不同磷条件对安氏伪镖水蚤的生长及摄食的影响

实验研究了海洋浮游动物桡足类安氏伪镖水蚤(Pseudodiaptomus annandalei)在分别喂食高磷和低磷扁藻(Platymonas subcordiformis)时的个体生长发育和摄食的情况。低磷扁藻(C/P=197或375,摩尔比)喂食安氏伪镖水蚤,安氏伪镖水蚤生长速率分别为g=(0.0437±0.0030)/d和g=(0.0339±0.0049)/d,高磷扁藻(C/P=78,摩尔比),安氏伪镖水蚤的生长速率为g=(0.1040±0.0395)/d。另外,从无节幼体到桡足幼体的发育所需的时间延长了1.5—2倍。就蚤体磷含量而言,在高磷条件下,个体的磷含量为干重的0.30%—0.33%,处于低磷喂养条件下的个体磷含量仅为0.08%—0.28%。结合生长速率和蚤体磷含量分析表明二者呈显著相关的线性关系。最后,在低磷条件下,无节幼体、桡足幼体和成体三个生长阶段的安氏伪镖水蚤个体的摄食率分别为150—370、695—703、648—733 cells/(ind?h),明显低于高磷条件下的790、720、728 cells/(ind?h)。据此得到结论:在磷限制条件下培养的饵料藻导致其营养价值发生了变化(C/P和C/N比值),对安氏伪镖水蚤的生长发育以及摄食率产生了显著负面影响,表明海洋桡足类安氏伪镖水蚤亦受到磷的限制。     

DDT对火腿伪镖水蚤（Pseudodiaptomus poplesia）急性和慢性毒性效应

为探讨内分泌干扰物DDT对海洋桡足类生物所产生的生物效应,研究了DDT对火腿伪镖水蚤(Pseudodiaptomus poplesia)的急性毒性作用及其在3.5、35、350ng.L-1浓度下对火腿伪镖水蚤变态率、存活率、繁殖力、性别比和体长的影响。结果表明:DDT对火腿伪镖水蚤48hLC50和96hLC50分别为6.75和3.53μg.L-1;各浓度DDT均引起火腿伪镖水蚤幼体变态率的降低;350ng.L-1DDT条件下,火腿伪镖水蚤的存活率、产卵率和抱卵雌体比率均显著降低;各浓度DDT对火腿伪镖水蚤的性别比和体长均未造成显著影响。依据本研究结果,结合目前所报道的近岸水体有机氯农药的污染状况,可以推断,DDT对近海桡足类生物造成的生态风险较高。     

三座南亚热带大型水库敞水区桡足类群落结构比较

于2000-2002年对南亚热带三座大型水库:新丰江水库、公平水库和飞来峡水库敞水区桡足类进行了定性定量采样.三座水库桡足类种类数较少,共检出桡足类4科10属14种,其中8种哲水蚤,5种剑水蚤和1种猛水蚤.主要分布在广东省水库的我国特有种舌状叶镖水蚤(Phyllodiaptomus tunguidus)和广布性种类右突新镖水蚤(Neodiaptomus schmackeri)是出现频率较高的哲水蚤种类,温中剑水蚤(Mesocyclops thermocyclopoides)、台湾温剑水蚤(Thermocyclops taihokuensis)和博平近剑水蚤(Tropocycl opsbopingi)是出现频率较高的剑水蚤种类.水库的流域面积、滞留时间、鱼类捕食和营养状态是影响水库桡足类种类组成和数量的重要因素.三座水库桡足类种类数与水库流域面积大小存在正相关关系.随着水库营养水平的升高,桡足类密度和生物量也相应增加,但是大型哲水蚤的优势度降低,而剑水蚤和桡足类幼体的优势度上升.水力滞留时间很短是引起飞来峡水库桡足类种群波动的主要因素.水库捕食性鱼类对甲壳浮游动物的选择性捕食,对桡足类种类影响较小,对桡足类成体密度影响较大.由于地区差异和鱼类捕食强度等因素的影响,桡足类种类多样性与水体营养状态之间存在不确定性.     

