Optimization of triploid induction by heat shock in Chinese shrimp Fenneropenaeus chinensis

Chromosome manipulation for commercially valuable marine animals plays an important role in aquaculture. The special reproductive characteristics of shrimp make it difficult to control fertilization and synchronize egg development, so research on chromosome manipulation in shrimp has proceeded very slowly. In the present study, triploid shrimp Fenneropenaeus chinensis were induced by heat shocks and the optimal-inducing condition was screened at different spawning temperatures. Level of triploid induction for each treatment was evaluated by flow cytometry at nauplius stage. The highest level of triploid induction reached to more than 90%. Starting time for each treatment was very crucial for triploid induction in shrimp. One optimal treatment condition for triploid induction was heat shock (29-32 degreesC), starting at 18-20 min for duration of 10 min. These conditions varied depending on the temperature at spawning. Triploid level at embryo stage and nauplius stage was not different, suggesting the same hatching rate between diploids and triploids. Heat shock is a very effective way to induce triploids in this species, and can be easily used on large scale without any harmful effect on the environment as compared with chemical treatment. (C) 2003 Elsevier Science B.V. All rights reserved.

Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis (vol 14, pg 307, 2002)

The geneswere cloned for the two apoprotein subunits, alpha and beta, of phycocyanin from the cyanobacterium Spirulina maxima (=Arthrospira maxima) strain F3. The alpha- and beta-subunit gene-coding regions contain 489 bp and 519 bp, respectively. The beta-subunit gene is upstream from the alpha-subunit gene, with a 111-bp segment separating them. Similarities between the alpha-subunits of S. maxima and nine other cyanobacteria were between 58% and 99%, as were those between the beta-subunits. The maximum similarity between the alpha- and beta-subunits from S. maxima was 27%.

Diversity of pigment-protein complexes of photosynthetic organisms.

PS I, PS II and light-harvesting complexes (LHC) in oxygen evolving photosynthetic organisms were reviewed. These organisms include cyanobacteria, red algae, brown algae, diatoms, chrysophytes, dinophytes, xanthophytes, crypophytes, green algae and green plants. The diversity of pigment-protein complexes that fuel the conversion of radiant energy to chemical bond energy was highlighted, and the evolutionary relationships among the LHC structural polypeptides and the characteristics of the fluorescence emission of PS I at 77 K was discussed.

Development of rRNA and rDNA-targeted probes for fluorescence in situ hybridization to detect Heterosigma akashiwo (Raphidophyceae)

Heterosigma akashiwo (Hada) is a fragile, fish-killing alga. Efforts to understand and prevent blooms due to this harmful species to mitigate the impact on aquaculture require the development of methods for rapid and precise identification and quantification, so that adequate warning of a harmful algal bloom may be given. Here, we report the development and application of rRNA and rDNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection and enumeration of H. akashiwo. The designed probes were species specific, showing no cross-reactivity with four common HAB causative species: Prorocentrum micans Ehrenberg, P. minimum (Pavillard) Schiller, Alexandrium tarmarense (Lebour) Balech, and Skeletonema costatum (Greville) Cleve, or with four other microalgae, including Gymnodinium sp. Stein, Platy-monas cordiformis (Karter) Korsch, Skeletonema sp.1 Greville and Skeletonema sp.2. The rRNA-targeted probe hybridized to cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, while cells labeled by rDNA-targeted probe exhibited exclusively fluorescent nucleus. The detection protocols were optimized and could be completed within an hour. For rRNA and rDNA probes, about a corresponding 80% and 70% of targeted cells could be identified and quantified during the whole growth circle, despite the inapparent variability in the average probe reactivity. The established FISH was proved promising for specific, rapid, precise, and quantitative detection of H. akashiwo. (C) 2008 Elsevier B.V. All rights reserved.

