Embryonic and genetic manipulation in fish

Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been thoroughly studied in China since 1960s. Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults. Most importantly, nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish. This was the first case of cloned fish with somatic cells. Based on the technique of microinjection, recombinant MThGH gene has been transferred into fish eggs and the first batch of transgenic fish were produced in 1984. The behavior of foreign gene was characterized and the onset of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis. This eventually led to the transgenic mosaicism. The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults. The transgenic common carp were more efficient in utilizing dietary protein than the controls. An "all-fish" gene construct CAgcGH has been made by splicing the common carp beta-actin gene (CA) promoter onto the grass carp growth hormone gene (gcGH) coding sequence. The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait. Combination of techniques of fish cell culture, gene transformation with cultured cells and nuclear transplantation should be able to generate homogeneous strain of valuable transgenic fish to fulfil human requirement in 21(st) century.

非洲爪蟾Brunol1 基因的功能以及文昌鱼肌动蛋白基因家族扩增的研究

BRUNOL 蛋白又称CELF（CUG-BP and ETR3 like factor）是一种典型的RNA 结合蛋白，它的N 端含有两个连续的RNA 识别结构域（RNA recognition motif，RRM)， C 端有一个RRM 结构域。主要参与对可变剪切、翻译、降解和编辑等基因表达转录后水平的调节。迄今在人类中已发现6 个Brunol 基因家族成员，即Brunol1-6；在非洲爪蟾中已发现了5 个：Brunol1-5。近期，我们克隆了爪蟾的Brunol1-5 并研究了它们在非洲爪蟾早期胚胎发育过程中的时空表达图式。结果显示，与以往研究结果一致， Brunol1 基因高量、特异地在神经管中表达，提示Brunol1 基因可能对于爪蟾的神经系统的发生和发育发挥着重要的作用。本实验利用Morpholino 和过表达等手段研究了爪蟾Brunol1 基因对于爪蟾早期胚胎发育的影响。结果显示，在下调和过表达Brunol 1 基因的情况下都会导致胚胎出现体轴弯曲，眼睛和头部发育不全等表型。而将 Brunol1 基因特异的Morpholino 与它的mRNA 共注射时可以明显挽救这一表型。我们通过原位杂交实验，检测了一些爪蟾神经系统的标记基因在Brunol1 过表达胚胎中的表达情况，结果发现过表达Brunol1 基因能显著地下调Krox-20, N-tubulin, Lhx2, Pax6 等的表达，而Sox2 和Otx2 的表达却未受影响。这说明Brunol1 的异常表达确实影响到了神经系统发育过程的信号调控网络，导致胚胎发育的畸形。该结果将有助于阐述Brunol1 基因对于脊椎动物神经系统发生的意义。肌动蛋白是一种分布广泛而且在进化上十分保守的蛋白，它是构成细胞骨架的关键组分。通常人们将肌动蛋白分成肌肉型和胞质型两种类型，它们各自行使着不同的功能。在此，我们通过对古老的脊索动物文昌鱼的肌动蛋白基因家族进行系统的分析发现，文昌鱼中该基因家族成员多达30 多个，而且它们中很多都有连锁现象；进化分析的结果显示，文昌鱼的肌动蛋白基因家族通过串联重复序列的复制发生扩增；从结构上看，它们的基因结构多样化, 包含2-7 个外显子；同时，我们还克隆了两个不同的文昌鱼肌肉型的肌动蛋白基因，并进一步比较了它们在文昌鱼早期胚胎中的表达图式。结果显示，这两个基因在表达上有着细微的差别，这提示文昌鱼肌动蛋白基因家族成员在功能上的分化。该结论将有助于阐述肌动蛋白基因家族的进化以及它们在脊索动物发育的中所扮演的功能。