浙江千岛湖桡足类的群落结构

1999年1－12月,研究了贫－中营养型的大型深水湖泊－浙江千岛湖浮游桡足类的群落结构,包括其种类组成、现存量动态及群落多样性。在一年的研究中,共观察到16种桡足类（5种哲水蚤、1种猛水蚤和10种剑水蚤）。根据各种桡足类（桡足幼体＋成体）的年平均密度,哲水蚤的优势种为右突新镖水蚤（Neodiaptomus schmack-eri)和球状许水蚤（Schmackeria forbesi);剑水蚤的优势种为一种中剑水蚤（Mesocyclops notius)和近邻剑水蚤（Cyclops vicinus)。桡足类群落平均密度46.81个／L,生物量0.126mg/L,物种多样性指数1.54。桡足类现存量的季节变化为夏＞秋＞冬＞春,8月份密度达98.50个／L,生物量0.333mg/L;5月份密度仅10.70个／L,生物量0.017mg/L。水平变化以Ⅱ号站现存量为最高,密度达80.10个／L,生物量0.289mg/L;V号站的密度最低,仅29.80个／L;Ⅳ号站生物量最低,0.066mg/L。多样性指数以I站为最高3.21,Ⅸ站最低,仅0.85。桡足类群落多样性随着水体透明度的增大呈下降的趋势。     

四川省的桡足类(下)

四川桡足类区系的特点由于四川的地形复杂,桡足类的种群繁多,目前已知53种,占全国已知206种的25.73%,占全国纯淡水种类的33.76%。凉山荡镖水蚤为我省特有种。53种桡足类中,以广温性种占绝对优势,其次是南方热带性种,北方寒带性种较少。这与四川地跨东洋界和古北界,全省气候比较温和有关。哲水蚤目虽是浮游性种类,但可能由于它的躯体较大,对环境、的适应性较弱,因此地域性较强。如我省特有种只在雷波马湖采得,其他地区还未发现。但也有一些分布延伸的现象,如近方蒙镖水蚤,原仅发现于西藏和新疆,海拔高度在1040—4000米,现在我省南溪县的池塘…     

浅水草型湖泊──扁担塘中桡足类的群落结构（英文）

本文研究了一浅水草型湖泊──扁担塘中桡足类的群落结构,包括种类组成、种群动态及现存量。在一周年的研究中,共发现14种浮游桡足类（9种剑水蚤和5种哲水蚤）。根据年平均密度,剑水蚤的优势种为：Mesocyclops notius,Cyclops vincinuis vincinus和Thermocyclops brevifurcatus,而哲水蚤的优势种为Meodiaptomus yantsekiangensis和Sinocalanus dorrii。通过比较长江沿岸的5个湖泊的桡足类的种类组成发现,桡足类的种类数并不与湖泊面积大小成正比,这与Dodson通过研究欧洲及北美湖泊中的枝角类得出的结论并不一致。此外,虽然这5个湖泊中的桡足类的种类数多达35种,但每个湖泊中的优势种都非常相似地集中到少数几个属或种,而其它均为偶尔性种类。     

摄食信息对球形棕囊藻和布氏双尾藻竞争结果的影响

浮游动物的摄食信息能增大棕囊藻囊体体积,囊体形成被认为是棕囊藻的诱导性防御机制。利用桡足类火腿伪镖水蚤和异养甲藻海洋尖尾藻释放的摄食信息,研究了诱导性防御对球形棕囊藻和布氏双尾藻的竞争的影响。结果表明,球形棕囊藻接收了火腿伪镖水蚤和海洋尖尾藻释放的摄食信息之后形成更大的囊体。防御启动后的球形棕囊藻比未接收摄食信息的球形棕囊藻更快地形成囊体,且囊体维持的时间更长。对照组和火腿伪镖水蚤摄食信息诱导的球形棕囊藻的生物体积比布氏双尾藻更高,且球形棕囊藻在竞争中占优势;而海洋尖尾藻摄食信息诱导的球形棕囊藻生物体积低于布氏双尾藻,且球形棕囊藻相对布氏双尾藻的竞争力下降。微型浮游动物海洋尖尾藻摄食信息导致球形棕囊藻相对硅藻布氏双尾藻的竞争力的下降,有利于解释硅藻先于棕囊藻发生藻华。     

东亚三角涡虫cDNA文库的构建及EST初步分析

为了快速高效地分离鉴定东亚三角涡虫的发育和再生相关基因,以总RNA为模板,借助CreatorTM SMARTTM cDNA Library Construction Kit和Advantage 2 PCR Kit成功构建了东亚三角涡虫的cDNA文库.经测定,cDNA文库原始库容为1.22×105个独立克隆,重组率超过98 %,插入片段平均大小为900 bp.从文库中随机挑选重组克隆测序,并在NCBI数据库中比对,结果获得208个rRNA基因,148个编码蛋白基因,78个染色体片段基因.该研究为我国涡虫发育和再生的深入研究奠定了坚实的分子基础.     