Phycobilisomes linker family in cyanobacterial genomes: divergence and evolution

Cyanobacteria are the oldest life form making important contributions to global CO2 fixation on the Earth. Phycobilisomes (PBSs) are the major light harvesting systems of most cyanobacteria species. Recent availability of the whole genome database of cyanobacteria provides us a global and further view on the complex structural PBSs. A PBSs linker family is crucial in structure and function of major light-harvesting PBSs complexes. Linker polypeptides are considered to have the same ancestor with other phycobiliproteins (PBPs), and might have been diverged and evolved under particularly selective forces together. In this paper, a total of 192 putative linkers including 167 putative PBSs-associated linker genes and 25 Ferredoxin-NADP oxidoreductase (FNR) genes were detected through whole genome analysis of all 25 cyanobacterial genomes (20 finished and 5 in draft state). We compared the PBSs linker family of cyanobacteria in terms of gene structure, chromosome location, conservation domain, and polymorphic variants, and discussed the features and functions of the PBSs linker family. Most of PBSs-associated linkers in PBSs linker family are assembled into gene clusters with PBPs. A phylogenetic analysis based on protein data demonstrates a possibility of six classes of the linker family in cyanobacteria. Emergence, divergence, and disappearance of PBSs linkers among cyanobacterial species were due to speciation, gene duplication, gene transfer, or gene loss, and acclimation to various environmental selective pressures especially light.

Molecular cloning, characterization and evolutionary analysis of phytoene desaturase (PDS) gene from Haematococcus pluvialis

Phytoene desaturase is one of the most important enzymes necessary for the biosynthesis of carotenoids in some cyanobacteria, green algae and plants. In this study, genomic DNA and cDNA of pds were cloned from unicellular green alga Haematococcus pluvialis strain323 using PCR and RT-PCR methods. The cDNA was cloned into plasmid pET-28a and efficiently expressed in Escherichia coli BL21. The complete genomic PDS gene of H. pluvialis, 3.3 kb in size, included eight exons and seven introns. To locate transcriptional regulation elements, an approximate 1 kb of 5'-flanking region was isolated by genome-walking method. Results of bioinformatic analysis showed several putative cis-elements e.g. the ABRE motif (abscisic acid responsive element), the C-repeat/DRE (dehydration responsive element) motif and the GCN4 motif were located in 5'-flanking region of pds. Results of phylogenetic analyses reveal that different sources of PDS genes form a separate clade, respectively, with 100% bootstrap support. Moreover, a maximum likelihood approach was employed to detect evidence of positive selection in the evolution of PDS genes. Results of branch-site model analysis suggest that 7.9% of sites along the green algal branch are under positive selection, and the PDS gene in green algae exhibits a different evolutionary pattern from its counterparts in cyanobacteria and plants.

The recombination and expression of the allophycocyanin beta subunit gene in the chloroplast of Chlamydomonas reinhardtii

A Chlamydomonas reinhardtii (C. reinhardtii) chloroplast expression vector, papc-B, containing the apc-B gene that encodes the beta subunit of the light-harvesting antenna protein allophycocyanin (APC) of cyanobacteria, was constructed and transferred to the chloroplast genome of C. reinhardtii by the biolistic method. The transformants were identified by Southern blot, Western blot and ELISA assays after selection on resistant medium. The recombinant APC beta subunit was expressed in the C. reinhardtii chloroplast and accounted for up to 2-3% (w/w) of the total soluble protein (TSP), suggesting a promising prospect of using C. reinhardtii chloroplasts to produce functional plant-derived proteins.

Evolutionary analysis of phycobiliproteins: Implications for their structural and functional relationships

Phycobiliproteins, together with linker polypeptides and various chromophores, are basic building blocks of phycobilisomes, a supramolecular complex with a light-harvesting function in cyanobacteria and red algae. Previous studies suggest that the different types of phycobiliproteins and the linker polypeptides originated from the same ancestor. Here we retrieve the phycobilisome-related genes from the well-annotated and even unfinished cyanobacteria genomes and find that many sites with elevated d(N)/d(S) ratios in different phycobiliprotein lineages are located in the chromophore-binding domain and the helical hairpin domains (X and Y). Covariation analyses also reveal that these sites are significantly correlated, showing strong evidence of the functional-structural importance of interactions among these residues. The potential selective pressure driving the diversification of phycobiliproteins may be related to the phycobiliprotein-chromophore microenvironment formation and the subunits interaction. Sites and genes identified here would provide targets for further research on the structural-functional role of these residues and energy transfer through the chromophores.