恒河猴TRIM5α 的组织分布及TRIMCyp 限制HIV-1 病毒颗粒产生的初步研究

三重基序蛋白TRIM5α(Tripartite motif protein 5 alpha)是哺乳动物细胞中一种重要的限制因子，广泛分布于各种哺乳动物细胞中。人类TRIM5α mRNA 广泛表达于人类各个组织中，并且I 型干扰素IFN-α/β/γ 均能与TRIM5α 基因启动子的ISRE 元件结合，上调TRIM5α mRNA 的表达。恒河猴(Macaca mulatta)TRIM5α 是恒河猴体内重要的限制因子。目前对恒河猴尤其是中国恒河猴TRIM5α 的组织分布以及在受到外界刺激时TRIM5α mRNA 表达量的变化研究还未见报道。本论文通过从中国恒河猴各组织中提取总RNA，以β-actin 基因作为内参照，通过逆转录PCR 检测各组织中TRIM5α mRNA 的表达。我们选择用HIV-GFP-VSVG 感染、用佛波脂(Phorbol myfismte acetate, PMA)+离子霉素(Ionomycin, Ion)，CD28 抗体+CD49d 抗体分别共刺激恒河猴PBMC，研究不同刺激对中国恒河猴TRIM5α mRNA 表达量的影响。研究发现：TRIM5α mRNA 广泛表达于恒河猴各组织中，在免疫系统和泌尿生殖系统各组织，如腹淋巴结、睾丸和附睾中表达量最高，而在神经系统各组织如大脑、脊髓中表达量比较少，在其他各组织中未见明显的表达差异。此外HIV-GFP-VSVG 感染、PMA+ Ion 与CD28 抗体+CD49d 抗体分别共刺激PBMC 均能促进PBMC TRIM5α mRNA 表达量的上调。 TRIM5α 作为恒河猴体内的最主要的限制HIV-1 感染的限制因子，除了可能通过促进HIV-1 的脱壳和阻止整合前复合物PIC(pre-integration complex)入核，恒河猴TRIM5α 还能限制HIV-1 病毒颗粒的产生。在这个过程中B30.2 结构域是非必需的，而B-box2 和Coiled-Coil 结构域起着决定性的作用。因为鹰猴(Aotes trivirgatus)TRIMCyp(omTRIMCyp) 蛋白和北平顶猴(Macaca leouina) TRIMCyp(npmTRIMCyp）蛋白的B-box2 和Coiled-Coil 结构域与恒河猴TRIM5α 的B-box2 和Coiled-Coil 具有很高的同源性，我们希望了解鹰猴TRIMCyp 蛋白和北平顶猴TRIMCyp 蛋白对HIV-1 病毒颗粒的产生是否有限制作用。本论文主要通过将质粒pNL4.3 分别与质粒pLPCX 、pLPCX-npmTRIMCyp-HA 、 pLPCX-omTRIMCyp-HA和pLPCX-rhTRIM5α-HA共转染293T细胞，通过western blot 检测细胞内Gag 蛋白和TRIM5 蛋白的表达情况，研究omTRIMCyp 蛋白和 npmTRIMCyp 蛋白对HIV-1 病毒颗粒产生的限制作用。结果表明：北平顶猴 TRIMCyp 蛋白、鹰猴TRIMCyp 蛋白都能不同程度的促进HIV-1 病毒Gag 蛋白的降解。

Quasi-3D Cytoskeletal Dynamics of Osteocytes under Fluid Flow

Osteocytes respond to dynamic fluid shear loading by activating various biochemical pathways, mediating a dynamic process of bone formation and resorption. Whole-cell deformation and regional deformation of the cytoskeleton may be able to directly regulate this process. Attempts to image cellular deformation by conventional microscopy techniques have been hindered by low temporal or spatial resolution. In this study, we developed a quasi-three-dimensional microscopy technique that enabled us to simultaneously visualize an osteocyte's traditional bottom-view profile and a side-view profile at high temporal resolution. Quantitative analysis of the plasma membrane and either the intracellular actin or microtubule (MT) cytoskeletal networks provided characterization of their deformations over time. Although no volumetric dilatation of the whole cell was observed under flow, both the actin and MT networks experienced primarily tensile strains in all measured strain components. Regional heterogeneity in the strain field of normal strains was observed in the actin networks, especially in the leading edge to flow, but not in the MT networks. In contrast, side-view shear strains exhibited similar subcellular distribution patterns in both networks. Disruption of MT networks caused actin normal strains to decrease, whereas actin disruption had little effect on the MT network strains, highlighting the networks' mechanical interactions in osteocytes.

