

栉孔扇贝抗氧化酶基因的克隆与表达分析

扇贝是我国海水养殖的重要品种，但自1994年以来，养殖扇贝陆续爆发的大规模死亡，不但造成了巨大的经济损失，而且直接威胁到现有产业的生存和发展。扇贝病害的不断爆发以及病因的多样性迫切要求制定新的疾病防治措施和开发新型的抗菌物质。因此，深入研究扇贝免疫防御机制，探讨提高机体抗病力的有效途径和方法，改良种质和培育抗病品系，无疑是解决目前困扰扇贝养殖业健康可持续发展的必经之路。 抗氧化酶可以清除活性氧，是维持机体内氧环境平衡，抵抗外界环境影响的重要免疫因子。本研究采用大规模 EST 测序方法和同源克隆的方法，结合 cDNA 末端快速扩增（RACE）技术，从栉孔扇贝中克隆到了超氧化物岐化酶(Superoxide dismutase，SOD)、过氧化氢酶(Catalase，CAT)、谷胱甘肽过氧化物酶（Glutathione peroxidase，GPX）等抗氧化酶基因的全长 cDNA序列，并对其基因结构进行了分析。同时，用实时定量PCR方法对这三个基因在健康扇贝血淋巴细胞、肾、鳃、肌肉、性腺等组织和在分别用鳗弧菌，溶壁微球菌和假丝酵母处理扇贝后不同时间段的表达差异情况进行了研究。 超氧化物歧化酶基因CfSOD的cDNA 全长为1022 bp，其中开放阅读框（Open Reading Frame, ORF）含有 459 bp，编码 153个氨基酸残基，无信号肽，为胞内蛋白。经BLASTP分析发现，CfSOD与其它动物具有较高的同源性。CfSOD中存在两个Cu/Zn-SOD的签名序列；另外Cu结合必须氨基酸(His-45，-47，-62 和-119)和Zn结合必须氨基酸(His-62，-70，-79和Asp-82)在CfSOD中保守。实时定量PCR 检测发现，CfSOD在鳃、血细胞和肾中有较高的表达。在鳗弧菌和溶壁微球菌刺激后，CfSOD的相对表达量逐渐下降，然后分别在32小时和16小时的时候恢复到刺激前的表达水平。在假丝酵母刺激后，CfSOD的mRNA表达没有显著差异。 栉孔扇贝过氧化氢酶基因CfCAT的cDNA全长为3146 bp，其中开放阅读框含有1521bp，编码507个氨基酸残基，无信号肽，为胞内蛋白。经BLASTP分析发现，CfCAT与其它动物具有较高的同源性。 CfCAT中存在过氧化氢酶近端活性位点和过氧化氢酶近端血红素配体签名序列，另外存在两个糖基化位点 NFS和 NFT，同时在CfCAT 的C末端存在过氧化物酶体定位信号AQL，为典型过氧化氢酶。实时定量PCR 检测发现，健康的扇贝中CfCAT在鳃和性腺中有较高的表达。CfCAT基因在鳗弧菌刺后表达升高，在4小时达到最高，约是刺激前表达量的6.8倍（P<0.05），后随着时间的变化而逐渐下降。在8小时表达量达到为刺激前表达量的1.3倍（P<0.05），在16和32小时略高于刺激前的水平。在溶壁微球菌刺激后CfCAT基因表达量也呈上升趋势，在刺激后4小时达到刺激前表达量的约2倍，然后有所下降，在16 小时又上升到刺激前表达量的2.9倍。CfCAT基因在假丝酵母刺激后的表达略有升高，4小时约是刺激前的1.2倍（P<0.05），在其他时间段变化不明显。 栉孔扇贝谷胱甘肽过氧化物酶基因CfGPX的cDNA 全长为1290 bp，其中开放阅读框含有705bp，编码235个氨基酸残基，有一个24核苷酸的信号肽序列。经BLASTP 分析发现，CfSOD与其它动物具有较高的同源性。CfGPX中发现谷胱甘肽过氧化物酶活性位点的签名序列， 另外发现硒半胱氨酸和硒半胱氨酸插入序列，为含硒型谷胱甘肽过氧化物酶。实时定量PCR 检测发现，未经处理的扇贝中CfGPX在性腺、肌肉、血和肾中有较高的表达。CfGPX基因在鳗弧菌刺后表达量快速上升，在6 小时的时候表达量达到最高，为刺激前的4.0倍（P>0.05），后随着时间的变化而逐渐下降。在溶壁微球菌刺激后CfGPX在前6小时表达略有降低，在6小时的时候表达量为刺激前的0.5倍（P<0.05），后随着时间的变化而逐渐升高。在16小时的时候表达量为刺激前的2.1倍（P<0.05）。在假丝酵母刺激后， CfGPX的表达量略有下降在8小时的时候表达量为刺激前的0.8倍（P<0.05）。 实验证明栉孔扇贝的超氧化物歧化酶基因CfSOD，过氧化氢酶基因CfCAT，谷胱甘肽过氧化物酶基因CfGPX基因在机体抵抗外界微生物刺激中起到了重要的作用。