Distribution and mobility of metals in agricultural soils near a copper smelter in South China

The distribution and mobility of heavy metals in the paddy soils surrounding a copper smelting plant in south China was investigated. We assessed the degree of metal contamination using an index of geoaccumulation. The metals were divided into two groups: (1) Cu, Zn, Pb and Cd, whose concentrations were heavily affected by anthropogenic inputs, and (2) Ni, Co and Cr, which were mainly of geochemical origin. Concentrations of Cu, Cd, Zn, and Pb in the polluted soils were higher than the Chinese soil quality criteria. The chemical partitioning patterns of Pb, Zn and Cu indicated that Pb was largely associated with the residual and NH2OH HCl extractable fractions. In contrast, Cd was predominantly associated with the MgCl2 extractable fraction. A large proportion of Cu was bound to the acidic H2O2 extractable fractions, while Zn was predominantly found in the residual phase. The fraction of mobile species, which potentially is the most harmful to the environment, was found to be elevated compared to unpolluted soils in which heavy metals are more strongly bound to the matrix. The mobility of the metals was studied by water extraction using a modification of Tessier's procedure, and the order of mobility was Zn > Cd > Cu > Ce > Pb.

东海大规模赤潮对微型浮游动物群落结构影响的研究

本文从赤潮发生前的现场模拟实验、赤潮发生时的现场培养实验和赤潮发生过程中的现场调查等方面，较为系统地研究了东海大规模赤潮对微型浮游动物群落结构的影响。 2005年长江口及邻近海区赤潮发生前，赤潮973MC2005－03航次在本海区进行了综合调查，期间分别于4月27日、5月4日和5月8日，在zzf1、zc18a和ra5三个站位利用现场船基培养的方法，研究了添加到赤潮密度106 cells L-1的东海原甲藻（Prorocentrum donghaiense）对微型浮游动物群落结构的影响。结果发现，赤潮密度的东海原甲藻对小型无壳纤毛虫的种群数量影响较大，而对中大型砂壳纤毛虫的影响较小，从而使得微型浮游动物群落有向中大型砂壳纤毛虫演替的趋势。在zzf1站位，小型无壳纤毛虫占绝对优势，添加东海原甲藻72 h后，优势种由管游虫（Cyrtostrombidium sp.）演替为另一种小型无壳纤毛虫急游虫4（Strombidium sp.4）；在以中型砂壳纤毛虫百乐拟铃虫（Tintinnospsis beroidea）为优势种的zc18a站位，仍然是以此为优势种，但其在群落中的优势度更加明显；ra5站位也是以小型无壳纤毛虫为主，添加东海原甲藻72 h后，其群落结构由急游虫2（Strombidium sp.2）向中大型砂壳纤毛虫纤毛虫百乐拟铃虫和亚速岛网纹虫（Favella azorica）演替。不同站位微型浮游动物群落结构变化的差异与其本身的群落结构组成有一定关系，也与浮游植物的群落组成有关。zzf1和ra5站位实验组中微型浮游动物的总丰度和总生物量都低于对照组，而zc18a站位变化不明显，这是由于前两个站位实验组中的优势种管游虫（zzf1站位）和急游虫2（ra5站位）的丰度和生物量迅速下降，而zc18a站位实验组的优势种百乐拟铃虫的丰度和生物量比较稳定造成的。 2005年长江口及邻近海区赤潮发生时，赤潮973 MC2005－04航次在本海区进行了综合调查，期间分别于5月26日、5月28日、5月28日和5月29日，在xzm1、srb、sra1和hb8a四个站位利用现场培养的方法，研究了东海大规模赤潮对微型浮游动物群落结构的影响。结果发现，东海大规模赤潮对微型浮游动物群落结构的影响与赤潮藻的密度密切相关：xzm1、srb和sra1站位是赤潮区，两种赤潮藻的总密度分别为，1.3×106 cells L-1、1.8×106 cells L-1和5.6×106 cells L-1，而hb8a站位位于非赤潮区，两种赤潮藻的总密度仅为5×105 cells L-1；实验进行72 h后，在以大型砂壳纤毛虫网纹虫和筒壳虫（Tintinnidium sp.）为主的xzm1、srb和sra1站位，仍然是以这些大型砂壳纤毛虫为主，且其在群落中的百分比显著增大，尤其在赤潮藻密度最高的的sra1站位，这种演替趋势表现的最明显；而在以小型无壳纤毛虫急游虫2为主的hb8a站位，微型浮游动物群落向中型砂壳纤毛虫真丁丁虫（Eutintinnus sp.）演替。培养过程中微型浮游动物总丰度的变化以及总生物量的变化都与赤潮藻的密度密切相关：赤潮藻密度较低的xzm1站位和srb站位变化不大；赤潮藻密度较高的sra1站位则快速下降；而非赤潮区的hb8a站位呈快速上升的趋势。以上结果进一步表明东海大规模赤潮能使微型浮游动物群落向中大型砂壳纤毛虫演替。 在2006年4月18日－5月30日，赤潮973项目MC2006航次在长江口及其邻近海区围绕东海原甲藻和米氏凯伦藻赤潮进行了综合调查。为了研究该过程中微型浮游动物群落结构的变化，我们在南北5个断面共计21个站位进行了取样，样品涉及赤潮发生前期、发生期和消退期。本次调查共发现纤毛虫66种，其中砂壳纤毛虫有8属37种，寡毛类无壳纤毛虫25种，另外还有前口类3属4种。在赤潮发生前之前，小型无壳纤毛虫的丰度普遍较高，平均值为1574 ind L-1，而中大型砂壳纤毛虫的丰度普遍偏低，平均值仅为14 ind L-1；赤潮发生以后，在大多数站位小型无壳纤毛虫的丰度出现不同程度的下降，平均值降为171 ind L-1，而中大型砂壳纤毛虫的丰度出现一定程度的上升，平均值增至216 ind L-1。赤潮消退时，小型无壳纤毛虫丰度的平均值降为109 ind L-1，而中大型砂壳纤毛虫的丰度继续增长，变为401 ind L-1。不同微型浮游动物百分比的统计结果也表明：微型浮游动物群落由小型无壳纤毛虫向中大型砂壳纤毛虫演替的趋势：小型无壳纤毛虫在群落中百分比的平均值由赤潮发生前的62 %变为赤潮发生时的32 %，至赤潮消退时又降为15 %；而中大型砂壳纤毛虫百分比的平均值从赤潮发生前的10 %变为赤潮发生时的24 %，至赤潮消退时增至50 %。典型站位za3、za5、zb7和zb9的结果再次表明了赤潮发生过程中微型浮游动物群落由小型无壳纤毛虫向中大型砂壳纤毛虫演替的趋势。 由以上赤潮发生前的现场模拟实验、赤潮发生时的现场培养实验和赤潮发生过程中的现场调查的结果均可见，东海大规模赤潮可以改变微型浮游动物的群落结构，呈现由小型无壳纤毛虫向中大型砂壳纤毛虫演替的趋势，进而有可能影响中大型浮游动物等摄食者的种群数量和群落结构，最终可能会影响整个海洋生态系统的结构和功能。