Molecularly imprinted solid-phase extraction sorbent for removal of nicotine from tobacco smoke

A sorbent showing specific affinity for nicotine was prepared by molecular imprinting technique, using nicotine as the template, methacrylic acid (MAA) as the functional monomer, ethyleneglycol dimethacrylate (EDMA) as the crosslinker and chloroform as the porogen. UV spectroscopic analysis in the molecular imprinting prepolymerization stage confirmed that nicotine could complex with the functional monomer by electrostatic interaction (ionic interaction and hydrogen bonding). The affinity and the binding properties of the imprinted polymer towards nicotine were investigated by equilibrium rebinding experiments. The results indicated the presence of nicotine-specific binding sites in the imprinted polymer, and that the imprinted polymer had a good capacity (90 mumol/g polymer) for nicotine. The elution conditions were optimized on the column packed with the imprinted polymer to elute nicotine quantitatively. The imprinted polymer was used as a solid-phase extraction (SPE) material for the removal of nicotine from tobacco smoke. The results obtained showed that the imprinted polymer was superior in terms of removing nicotine in tobacco smoke, compared with the commercial filter tip.

Synthesis and characterization of network liquid crystal elastomers and thermosets

A new series of network liquid crystal polymers were synthesized by graft copolymerization of the difunctional mesogenic monomer 4-allyloxy-benzoyloxy-4'-allyloxybiphenyl (M) upon polymethylhydrosiloxane (PMHS). Monomer M acted not only as a mesogenic unit but also as a crosslinker for the network polymers. The chemical structures of the polymers were confirmed by IR spectroscopy. DSC, TGA, and X-ray scattering were used to measure their thermal properties and mesogenic properties. The glass transition temperature (T-g) of these network liquid crystal polymers was increased when the monomer was increased, and T-d (temperature of 5% weight loss) at first went up and reached a maximum at P, then went down. The slightly crosslinked polymers (P, P,) show rubber-like elasticity, so it was called liquid-crystal elastomer. Network polymers will lose elasticity property with a highly crosslinked degree, and turn into thermosetting polymers (P-4, P-5). All polymers exhibited a smectic texture by X-ray scattering.

Comparative proteomic profiles of the hepatopancreas in Fenneropenaeus chinensis response to hypoxic stress

Hypoxia, as one suboptimal environmental condition, can affect the physiological state of shrimp during pond aquaculture. To better understand the mechanism of response to hypoxic stress in Chinese shrimp Fenneropenaeus chinensis, proteome research approach was utilized. Differentially expressed proteins of hepatopancreas in adult Chinese shrimp between the control and hypoxia-stressed groups were screened. By 2-DE analysis, 67 spots showed obvious changes after hypoxia. Using LC-ESI-MS/MS, 51 spots representing 33 proteins were identified including preamylase, arginine kinase, phosphopyruvate hydratase, citrate synthase, ATP synthase alpha subunit, chymotrypsin BI, chitinase, ferritin, C-type lectin receptors, transketolase, formylglutathione hydrolase, formyltetrahydrofolate dehydrogenase, aldehyde dehydrogenase, glutathione peroxidase, cytosolic manganese superoxide dismutase, protein disulfide isomerase, beta-actin, oncoprotem nm23, crustacyanin-Cl and so on. These proteins could be functionally classified into several groups such as proteins related to energy production, metabolism-related proteins, immune-related proteins, antioxidant proteins, chaperones, cytoskeleton proteins and ungrouped proteins. The transcription levels of ten selected genes encode the identified proteins were analyzed by real-time PCR at different sampling times of hypoxia. This study is the first analysis of differentially expressed proteins in the hepatopancreas of shrimp after hypoxia and provides a new insight for further study in hypoxic stress response of shrimp at the protein level.