长江口桡足类数量分布与变动

于1999年枯水期(2～3月份)、丰水期(8月份)、2000年枯水期(2～3月份)对长江河口浮游动物桡足类采样调查,研究了长江河口桡足类的数量分布与变动.结果表明:1999年枯水期,桡足类在整个长江河口区的平均数量相对不大,为76ind/m3,但却占同期浮游动物平均数量的95.61%;1999年丰水期桡足类平均数量为254ind/m3,占同期浮游动物平均数量的84.29%;2000年枯水期桡足类平均数量为97ind/m3,占同期浮游动物总数量的84.46%.从优势度看,1999、2000年枯水期主要优势种为华哲水蚤(Sinocalanus sinensis);1999年丰水期主要优势种为火腿许水蚤(Schmackeria poplesia).虫肢歪水蚤(Tortanus vermiculus)、真刺唇角水蚤(Labidocera euchaeta)则在枯、丰水期均为优势种.从种类数看,桡点类在1999年、2000年枯水期均为14种,1999年丰水期为枯水期的近2倍,达25种.对于长江河口主要桡足类而言,华哲水蚤的季节变化明显,适宜生活在盐度较低水域.虫肢歪水蚤数量年际变化较大,其适盐范围比华哲水蚤较宽、较高.真刺唇角水蚤的适盐范围与虫肢歪水蚤相似且更高一些,但该种在枯水期数量较少,丰水期数量较多,变化显著,更适宜在较高温时生长.火腿许水蚤适盐范围宽,能适应很大范围盐度变化,枯水期数量少,丰水期数量大,较高温度生长良好.     

水体化学要素对分叉小猛水蚤(Tisbe furcata)和小拟哲水蚤(Paracalanus parvus)室内养殖种群密度的影响

以分叉小猛水蚤(Tisbe furcata)和小拟哲水蚤(Paracalanus parvus)为研究对象, 对比室内养殖条件下两种桡足类的种群密度差异, 并分析水体化学要素亚硝态氮、硝态氮、氨氮和磷酸盐与桡足类种群密度的关系。结果表明: 分叉小猛水蚤较小拟哲水蚤种群密度优势明显(P<0.01), 更适合北方室内养殖, 盐度可能起主要作用; 水体亚硝态氮最为重要, 在桡足类种群的快速增长期, 其浓度与两种桡足类的种群密度均呈极显著正相关性; 水体磷酸盐浓度与两种桡足类的种群密度均呈极显著正相关性; 通过部分换水的方式可以实现分叉小猛水蚤的可持续培养, 换水量不要超过水体总量的1/3; 本研究所采用复合饲料和养殖方式可以实现分叉小猛水蚤的北方室内大规模、高密度和可持续养殖。     

Differential gene expression profile of the calanoid copepod, Pseudodiaptomus annandalei, in response to nickel exposure

To better understand the underlying mechanisms of reactions of copepods exposed to elevated level of nickel, the suppression subtractive hybridization (SSH) was used to elucidate the response of the copepod Pseudodiaptomus annandalei to nickel exposure at the gene level. P. annandale is one of a few copepod species that can be cultured relatively easy under laboratory condition, and it is considered to be a potential model species for toxicity study. In the present study, P. annandalei were exposed to nickel at a concentration of 8.86 mg L^− 1 for 24 h, after which the RNA was prepared for SSH using unexposed P. annandalei as drivers. A total of 474 clones on the middle scale in the SSH library were sequenced. Among these genes, 129 potential functional genes were recognized based on the BLAST searches in NCBI and Uniprot databases. These genes were then categorized into nine groups in association with different biological processes using AmiGO against the Gene Ontology database. Of the 129 genes, 127 translatable DNA sequences were predicted to be proteins, and the putative amino acid sequences were searched for conserved domains (CD) and proteins using the CD-Search service and BLASTp. Among 129 genes, 119 (92.2%) were annotated to be involved in different biological processes, while 10 genes (7.8%) were classified as an unknown-function gene group. To further confirm the up-regulation of differentially expressed genes, the quantitative real time PCR were performed to test eight randomly selected genes, in which five of them, i.e. α-tubulin, ribosomal protein L13, ferritin, separase and Myohemerythrin-1, exhibited clear up-regulation after nickel exposure. In addition, MnSOD was further studied for the differential expression pattern after nickel exposure and the results showed that MnSOD had a time- and dose-dependent expression pattern in the copepod after nickel exposure. To the best of our knowledge, this is the first attempt to investigate the toxicity effects of nickel on a copepod at molecular level.     