应用PCR-DGGE技术分析长江口低氧区和黄海冷水团的细菌群落组成

利用PCR-DGGE技术对长江口外低氧区海域和黄海冷水团海域的细菌群落组成进行了分析。 长江口外低氧区海域的细菌群落组成分析结果为：对获得的25条DGGE条带进行了克隆、测序，所得到的序列进行了系统进化分析（细菌16S rRNA基因V3区序列），分别归属于4个细菌类群：变形菌门(Proteobacteria)、拟杆菌门(Bacteroides)、厚壁菌门(Firmicutes)和蓝细菌门(Cyanobacteria)。其中有16条分别与变形细菌亚群的γ和δ-Proteobacteria相似。通过时空分析发现，低氧水体的细菌群落组成与非低氧水体的组成是不同的。低氧水体的优势菌群是拟杆菌门(Bacteroides)中的Flavobacteria。 黄海冷水团海域的细菌群落组成和优势菌群分析结果为：细菌16S rDNA V3区特征片段经DGGE分离、条带切割，共得到24条DGGE条带，克隆、测序后，将所得序列进行系统进化分析，分别归属于2个细菌类群：变形细菌门(Proteobacteria)和拟杆菌门(Bacteroides)。在24条序列中有16条分别与变形细菌亚群的γ和δ-Proteobacteria相似，有5条与拟杆菌门相似。通过时空分析发现，10月份（冷水团存在期），冷水团内部水体的细菌群落组成包括γ-Proteobacteria、δ-Proteobacteria和Bacteroides，而冷水团外部的水体的细菌群落组成包括γ-Proteobacteria和Bacteroides。冷水团内部水层的优势菌群为γ-Proteobacteria。4月份虽然冷水团没有形成，但是所调查的海域海水温度都不高，在7℃－12℃范围内，所以4月份所有站位，不管是底层的还是总的的细菌群落组成都与10月份冷水团内部（海水温度低于10℃）水体的相同，与10月份冷水团外部（海水温度大于19℃）的不同。