Transfer of paralytic shellfish toxins via marine food chains: A simulated experiment

Objective To study the transfer of paralytic shellfish toxins (PST) using four simulated marine food chains: dinoflagellate Alexandrium tamarense -> Arterriia Artemia salina -> Mysid shrimp Neomysis awatschensis; A. tamarense-N. awatschensis: A. taniarense A. salina -> Perch Lateolabrax japonicus; and A. tamarense -> L. japonicus. Methods The ingestion of A. tamarense, a producer of PST, by L. japonicus, N. awatschensis, and A. salina was first confirmed by microscopic observation of A. tamarense cells in the intestine samples of the three different organisms, and by the analysis of Chl.a levels iii the samples. Toxin accumulation in L. japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly ibrough the vector of A. salina was then studied. The toxicity of samples was measured using the AOAC mouse bioassay method, and the toxin content and profile of A. tamarense were analyzed by the HPLC method. Results Both A. salina and N. awatschensis could ingest A. tamarense cells. However, the ingestion capability of A. salina exceeded that of N. awatschensis. After the exposure to the culture of A. tamarense (2 000 cells(.)mL(-1)) for 70 minutes, the content of ChLa in A. salina and N. awatschensis reached 0.87 and 0.024 mu g-mg(-1), respectively. Besides, A. tamarense cells existed in the intestines of L. japonicus, N. awatschensis and A. salina by microscopic observation. Therefore, the three organisms could ingest A. tamarense cells directly. A. salina could accumulate high content of PST, and the toxicity of A. salina in samples collected on days 1, 4, and 5 of the experiment was 2.18, 2.6, and 2.1 MU(.)g(-1), respectively. All extracts from the samples could lead to death of tested mice within 7 minutes, and the toxin content in arternia sample collected on the 1st day was estimated to be 1.65x10(-5) pg STX equa Vindividual. Toxin accumulation in L. japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly froin the vector of A. salina was also studied. The mice injected with extracts from L. japonicus and N. awatschensis samples that accumulated PST either directly or indirectly showed PST intoxication symptoms, indicating that low levels of PST existed in these samples. Conclusion Paralytic shellfish toxins can be transferred to L. japonicus, N. awatschensis, and A. salina from A. taniarense directly or indirectly via the food chains.

模拟增温效应对黑褐苔草（Carex alrofusca）生长特征的影响

在典型矮蒿草草甸上,按大小梯度设置温棚（由小到大依次为A、B、C、D、E）进行模拟增温效应对黑褐苔草（Carex alrofusca）的分蘖数、叶片数和高度影响的研究.结果表明：温度（地表温度和地温）随温室的减小而升高,其中,温室A内地表温度和地温最高,比对照分别提高了2.35℃和2.13℃.温室D的分蘖数增幅最大,温室D与温室A间分蘖数变异达到显著水平（P〈0.05）,温室A至D中黑褐苔草的分蘖数变异随温度的升高而降低,并与温度呈负相关关系（P地表〈0.05;P地温〈0.01）;叶片数的变化趋势与分蘖数基本一致;黑褐苔草平均高度随温度的增加而升高,温室A与对照间达到极显著水平（P〈0.01）,各处理黑褐苔草高度与温度呈正相关且达到极显著水平（P〈0.01）.

甘西鼠尾草一新变型

2004年8月份,我们在巴颜喀拉山南坡考察时,在四川省的壤塘县发现了甘西鼠尾草的一个白花变型,生境为海拔3 350 m的沟谷林缘灌丛,现报道如下.

青藏铁路沿线高寒草原生态质量评价指标体系初探

为了全面和客观评价青藏铁路沿线高寒草原生态环境质量以及生态恢复工程的质量,在实地测定大量样方和统计分析的基础上,选取了盖度、物种多样性指数、丰富度指数、物种数、优势物种分盖度、土壤有机质含量、土壤深度等指标作为评价指标,制定了青藏铁路沿线高寒草原生态环境质量与生态恢复质量的评价指标值.该方法既反映了植被生态环境的量化指标,也考虑了与环境密切相关的植物生长立地表征指标,各项指标相互联系和相关,具有易获得性和实用性,符合当地生态环境质量现状,可以科学、客观地评价区域生态环境质量,对于指导青藏铁路建设中的生态建设与生态建设工程的质量具有实际的指导意义.

