Discovery of the genes in response to white spot syndrome virus (WSSV) infection in Fenneropenaeus chinensis through cDNA microarray

We used microarray technology to study differentially expressed genes in white spot syndrome virus (WSSV)-infected shrimp. A total of 3136 cDNA targets, including 1578 unique genes from a cephalothorax cDNA library and 1536 cDNA clones from reverse and forward suppression subtractive hybridization (SSH) libraries of Fenneropenaeus chinensis, plus 14 negative and 8 blank control clones, were spotted onto a 18 x 18 mm area of NH2-modified glass slides. Gene expression patterns in the cephalothorax of shrimp at 6 h after WSSV injection and moribund shrimp naturally infected by WSSV were analyzed. A total of 105 elements on the arrays showed a similar regulation pattern in artificially infected shrimp and naturally infected moribund shrimp; parts of the results were confirmed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The up-regulated expression of immune-related genes, including heat shock proteins (HSP70 and HSP90), trehalose-phosphate synthase (TPS), ubiquitin C, and so forth, were observed when shrimp were challenged with WSSV. Genes including myosin LC2, ATP synthase A chain, and arginine kinase were found to be down-regulated after WSSV infection. The expression of housekeeping genes such as actin, elongation factor, and tubulin is not stable, and so these genes are not suitable as internal standards for semiquantitative RT-PCR when shrimp are challenged by WSSV. As a substitute, we found that triosephosphate isomerase (TPI) was an ideal candidate of interstandards in this situation.

Characterization and mapping of expressed sequence tags isolated from a subtracted cDNA library of Litopenaeus vannamei injected with white spot syndrome virus

A large number of polymorphic simple sequence repeats (SSRs) or microsatellites are needed to develop a genetic map for shrimp. However, developing an SSR map is very time-consuming, expensive, and most SSRs are not specifically linked to gene loci of immediate interest. We report here on our strategy to develop polymorphic markers using expressed sequence tags (ESTs) by designing primers flanking single or multiple SSRs with three or more repeats. A subtracted cDNA library was prepared using RNA from specific pathogen-free (SPF) Litopenaeus vannamei juveniles (similar to 1 g) collected before (0) and after (48 h) inoculation with the China isolate of white spot syndrome virus (WSSV). A total of 224 clones were sequenced, 194 of which were useful for homology comparisons against annotated genes in NCBI nonredundant (nr) and protein databases, providing 179 sequences encoded by nuclear DNA, 4 mitochondrial DNA, and 11 were similar to portions of WSSV genome. The nuclear sequences clustered in 43 groups, 11 of which were homologous to various ESTs of unknown function, 4 had no homology to any sequence, and 28 showed similarities to known genes of invertebrates and vertebrates, representatives of cellular metabolic processes such as calcium ion balance, cytoskeleton mRNAs, and protein synthesis. A few sequences were homologous to immune system-related (allergens) genes and two were similar to motifs of the sex-lethal gene of Drosophila. A large number of EST sequences were similar to domains of the EF-hand superfamily (Ca2+ binding motif and FRQ protein domain of myosin light chains). Single or multiple SSRs with three or more repeats were found in approximately 61 % of the 179 nuclear sequences. Primer sets were designed from 28 sequences representing 19 known or putative genes and tested for polymorphism (EST-SSR marker) in a small test panel containing 16 individuals. Ten (53%) of the 19 putative or unknown function genes were polymorphic, 4 monomorphic, and 3 either failed to satisfactorily amplify genomic DNA or the allele amplification conditions need to be further optimized. Five polymorphic ESTs were genotyped with the entire reference mapping family, two of them (actin, accession #CX535973 and shrimp allergen arginine kinase, accession #CX535999) did not amplify with all offspring of the IRMF panel suggesting presence of null alleles, and three of them amplified in most of the IRM F offspring and were used for linkage analysis. EF-hand motif of myosin light chain (accession #CX535935) was placed in ShrimpMap's linkage group 7, whereas ribosomal protein S5 (accession #CX535957) and troponin I (accession #CX535976) remained unassigned. Results indicate that (a) a large number of ESTs isolated from this cDNA library are similar to cytoskeleton mRNAs and may reflect a normal pathway of the cellular response after im infection with WSSV, and (b) primers flanking single or multiple SSRs with three or more repeats from shrimp ESTs could be an efficient approach to develop polymorphic markers useful for linkage mapping. Work is underway to map additional SSR-containing ESTs from this and other cDNA libraries as a plausible strategy to increase marker density in ShrimpMap.

