肽核酸探针技术在赤潮生物检测中的应用

国家重点基础研究发展项目973(2001CB409704);; 863计划(2003AA635060);; 2004年度厦门市科技创新基金项目(3502Z20041059);; 福建省海洋与渔业局科技项

基于培养基压力控制的组织工程化生物膜动态培养系统的研究

目的研究压力大小可控的培养系统,以实现组织工程化生物膜的动态培养。方法根据帕斯卡定律的基本原理,设计培养基压力的控制方法,建立由培养皿、蠕动泵、压力控制模块、主控器与检测模块等组成的组织工程化生物膜动态培养系统。结果该系统可实现培养基于0.12～19.76 mmHg压力范围内的控制。结论该系统为适宜压力范围内的生物膜培养提供所需的压力环境,为组织工程化生物膜动态培养技术提供新思路

赤潮生物毒素的药理作用研究进展

20 0 4年度厦门市科技创新基金项目 ;; 2 0 0 4年福建省海洋与渔业局科技项目 ;; 国家自然科学基金 (40 0 760 31 ;40 3760 32 )资

Simultaneous determination of 7 nucleosides in Asterias rollestoni using reversed-phase high performance liquid chromatography

建立了罗氏海盘车中7种核苷化合物的反相高效液相色谱分析测定方法。采用超声波辅助提取,选用两根不同的C18色谱柱串联,以甲醇和0.2%(体积分数)乙酸/水溶液为流动相梯度洗脱分离。优化的色谱条件为:柱温为室温,检测波长为260nM,流速为0.8Ml/MIn,进样量为20μl。结果表明,7种核苷化合物在一定的浓度范围内线性关系良好,次黄嘌呤和胸苷的线性范围为0.65~40Mg/l,尿苷、黄嘌呤和肌苷的线性范围为0.80~40Mg/l,胸腺嘧啶的线性范围为1.15~40Mg/l,鸟苷的线性范围为0.50~40Mg/l。样品中7种核苷化合物的加标回收率为90.00%~105.00%,相对标准偏差为0.72%~3.23%。该方法操作简便、灵敏度高、重复性好,回收率高,适用于罗氏海盘车中7种核苷类成分的同时分析,也可用于罗氏海盘车的质量控制和综合评价。A method for the simultaneous determination of 7 nucleosides in Asterias rollestoni was devel-oped using reversed-phase high performance liquid chromatography ( RP-HPLC) .Analytes were extracted by ultrasonic-assisted extraction and separated on two different C18 columns,which were connected in se-ries,under the gradient elution with the mobile phases of methanol and 0.2% ( v/v) acetic acid/water at room temperature.The chromatographic conditions were as follows: flow rate,0.8 mL/min; detection wavelength,260 nm; injection volume,20 μL.Under the optimized conditions,good linear relationships between the values of mass concentrations and the peak areas of hypoxanthine,uridine,xanthine,thy-mine,inosine,guanosine and thymidine were observed in the ranges of 0.65-40,0.80-40,0.80-40,1.15-40,0.80-40,0.50-40,and 0.65-40 mg/L,respectively.The relative standard devia-tions were around 0.72%-3.23% and the recoveries were around 90.00%-105.00%.The results showed that the developed method is sensitive,accurate and reproducible.It is suitable for the analysis of nucleosides in Asterias rollestoni with high recoveries and it is expected to be used for the quality control and evaluation of Asterias rollestoni.国家自然科学基金项目(20905017);海洋公益性行业科研专项(200705011;200805039);海洋局青年基金项目(2010140);海洋一所基本科研业务专项(GY-022008T32;2010G25);中国科学院实验海洋生物学重点实验室开放基金课

1950-2007年黄河入海水沙通量变化趋势及突变特征

基于1950-2007年黄河利津站水沙数据,采用Mann-Kendall检验以及Mann-Whitney-Pettitt(MWP)与贝氏变点分析方法来分析黄河入海水沙通量变化规律,结果表明:黄河全年入海径流通量与泥沙通量分别以-8.1139亿m~3/a和-0.2285亿t/a速率显著减少,汛期变化幅度大于非汛期,尤以泥沙通量为甚;全年以及汛期和非汛期入海径流通量与泥沙通量时序均存在显著转折,且各自变点出现时间不完全一致,全年入海径流通量与泥沙通量时序转折分别发生于1968年、1985年、2002年和1968年、1985年、1996年;入海水沙通量变化趋势与时序变点与流域自然因素变化与人类活动影响密切相关,部分变点出现时间与人类活动介入相吻合

Long-term trend and change point analysis on runoff and sediment fluxes into the sea from the Yellow River during the period of 1950-2007