不同强度的UV-B辐射对高山植物麻花艽光合作用及暗呼吸的影响

以人工种植的多年生高山植物麻花艽( Gentiana straminea )为材料,在3个不同强度的UV-B辐射处理下,定时测定处理和对照叶片的净光合速率、表观量子效率和暗呼吸的变化.结果显示:UV-B处理对麻花艽叶片的光合作用在短期内有一定的抑制作用,但随着处理时间的增加,该高山植物能很快地适应强UV-B辐射的处理.表明麻花艽这种青藏高原常见的高山植物在长期的自然选择过程中可能已经形成了适应UV-B辐射的特有生理机制.暗呼吸的实验结果亦表明:在3种强度的UV-B辐射处理下,麻花艽叶片的呼吸作用从一开始就未受到抑制;随着UV-B辐射时间的增加,UV-B辐射强度越高,呼吸强度越强;这可能是 UV-B辐射并未引起麻花艽呼吸机构的破坏所致.

人工食物对高原鼠兔稳定性碳和氮同位素组成的影响

以玉米面、小米面和绿豆粉(质量比为5：3：2)的混合食物饲喂青藏高原地区特有种高原鼠兔，40 d后断头杀死，取其后腿肌肉测定稳定性碳和氮同位素组成。结果表明，用混合食物饲喂后，高原鼠兔的稳定性碳同位素比值显著升高，由对照的(一24．660．25)‰上升为(一19．050．09)％。；稳定性氮同位素的组成变化较小，仅由对照的(3 280．13)‰增加为饲喂后的(3．6l0．32)‰。经人工食物饲喂后，高原鼠兔与其食物间稳定性碳同位素的分馏效应为4．73‰o，稳定性氮同位素的分馏效应为2．79％e。c。组分食物的加入影响高原鼠兔的稳定性同位素(特别是氮同位素)代谢模式，其稳定性同位素代谢周转率可能高于原来预测的40 d。

青海湖鸬鹚繁殖成功率初报

2000年3～8月对青海湖西山鸬鹚岛鸬鹚（Phalacrocorax carbo）不同繁殖阶段的成功率做了初步观察。鸬鹚卵的窝卵数为3.47枚，孵化率为37.9%，雏鸟成活率为 61.1%，繁殖成功率为23.2%。孵化期的前3d，鸬鹚巢的损失率高达36.1%；育雏期前10d雏鸟损失率为25%。这些数据可看作鸬鹚种群-青海湖湿地生态系统的一个重要组分——动态监测的起始数据之一，可在未来进一步的比较分析中得到应用。

基于UKF的移动机器人主动建模及模型自适应控制方法

利用基于无色卡尔曼滤波(UnscentedKalmanFilter,UKF)的状态和参数联合估计方法对移动机器人进行在线主动建模,基于该主动模型的逆动力学控制方法,实现了移动机器人对其自身不确定因素的自主性.在针对全方位移动机器人的仿真实验中,验证了UKF对时变的状态和参数的收敛性和跟踪能力,并给出了不确定界.基于主动建模的逆动力学控制方法与常值PID控制方法的比较结果,验证了该方法的有效性.

CIMS递阶控制系统设计

本文首先对CIMS递阶控制结构研究的现状进行了综述及分析,在此基础上, 发展了一种新的CIMS递阶结构参考模型CIMCSA．并进一步提出了自顶向下和自下向上相结合的CIMS递阶控制系统设计方法。

岩溶水系统对大气CO2的潜在影响——基于热力学的研究

认识不同条件下岩溶水释放或吸收CO2 的反应过程是研究碳酸盐岩对碳循环响应的前提和基础。本文从吉布斯自由能的热力学原理出发,对全球不同岩溶地区162 组岩溶水(河水、溪水、湖水等) 进行了热力学研究,结果显示:1) 河水、溪水、湖水和洞穴滴水等岩溶水所处的环境因方解石矿物沉积而释放CO2 成为大气CO2 一个潜在的源;2) 地下水在所处的环境下由于方解石的溶解而吸收CO2 ,成为大气CO2 一个潜在的汇;3) 少数出露点的泉水所处的环境既可发生方解石的溶解而吸收CO2 ,成为大气CO2 的潜在汇,也可发生方解石的沉积而释放CO2 ,成为大气CO2 的潜在源;4) 在洪水期,泉水的水化学特征变化并未导致对大气CO2 潜在贡献在源汇之间的跨跃性转变。162 组岩溶水数据中,所有河水与溪水皆无一例外地在释放CO2 。结果表明,从吉布斯自由能的热力学原理出发,研究岩溶水系统对大气CO2 潜在源汇的贡献,没有条件约束,是一种较好的途径。