栉孔扇贝与海湾扇贝免疫学比较研究

扇贝是我国重要的海水养殖品种，但自1997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。引起扇贝大规模死亡原因是多方面的，其主要原因是病原滋生、养殖环境恶化和种质衰退。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，促进我国扇贝养殖业的健康、可持续发展。本文通过比较栉孔扇贝和海湾扇贝正常状态和重金属污染胁迫下相关免疫指标的变化及HSP70和HSP90在重金属胁迫后表达规律，以期更好的了解贝类的防御机制，为扇贝病害防治提供资料。 本研究采用流式细胞仪技术对栉孔扇贝和海湾扇贝血细胞死亡率、细胞吞噬率和呼吸爆发进行了测定，发现正常状态下两种扇贝的细胞死亡率相差不大，呼吸爆发的基础值相差也不大，而海湾扇贝细胞吞噬率显著高于栉孔扇贝。在血淋巴和肝胰腺中，海湾扇贝SOD、ACP酶活性均显著高于栉孔扇贝；海湾扇贝的MDA含量也高于栉孔扇贝，但差异不显著。鳗弧菌感染实验发现，海湾扇贝累积死亡率远低于栉孔扇贝。上述结果表明，海湾扇贝对细菌等异物的吞噬和杀灭能力以及机体自身的抗氧化能力高于栉孔扇贝，这为海湾扇贝比栉孔扇贝具有更高的抗逆性提供了证据。 不同浓度Pb2+刺激后，海湾扇贝血细胞的死亡率明显低于栉孔扇贝。海湾扇贝细胞吞噬率和呼吸爆发均高于栉孔扇贝。对两种扇贝体液免疫指标的测定发现，海湾扇贝的SOD、ACP活性和MDA含量均高于栉孔扇贝。结果表明，在Pb2+刺激下两种扇贝都处于重金属氧化胁迫下，其免疫系统均受到了影响，但相同剂量的Pb2+对两种扇贝的毒害程度不同，海湾扇贝受到的影响要低于栉孔扇贝。 采用同源克隆和cDNA 末端快速扩增（RACE）技术，从海湾扇贝血淋巴中克隆到了热休克蛋白90（AiHSP90）基因。AiHSP90的cDNA序列全长为2669bp，其中5' 非编码区（UTR）为90bp，3' UTR为44bp，开放阅读框（Open Reading Frame, ORF）包括2175bp，2524-2528位AATAA为推测的AiHSP90基因的 mRNA 多聚腺苷酸加尾信号（Polyadenylation Signal）。开放阅读框编码724个氨基酸残基，推测的理论分子量为83.08 kDa，等电点为4.81。 利用荧光实时定量PCR 技术检测了AiHSP90基因在革兰氏阴性菌鳗弧菌和革兰氏阳性菌藤黄微球菌感染后不同时间点相对于β-actin基因的表达。在鳗弧菌和藤黄微球菌刺激后，除72h外AiHSP90均上调表达，在9h其mRNA表达达到最大值，分别约为空白组的6.8倍和3倍；随后随着时间的推移，其表达下降，在48h恢复到接近空白组的水平；到72h时降到最低点。鳗弧菌对AiHSP90的诱导比藤黄微球菌要高，这也反映出鳗弧菌对海湾扇贝有更强的毒力，诱导的HSP90表达也更强。本研究首次揭示出在软体动物中HSP90在细菌感染条件下的表达情况，结果表明海湾扇贝AiHSP90参与了机体对细菌感染的应答。 采用荧光实时定量PCR技术，对栉孔扇贝和海湾扇贝在不同浓度、不同种类重金属胁迫后HSP70和HSP90基因表达规律进行了比较。结果显示，镉和铅对栉孔扇贝处理10天后即诱导HSP70以较高水平表达，20天后镉处理组HSP70恢复到与对照接近的水平，而在铅处理组也呈现出下降的趋势。而海湾扇贝HSP70基因表达与栉孔扇贝恰好相反，处理20天后其HSP70表达才出现较高水平。铜离子处理对诱导两种扇贝HSP70的影响相似，对栉孔扇贝HSP70的诱导强度更大。对于同一种重金属，不同的浓度诱导的HSP70表达也各不相同。而对于HSP90，海湾扇贝在受到三种重金属刺激后HSP90的表达量均高于栉孔扇贝，而且长时间的刺激（20天）诱导了更高的HSP90基因表达。不同的浓度的同一种重金属离子诱导的HSP90表达也各不相同。这些结果表明，在重金属胁迫下HSP70以及HSP90在栉孔扇贝和海湾扇贝中均表现出不同的应答方式。结合前面海湾扇贝抗逆性较强的综合结果，两种扇贝热休克蛋白基因表达的比较提示我们栉孔扇贝HSP70的激烈反应预示着抗逆能力的不足，而海湾扇贝HSP90的高表达却预示着更强的抗逆能力，有待于更多应激实验如热刺激、病原刺激等的进一步验证。