应用抑制性差减杂交法分离法分离阜豆苗期根系镉胁迫诱导表达的基因

以1/2Hoagland溶液培养的阜豆幼苗根系为对照群体,含Cd2+浓度为100μM营养液处理的为目标群体,进行抑制差减杂交。用经过对照组cDNA差减目标组cDNA构建了一个含有大约600个独立克隆的差减文库。随机挑取部分克隆进行菌落PCR鉴定,表明插入的片段大小均在250～800 bp。对已确认的6个阳性克隆测序,序列分析和同源性比较,表明它们与镉胁迫有关。     

Single subunit type of ferritin from visceral mass of Saccostrea cucullata: cloning, expression and cisplatin-subunit analysis

Ferritin, the iron storage protein, plays a key role in iron metabolism. Here, we have cloned an inducible ferritin cDNA with 516 bp within the open reading frame fragment from the visceral mass of Saccostrea cucullata. The subunit sequence of the ferritin was predicted to be a polypeptide of 171 amino acids with a molecular weight (MW) of 19.9182 kDa and an isoelectric point of 5.24. The cDNA sequence of S. cucullata ferritin was constructed into a pET-32a expression system for expressing its relative protein efficiently in the Escherichia coli BL21 strain under isopropyl-β-D-thiogalactoside (IPTG) induction. The recombinant ferritin, which was further purified on a Ni-NTA resin column and digested with enterokinase, was detected as a single subunit of approximately MW 20 kDa using both SDS-PAGE and mass spectrometry. S. cucullata ferritin (ScFer) showed 98% identity with Crassostrea gigas ferritin at the amino acid level. The secondary structure and phosphorylation sites of deduced amino acids were predicted with ExPASy proteomics tools and the NetPhos 2.0 server, respectively, and the subunit space structure of recombinant S. cucullata ferritin (rScFer) was built using the molecular operating environmental software system. The results of both in-gel digestion and identification using MALDI-TOF MS/MS showed that the recombinant protein was ScFer. ICP-MS indicated that rScFer subunit can directly bind to cisplatin[cis-Diaminedichloroplatinum(CDDP)], giving approximately 22.9 CDDP/ferritin subunit for forming a novel complex of CDDP-subunit, which suggests that it constructs a nanometer CDDP core-ferritin for developing a new drug of anti-cancer. The results of both the real-time PCR and Western blotting showed that the expression of ScFer mRNA was up-regulated in the oyster under the stress of Cd(2+). In addition, the expression increment of ScFer mRNA under bacterial challenge indicated that ferritin participated in the immune response of S. cucullata. The recombinant ScFer should prove to be useful for further study of the structure and function of ferritin in S. cucullata.     

Cloning analysis of ferritin and the cisplatin-subunit for cancer cell apoptosis in Aplysia juliana hepatopancreas

Ferritin, an iron storage protein, plays a key role in iron metabolism in vivo. Here, we have cloned an inducible ferritin cDNA with 519 bp within the open reading frame fragment from the hepatopancreas of Aplysia juliana (AJ). The subunit sequence of the ferritin was predicted to be a polypeptide of 172 amino acids with a molecular mass of 19.8291kDa and an isoelectric point of 5.01. The cDNA sequence of hepatopancreas ferritin in AJ was constructed into a pET-32a system for expressing its relative protein efficiently in E. coli strain BL21, under isopropyl-β-d-thiogalactoside induction. The recombinant ferritin, which was further purified on a Ni-NTA resin column and digested with enterokinase, was detected as a single subunit of approximately 20 kDa mass using both SDS-PAGE and mass spectrometry. The secondary structure and phosphorylation sites of the deduced amino acids were predicted using both ExPASy proteomic tools and the NetPhos 2.0 server, and the subunit space structure of the recombinant AJ ferritin (rAjFer) was built using a molecular operating environment software system. The result of in-gel digestion and identification using MALDI-TOF MS/MS showed that the recombinant protein was AjFer. ICP-MS results indicated that the rAjFer subunit could directly bind to cisplatin[cis-Diaminedichloroplatinum(CDDP)], giving approximately 17.6 CDDP/ferritin subunits and forming a novel CDDP-subunit. This suggests that a nanometer CDDP core-ferritin was constructed, which could be developed as a new anti-cancer drug. The flow cytometry results indicated that CDDP-rAjFer could induce Hela cell apoptosis. Results of the real-time PCR and Western blotting showed that the expression of AjFer mRNA was up-regulated in AJ under Cd(2+) stress. The recombinant AjFer protein should prove to be useful for further study of the structure and function of ferritin in Aplysia.     