WSSV囊膜蛋白VP28基因在蓝藻中克隆和表达

该研究通过蔗糖密度梯度离心分离纯化WSSV-中国株,在电镜观察下观察到不同病毒组分的大小、形态及负染后显示的精细结构;通过SDS-PAGE鉴定在囊膜组分、完整病毒粒子组分中的VP28蛋白.设计一对特异性PCR扩增引物.PCR法从WSSV-中国株基因组DNA中扩增得到vp28基因片段640bp,并在起始密码子ATG前,填加了适合于蓝藻表达系统高效表达的SD序列.进一步将vp28基因正向连接到海藻穿梭表达载体pRL-489上的启动子PpsbA下游,酶切鉴定连接正确.通过PCR扩增在DNA水平上验证vp28基因在两种鱼腥藻Anabaenasp.PCC7120中均以质粒形式存在,在聚球藻Synechococcussp.PCC7002中以整合形式存在于染色体DNA上.用制备的抗WSSV的抗血清,通过WesternBlotting在蛋白水平上证明vp28基因在两种鱼腥藻Anabaenasp.PCC7120中均得到了表达,分子量为28kD.该论文的研究目的是通过基因工程获得WSSV囊膜蛋白VP28的转基因蓝藻,期望有助于揭示WSSV感染对虾的分子机一,确定VP28的功能.

两种蓝藻真核型Ser/Thr激酶基因功能的初步验证

Ser/Thr蛋白激酶（serine/threonine kinases，STK）在真核生物的信号转导通路中具有重要作用，而且已经成为对抗肿瘤、结核等多种人类疾病的药物作用靶点。上世纪九十年代，有研究发现STK在原核生物的信号转导中也发挥重要作用。本论文以聚球藻PCC7942（Synechococcus sp. PCC7942）和钝顶螺旋藻（Spirulina platensis）为材料，对几个真核型的Ser/Thr蛋白激酶基因的功能进行了初步验证。 蓝藻兼具细菌和植物的特点，具有成熟的转化体系，为真核生物基因功能的研究提供了新的模式宿主。聚球藻PCC7942是一种单细胞的淡水蓝藻，具有天然的外源DNA转化系统，是蓝藻分子遗传学研究的模式生物。通过基因敲除及表达差异分析发现聚球藻PCC7942中的Ser/Thr蛋白激酶stk196参与高温胁迫的信号传递。钝顶螺旋藻是原核丝状蓝藻，由于其蕴涵高品质营养成分而成为一类具有重要经济价值的微藻，该研究利用半定量RT-PCR方法，分析四个具有跨膜结构域的Ser/Thr蛋白激酶在正常生长温度下和经低温、高温诱导后表达量的变化情况，发现stk2103在低温诱导后的表达量降低，高温诱导后的表达量升高，提示该基因的表达可能参与了钝顶螺旋藻对温度的适应。 蓝藻中真核型Ser/Thr蛋白激酶功能的研究为我们进一步研究真核生物的Ser/Thr蛋白激酶功能提供了借鉴，并对植物抗逆胁迫的研究提供重要的理论依据。