利用海带配子体营养增殖体系实现外源基因表达

1993年以来，本实验室在借鉴高等植物基因工程原理和方法的基础上，利用海带世代交替生活史，建立了海带遗传转化体系,并申请国家专利PCT/CN03/00534（Trends in Biotechnology, 2005, 23,264-268.）。海带遗传转化体系是建立在基因枪转化海带配子体，经孤雌或受精途径再生幼孢子体后用氯霉素筛选以及海上安全栽培等一系列技术平台上的新型体系。目前该体系已经成功实现报告基因（β-半乳糖甘酶基因，LacZ）和目的基因（乙肝表面抗原基因，HBsAg）在孢子体阶段的稳定表达。由于应用该体系表达目的基因须经过孢子体再生以及海上栽培等阶段，周期较长，因此本论文拟通过以下5方面工作初步建立以配子体营养增殖为基础的新型海带表达体系，并尝试在配子体阶段实现转化、筛选、增殖及产物检测等操作。1)确立针对配子体的选择标记。2)利用光生物反应器增殖配子体。3)拓展新的报告基因种类。4)克隆内源高效启动子。5)利用该体系表达rt-PA基因（编码价格昂贵的特效溶栓药，瑞替普酶）。本论文研究结果为1)升高温度能够显著增强草丁膦的筛选效果，根据敏感性实验统计结果确定了草丁膦作为海带配子体遗传转化选择压力的筛选方案为40mg/l连续处理7d×3次。2)利用室内光生物反应器营养增殖海带配子体技术，经草丁膦筛选，实现了SV40启动子驱动草丁膦抗性基因bar在配子体细胞中的稳定表达。3)采用调整配子体营养生长状态的方法减低背景荧光，从而首次观察到增强型绿色荧光蛋白基因（egfp）在海带配子体细胞中的瞬间表达。4)克隆了海带肌动蛋白基因（actin）的基因组序列，这是褐藻肌动蛋白基因组序列克隆的首次报道，通过染色体步移获得海带actin基因上游约450bp的片段。5)在上述工作基础上，与中国药科大学合作构建了瑞替普酶基因（rt-PA）的海带表达载体，并实现该基因在海带配子体细胞中的稳定表达，产物具有正确的生物学活性，平均表达量达到0.159μg/mg可溶蛋白。这是该基因在植物中表达的首次报道。研究结果提示在光生物反应器营养增殖体系基础上构建海带配子体表达系统的方案是可行的。

Restructuring of a zooplankton community by perturbation from a wind-forced coastal jet

The impact of transient wind events on an established zooplankton community was observed during a, field survey in a, coastal region off northern Norway in May 2002. A transient wind event induced a coastal jet/filament intrusion of warm, saline water into our survey area where a semi-permanent eddy was present. There was an abrupt change in zooplankton community structure within 4-7 days of the wind event, with a change in the size structure, an increase in lower size classes less than 1 mm in equivalent spherical diameter (ESD) and a decrease in larger size classes greater than 1.5 mm in ESD. The slope of zooplankton biovolume spectra changed from -0.6 to -0.8, consistent with the size shifting towards smaller size classes. This study shows that even well established zooplankton communities are susceptible to restructuring during transient wind events, and in particular when wind forcing induces horizontal currents or filaments.

Identification of a silicatein(-related) protease in the giant spicules of the deep-sea hexactinellid Monorhaphis chuni

Silicateins, members of the cathepsin L family, are enzymes that have been shown to be involved in the biosynthesis/condensation of biosilica in spicules from Demospongiae (phylum Porifera), e. g. Tethya aurantium and Suberites domuncula. The class Hexactinellida also forms spicules from this inorganic material. This class of sponges includes species that form the largest biogenic silica structures on earth. The giant basal spicules from the hexactinellids Monorhaphis chuni and Monorhaphis intermedia can reach lengths of up to 3 m and diameters of 10 mm. The giant spicules as well as the tauactines consist of a biosilica shell that surrounds the axial canal, which harbours the axial filament, in regular concentric, lamellar layers, suggesting an appositional growth of the spicules. The lamellae contain 27 kDa proteins, which undergo post-translational modification (phosphorylation), while total spicule extracts contain additional 70 kDa proteins. The 27 kDa proteins cross-reacted with anti-silicatein antibodies. The extracts of spicules from the hexactinellid Monorhaphis displayed proteolytic activity like the silicateins from the demosponge S. domuncula. Since the proteolytic activity in spicule extracts from both classes of sponge could be sensitively inhibited by E-64 (a specific cysteine proteinase inhibitor), we used a labelled E-64 sample as a probe to identify the protein that bound to this inhibitor on a blot. The experiments revealed that the labelled E-64 selectively recognized the 27 kDa protein. Our data strongly suggest that silicatein(-related) molecules are also present in Hexactinellida. These new results are considered to also be of impact for applied biotechnological studies.