疫霉侵染下辣椒幼苗消减文库的构建与初步分析

为了分离和鉴定辣椒中疫霉诱导基因,以高抗疫霉病辣椒品种L11为材料,以接种辣椒疫霉菌的幼嫩叶片为处理（tester）,以未接种自然生长的幼嫩叶片为对照(driver),利用抑制性消减杂交技术(suppression subtractive hybridization,SSH)构建了疫霉侵染下辣椒幼苗的消减文库。从消减文库中随机挑取30个阳性克隆,提取质粒进行PCR鉴定,显示插入片段大小大部分集中在200～1 000 bp之间,文库质量良好。随机挑取40个克隆进行测序,共获得35个有效EST序列。经Blastx分析表明：有30个EST与GenBank中其他序列有同源性,5个EST为未知功能序列。已知功能的EST序列分别编码NAC转录因子、丝氨酸/苏氨酸蛋白激酶、P450单加氧酶、叶绿素a/b结合蛋白、谷胱甘肽转移酶、几丁质酶等,这些蛋白涉及抗病信号传递、抗氧化作用、转录调控及光合作用等多种生理过程。本研究为抗病基因克隆和系统研究疫霉侵染下辣椒基因的表达奠定了重要的理论基础。     

Characterization, cloning and expression of the ferritin gene from the Korean polychaete, Periserrula leucophryna

Ferritin is a major eukaryotic protein and in humans is the protein of iron storage. A partial gene fragment of ferritin (255 bp) taken from the total RNA of Periserrula leucophryna, was amplified by RT-PCR using oligonucleotide primers designed from the conserved metal binding domain of eukaryotic ferritin and confirmed by DNA sequencing. Using the 32P-labeled partial ferritin cDNA fragment, 28 different clones were obtained by the screening of the P. leucophryna cDNA library prepared in the Uni-ZAP XR vector, sequenced and characterized. The longest clone was named the PLF (Periserrula leucophryna ferritin) gene and the nucleotide and amino acid sequences of this novel gene were deposited in the GenBank databases with accession numbers DQ207752 and ABA55730, respectively. The entire cDNA of PLF clone was 1109 bp (CDS: 129-653), including a coding nucleotide sequence of 525 bp, a 5'-untranslated region of 128 bp, and a 3'-noncoding region of 456 bp. The 5'-UTR contains a putative iron responsive element (IRE) sequence. Ferritin has an open reading frame encoding a polypeptide of 174 amino acids including a hydrophobic signal peptide of 17 amino acids. The predicted molecular weights of the immature and mature ferritin were calculated to be 20.3 kDa and 18.2 kDa, respectively. The region encoding the mature ferritin was subcloned into the pT7-7 expression vector after PCR amplification using the designed primers and included the initiation and termination codons; the recombinant clones were expressed in E. coli BL21(DE3) or E. coli BL21(DE3)pLysE. SDS-PAGE and western blot analysis showed that a ferritin of approximately 18 kDa (mature form) was produced and that by iron staining in native PAGE, it is likely that the recombinant ferritin is correctly folded and assembled into a homopolymer composed of a single subunit.     

利用抑制性扣除杂交技术克隆水稻磷饥饿诱导基因

磷素是植物生长所必需的重要元素。在缺磷环境中,植物能够调节自身的形态、生理生化和基因表达水平来适应环境的变化。为研究水稻(Oryzn sativa L.)耐低磷胁迫的分子机理,采用抑制性扣除杂交技术(SSH)构建磷饥饿诱导的水稻根系扣除cDNA文库。通过文库筛选和测序获得18个已知基因和47个功能未知基因。这些基因参与了不同的代谢过程,包括磷吸收和转运、信号传导、蛋白质合成和降解、碳水化合物代谢和胁迫反应。Northern杂交结果表明,在磷饥饿胁迫下这些基因呈现不同的表达模式,并且不同代谢过程中的基因对磷饥饿有着不同的反应。     

中间锦鸡儿CiABR17基因的克隆及表达分析

该研究采用blastn比对方法,从中间锦鸡儿干旱胁迫抑制消减杂交文库筛选出ABA响应蛋白17基因(ABR17)的EST序列。RACE扩增得到cDNA序列594bp,其中ORF为471bp,编码157个氨基酸,预测蛋白质分子量为16.44kD,等电点为4.93,平均疏水指数为-0.126,是一种亲水蛋白。实时荧光定量PCR检测发现,中间锦鸡儿CiABR17基因在脱水、NaCl和热胁迫下均有不同程度表达,说明CiABR17基因与中间锦鸡儿响应逆境胁迫有关;过表达CiABR17拟南芥株系的鲜重、干重和莲座直径均极显著高于野生型株系。