基于1950-2007年黄河利津站水沙数据,采用Mann-Kendall检验以及Mann-Whitney-Pettitt(MWP)与贝氏变点分析方法来分析黄河入海水沙通量变化规律,结果表明:黄河全年入海径流通量与泥沙通量分别以-8.1139亿m~3/a和-0.2285亿t/a速率显著减少,汛期变化幅度大于非汛期,尤以泥沙通量为甚;全年以及汛期和非汛期入海径流通量与泥沙通量时序均存在显著转折,且各自变点出现时间不完全一致,全年入海径流通量与泥沙通量时序转折分别发生于1968年、1985年、2002年和1968年、1985年、1996年;入海水沙通量变化趋势与时序变点与流域自然因素变化与人类活动影响密切相关,部分变点出现时间与人类活动介入相吻合

基于GIS空间分析的海底表层沉积物粒度分布特征插值研究

为了对比研究不同空间插值方法在沉积物粒度空间分布表述上的精度及适用性,在2004年11月荣成宁津小型海湾-黑泥湾综合调查的基础上,探讨了表层沉积物粒度参数的空间插值,比较了IDW(反距离加权法)、Kriging(克里金插值法)、Spline(样条插值法)与NN(自然邻域法)4种GIS空间分析方法的特征、差异及实效性,并对影响插值结果准确性的因素进行分析。结果显示,在黑泥湾表层沉积物粒度结果的空间插值计算中,从插值准确性和空间表达能力两方面考虑,IDW,Spline,Kriging和NN中以IDW法较为适宜,但要考虑到"牛眼效应"的出现会与局部实际情况存在差异;Kriging法和NN法的插值结果准确性较高,但其空间表达能力稍逊;Spline法在近岸带表层沉积物粒度特征插值中的应用性相对较差。海洋调查要素空间分布的内在规律性是控制插值结果的主要因素,表层沉积物分布以长期稳定的潮流、地形特征为主导因素,呈现由岸向海条带状分布的特征。数据均匀分布区域的插值结果要优于边界区和突变区;另外,在选取的3个观察尺度上,不同插值方法的误差均与野外取样网格间距呈显著线性正相关。

Comparative Study on Method of Amplifying the rRNA of the Genomes from Gymnodinium sp. and Prorocentrum minimum by PCR

以赤潮种裸甲藻(Gymnodiniumsp.)和微小原甲藻(Prorocentrumminimum)为试验材料,以不同方法提取 其基因组并进行纯化,然后采用PCR方法扩增其rRNA基因,包括18S,28S和ITS片断,并进行扩增条件的比较和优化,得到两种藻的最佳DNA提取条件和PCR扩增条件.裸甲藻和微小原甲藻的DNA提取宜采用改良的CTAB 方法;并需对粗提取的DNA用CTAB方法进行纯化.两种藻的最适模板浓度为纯化后模板1 0～2.0μL;最适Mg2+ 浓度为2 0μL(25mmol/L);ITS引物PCR扩增的退火温度为50℃,而18S三对引物的退火温度均为55℃,28S的退 火温度为54℃最为适宜.The genomes from bloom-forming species, Gymnodinium sp.and Prorocentrum minimum were extracted and purified by a series of methods, and the gene fragments of 18S,28S and ITS (Internal Transcribed Spacer) of Gymnodinium sp.and Prorocentrum minimum genomic DNA were amplified by polymerase chain reaction(PCR).All of the extracting methods and PCR condition were optimized by comparing its advantages, and the preferable methodological conditions were achieved. The advisable method of extracting and purifying genome was the improved method of CTAB, and the genomes should be purified by CTAB also.The better concentration of DNA template was 1.0～2.0μL of purified genomes from Gymnodinium sp.and Prorocentrum minimum, and the concentration of Mg~(2+) was 2.0μL of 25 mmol/L in 25μL PCR systems.The advisable annealing temperature was 50℃ to primer pair of ITS, 55℃ to 18S, and 54℃ to 28S respectively in PCR program.国家重点基础研究发展规划973项目(G1999043706);; 国家自然科学基金(40076031;40376032)资助项目

Observation of harmful algal blooms caused by Prorocentrum and Gymnodinium species in the western Xiamen Harbour