The unique skeleton of siliceous sponges (Porifera; Hexactinellida and Demospongiae) that evolved first from the Urmetazoa during the Proterozoic: a review

Sponges (phylum Porifera) had been considered as an enigmatic phylum, prior to the analysis of their genetic repertoire/tool kit. Already with the isolation of the first adhesion molecule, galectin, it became clear that the sequences of sponge cell surface receptors and of molecules forming the intracellular signal transduction pathways triggered by them, share high similarity with those identified in other metazoan phyla. These studies demonstrated that all metazoan phyla, including Porifera, originate from one common ancestor, the Urmetazoa. The sponges evolved prior to the Ediacaran-Cambrian boundary (542 million years ago [myr]) during two major "snowball earth events", the Sturtian glaciation (710 to 680 myr) and the Varanger-Marinoan ice ages (605 to 585 myr). During this period the ocean was richer in silica due to the silicate weathering. The oldest sponge fossils (Hexactinellida) have been described from Australia, China and Mongolia and are thought to have existed coeval with the diverse Ediacara fauna. Only little younger are the fossils discovered in the Sansha section in Hunan (Early Cambrian; China). It has been proposed that only the sponges possessed the genetic repertoire to cope with the adverse conditions, e.g. temperature-protection molecules or proteins protecting them against ultraviolet radiation. The skeletal elements of the Hexactinellida (model organisms Monorhaphis chuni and Monorhaphis intermedia or Hyalonema sieboldi) and Demospongiae (models Suberites domuncula and Geodia cydonium), the spicules, are formed enzymatically by the anabolic enzyme silicatein and the catabolic enzyme silicase. Both, the spicules of Hexactinellida and of Demospongiae, comprise a central axial canal and an axial filament which harbors the silicatein. After intracellular formation of the first lamella around the channel and the subsequent extracellular apposition of further lamellae the spicules are completed in a net formed of collagen fibers. The data summarized here substantiate that with the finding of silicatein a new aera in the field of bio/inorganic chemistry started. For the first time strategies could be formulated and experimentally proven that allow the formation/synthesis of inorganic structures by organic molecules. These findings are not only of importance for the further understanding of basic pathways in the body plan formation of sponges but also of eminent importance for applied/commercial processes in a sustainable use of biomolecules for novel bio/inorganic materials.

青海家牦牛HIF-1α基因组织特异性表达

[目的]探究青海家牦牛HIF-1α基因组织的特异性表达。[方法]应用半定量反转录PCR和实时定量反转录PCR（SYBRGreen）技术对青海家牦牛HIF-1α基因的组织特异性表达进行检测。通过提取不同组织总RNA，经DNase I消化后，用随机引物进行反转录合成cDNA，采用特异性引物分别对HIF-1α和β-actin基因进行RT-PCR和Real Time RT-PCR扩增。[结果]结果表明，HIF-1α基因在心、肝、脾、肺、肾、脑、肌肉、睾丸组织中均有表达，其中以睾丸和脾中HIF-1α基因表达量最高，肌肉的表达量最低。[结论]该研究为进一步揭示HIF-1α在高原土著动物低氧适应过程中的分子机制有着重要的意义。

高原鼠兔ob基因的组织表达特征

ob基因编码的leptin蛋白在调节生物体能量平衡中起到重要作用。本研究应用Taqman探针real time PCR技术对高原鼠兔ob基因的组织分布进行检测。通过提取不同组织总RNA，经DNase I消化后，用随机引物进行反转录合成cDNA，采用特异性Taqman探针和引物分别对ob基因及β-actin基因进行实时定量PCR扩增，对不同组织中ob基因和β-actin基因的初始拷贝数之比进行比较。结果表明ob基因在脑、心脏、肺、肝脏、脾脏、肾脏、骨骼肌、脂肪组织中均有表达，其中以白色脂肪组织中ob基因表达量最高，其次为心脏和肺，表达量最低的是肝脏和肾脏。