胶州湾典型增养殖海域有害赤潮生消过程及关键因子研究

有害赤潮（Harmful algal blooms, HAB）已成为全球性的海洋灾害，对我国近海养殖业危害尤为巨大，研究赤潮发生、发展的主要环境影响因子，对于控制赤潮的发生以及加强赤潮的防治具有十分重要的意义。本文根据国家自然科学基金“九五”重大项目“中国沿海典型增养殖区有害赤潮发生动力学防治对策研究”项目组近年来对胶州湾东北部养殖海域的监测资料，分析了该海区营养状况，结合该区自然环境特点，探讨了发生于1998年7月和1999年6月两次硅藻赤潮的主要影响因子；并应用胶州现场海水进行了室内模拟培养实验，对影响有害赤潮生消过程的关键环境因子进行了分析探讨。对1998和1999年赤潮多发季节胶州湾东北部增养殖水域海水的营养状况进行了分析，并参照潜在性富营养化的概念，应用NQI指数对该海域的富营养化水平进行了评价。结果表明：（1）胶州湾东北部增养殖海域赤潮多发季节TIN、DIP和活性硅酸盐的含量均较高，远高于同期胶州湾大面观测站各营养盐的平均含量；该海域有机污染物含量相对营养盐含量不高。（2）该海域春季氮、磷营养盐含量充足，水域属富营养化平；夏季该海域具有较高的生物具，磷酸盐水平相对较低，海域多数时间属于磷限制潜在性富营养化水平；但这种限制极易为外界补充所改变，如陆源输入、养殖废水的排放等。（3）该海域为典型增养殖区，养殖废水对海域营养盐的补充有重要意义，因此应当加强对该海域养殖业的综合管理。对分别于1998年7月和1999年6月在胶州湾东北部增养殖海域发生的中肋骨条藻（Skeletonema costatum）赤潮和浮动弯角藻（Eucampia zoodiacus）赤潮生消过程中环境因子的变化进行了探讨。发生赤潮生物量的升高常伴随海水pH值、溶解氧（DO）含量的升高以及各营养盐（氮、磷、硅）含量的降低；认为在该海域较高营养盐含量和较高生物量的情况下，赤潮发生前大规模的降雨过程及随后的气候条件为赤潮生物的快速生长繁殖创造了有利条件；无机磷和活性硅酸盐含量相对于无机氮显得较低，对该海域硅藻赤潮的生消过程起着控制作用。采集胶州湾现场海水进行了实验室模拟培养实验，对影响赤潮生消过程的主要环境因素进行了研究和探讨。结果表明，现场海水进行模拟培养时，排除了潮流、风以及水团间交流混合等因素对水体中浮游生物的影响，培养水体中的浮游植物生物量都存在增高的现象；在氮、磷、硅等营养盐含量充足的条件下，浮游植物可快速进入指数增长期，并较快发展形成赤潮，高浓度的营养盐常导致培养水体中浮游植物的较高的生长速率，浮游植物对氮磷营养盐的吸收受到环境中营养盐含量的影响；初始环境下营养盐的限制可抑制浮游植物的进一步增殖，发生赤潮水体中某种营养盐的含量降低最终导致了赤潮的消亡。

Relationships between nitrogen and phosphorus forms and ratios and the development of dinoflagellate blooms in the East China Sea

During late spring and early summer of 2005, large-scale (> 15 000 km(2)), mixed dinoflagellate blooms developed along the the coast of the East China Sea. Karenia mikimotoi was the dominant harmful algal bloom species in the first stage of the bloom (late May) and was succeeded by Prorocentrum donghaiense approximately 2 wk later. Samples were collected from different stations along both north-south and west-east transects, from the Changjiang River estuary to the south Zhejiang coast, during 3 cruises of the Chinese Ecology and Oceanography of Harmful Algal Blooms Program, before and during the bloom progression. Nitrogen isotope tracer techniques were used to measure rates of NO3-, NH4+, urea, and glycine uptake during the blooms. High inorganic nitrogen (N), but low phosphorus (P) loading from the Changjiang River led to high dissolved inorganic N:dissolved inorganic P ratios in the sampling area and indicate the development of P limitation. The rates of N-15-uptake experiments enriched with PO43- were enhanced compared to unamended samples, suggesting P limitation of the N-uptake rates. The bloom progression was related to the change in availability of both organic and inorganic N and P. Reduced N forms, especially NH4+, were preferentially taken up during the blooms, but different bloom species had different rates of uptake of organic N substrates. K mikimotoi had higher rates of urea uptake, while P. donghaiense had higher rates of glycine uptake. Changes in the availability of reduced N and the ratios of N:P in inorganic and organic forms were suggested to be important in the bloom succession. Nutrient ratios and specific uptake rates of urea were similar when compared to analogous blooms on the West Florida Shelf.