对近年来发生在厦门海域的裸甲藻(Gymnodinuum)和原甲藻(Prorocentrum)赤潮的发生情况进行了监测分析,采用了采样、分离、单种培养、显微镜和扫描电镜观察r、DNA序列分析等系列监测、分离培养和赤潮生物鉴定技术,重点观察并确证了厦门海域存在的赤潮原因种为微小原甲藻(Prorocentrum minimum)、Takayama pulchellum、无纹环沟藻(Gyrodinium instriatum)。光学显微镜观察表明,赤潮发生海域存在着许多原甲藻和裸甲藻种类,但不能进一步确认到种。利用电子显微镜观察,可根据微小原甲藻体表规则的花纹等特征,根据Takayama pulchellum具有明显的特异性反S形顶沟等特征分别对它们进行有效地分类鉴定。分子分类学分析表明,T.pulchellum(株名为TPXM)28S rDNA D1-D2区序列长度为721 bp,与基因库中同种相似株的同源性大于99%;微小原甲藻(株名PMDH)的ITS和28S rDNA序列与基因库中同种序列的同源性高达99%;无纹环沟藻(株名GIXM)的ITS与基因库中登记的分离自中国深圳海域的4株同种藻的同源性也高达99%。用ITS序列和28S rDNA序列建立的系统进化树也能很好地显示微小原甲藻、Takayama pulchellum、无纹环沟藻之间以及它们与其他藻之间的亲缘关系。将上述结果结合文献记录和环境条件进行了分析,证实这3种赤潮种类Takayama pulchellum、无纹环沟藻、微小原甲藻是厦门海域较为常见的赤潮原因种。对上述检测和鉴定方法的系统应用也表明,这些方法可应用于对现场赤潮生物进行有效监测。Harmful algal blooms(HABs) is increasing globally,and HABs is one of the major disasters along the coast of Xiamen Harbour.Further investigations and researches on the main types of HABs and the algal species causing HAB in coast water of Xiamen Harbour should be made;the environmental monitoring networks and methods along the coast of Xiamen Harbour should be set up and consummated;science researches with local characteristics should be fully developed,and the exotic HAB species from the cabin seawater of foreign ships should also be studied.Aiming directly at rapid identification and regular monitoring of HABs species,this paper tried to develop essential methods of identificating and monitoring HABs species,and evaluated systemic methods for rapidly monitoring and classifying HABs species in morphology and molecular taxonomy.Meanwhile,series of studies were also undertaken to evaluate in the field and tried to find these important HABs causing species along the coast water of western Xiamen Harbour.The harmful algal bloom events caused by Prorocentrum and Gymnodinium species in coast water of western Xiamen Harbour were monitored during recent years.A series of detecting methods based on the analysis of environmental factors,sampling,cell isolation and unicellular culture of these HABs species,observation under microscope and scanning electron microscope(SEM),and sequence analysis of rDNA were used together to monitor and identify these harmful algae.The methods used for collecting samples and establishing specific HAB unicellular cultures were described in detail.Methods of SEM(scanning electron microscope) and ESEM(environmental scanning electron microscope) for Gymnodinium were developed and optimized.These important species that caused harmful algal bloom in Xiamen Harbour were observed and identified as Takayama pulchellum(strain name:TPXM),Gyrodinium instriatum(strain name:GIXM) and Prorocentrum minimum(strain name:PMXM).Algal morphology was observed under LM,and the results suggested that there were many Prorocentrum and Gymnodinium species in coast water of western Xiamen Harbour,and these species could not be identified as specific species under LM.Two strain of Prorocentrum and Gymnodinium were isolated during blooms in Xiamen Harbor and their morphology were observed under SEM.The results showed that there were regular patterns and other characteristics on Prorocentrum cells,which indicated that PMXM was Prorocentrum minimum,and cells of TPXM were broadly oval with a conspicuous and well-defined sigmoid apical groove present on the epitheca,and the apical groove was a characteristic reversed S-shape.These characteristics showed that TPXM was Takayama pulchellum.Molecular biological analyses of rDNA sequences of Prorocentrum and Gymnodinium showed that the D1-D2 region of partial LSU(large subunit) rDNA of T.pulchellum(TPXM) had 721bp and shared more than 99% similarity to the same species whose data was deposited in the GenBank,the ITS and D1-D2 region of partial LSU of P.minimum(PMXM) also shared more than 99% similarity to the same species in the GenBank,and ITS region of G.instriatum(GIXM) shared more than 99% similarity to the same species isolated from Shenzhen Bay in China and its sequences were deposited in the GenBank,too.The analysis of rDNA sequences and phylogenetic trees could be an appropriate taxonomic evidence,especially the length and whole sequences analysis of ITS and variable regions D1-D2 of 28S rDNA could be more reliable and could provide more exact taxonomic informations than other methods.The phylogenetic trees constructed from ITS and partial 28S rDNA could show the relationships of T.pulchellum(TPXM),G.instriatum(GIXM) and P.minimum(PMXM) and their correlative species,and could also separate these species from their disrelated species clearly.According to above results,the characteristics and causation of HABs in western Xiamen Harbour caused by the three species were discussed based on the analysis of related references and environmental conditions.All the results suggested that the three HABs causing species,such as T.pulchellum(TPXM),G.instriatum(GIXM),P.minimum (PMXM) were common HAB species in coast water of western Xiamen Harbour.The results of methodological application in field indicated that these methods and protocols might become useful,feasible and reliable tools to monitor in situ distribution of bloom-forming taxa,such as Gymnodinium and Prorocentrum in natural water.国家重点基础研究发展规划973项目(CEOHAB2001CB409704);; 厦门市创新科技基金项目(3502Z20041059);; 中国博士后基金项目(20060400854);; 农业部淡水鱼类种质资源与生物技术重点实验室开放课题(LFB20070611