Cortactin mediated morphogenic cell movements during zebrafish (Danio rerio) gastrulation

Cell migration is essential to direct embryonic cells to specific sites at which their developmental fates are ultimately determined. However, the mechanism by which cell motility is regulated in embryonic development is largely unknown. Cortactin, a filamentous actin binding protein, is an activator of Arp2/3 complex in the nucleation of actin cytoskeleton at the cell leading edge and acts directly on the machinery of cell motility. To determine whether cortactin and Arp2/3 mediated actin assembly plays a role in the morphogenic cell movements during the early development of zebrafish, we initiated a study of cortactin expression in zebrafish embryos at gastrulating stages when massive cell migrations occur. Western blot analysis using a cortactin specific monoclonal antibody demonstrated that cortactin protein is abundantly present in embryos at the most early developmental stages. Immunostaining of whole-mounted embryo showed that cortactin immunoreactivity was associated with the embryonic shield, predominantly at the dorsal side of the embryos during gastrulation. In addition, cortactin was detected in the convergent cells of the epiblast and hypoblast, and later in the central nervous system. Immunofluorescent staining with cortactin and Arp3 antibodies also revealed that cortactin and Arp2/3 complex colocalized at the periphery and many patches associated with the cell-to-cell junction in motile embryonic cells. Therefore, our data suggest that cortactin and Arp2/3 mediated actin polymerization is implicated in the cell movement during gastrulation and perhaps the development of the central neural system as well.

Expression of muscle-related genes and two MyoD genes during amphioxus notochord development

The notochord is one of the diagnostic features of the phylum Chordata. Despite the similarities in the early morphogenetic patterns of the notochords of various chordates, they are strikingly distinct from one another at the histological level. The amphioxus notochord is one example of an evolutionary novelty because it is made up of muscle cells. Our previous expressed sequence tag analysis, targeting messenger RNAs expressed in the adult amphioxus notochord, demonstrated that many muscle-related genes are expressed there. To characterize amphioxus notochord cells and to gain insights into the myogenic program in the notochord, we determined the spatial and temporal expression patterns of these muscle-related genes during amphioxus development. We found that BbNA1 (notochord actin), Amphi-Trop I (troponin I), Amphi-TPmyosin (tropomyosin), Amphi-MHC2 (myosin heavy chain), Amphi-nMRLC (notochord-specific myosin regulatory light chain), AmphinTitin/MLCK (notochord-specific titin/myosin light chain kinase), Amphi-MLP/CRP3 (muscle LIM protein), and Amphi-nCalponin (notochord-specific calponin) are expressed with characteristic patterns in notochord cells, including the central cells, dorsally located cells, and ventrally located cells, suggesting that each notochord cell has a unique molecular architecture that may reflect its function. In addition, we characterized two MyoD genes (Amphi-MyoD1 and Amphi-MyoD2) to gain insight into the genetic circuitry governing the formation of the notochord muscle. One of the MyoD genes (Amphi-MyoD2) is expressed in the central notochord cells, and the coexistence of Amphi-MyoD2 transcripts along with the Amphi-MLP/CRP3 transcripts implies the participation of Amphi-MyoD2 in the myogenic program in the notochord muscle.

Molecular coordinated regulation of gene expression during ovarian development in the penaeid shrimp

To understand the molecular events of ovarian development in penaeid shrimp, RNA arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify differentially expressed genes during ovarian maturation in Metapenaeus ensis. From a screening of 700 clones in a cDNA library of the shrimp ovary by the products of RAP-PCR of different maturation stages, 91 fragments with differentially expressed pattern as revealed by dot-blot hybridization were isolated and sequenced. Forty-two of these fragments show significant sequence similarity to known gene products and the differentially expressed pattern of 10 putative genes were further characterized via Northern hybridization. Putative glyceraldehyde-3-phosphate dehydrogenase and arginine kinase are related to provision of energy for active cellular function in oocyte development. Translationally controlled tumor protein, actin, and keratin are related to the organization of cytoskeleton to accomplish growth and development of oocytes. High mobility group protein DSP1, heat shock protein 70, and nucleoside diphosphate kinase may act as repressors before the onset of ovarian maturation. Peptidyl-prolyl cis-trans isomerase and glutathione peroxidase are related to the stabilization of proteins and oocytes. This study provides new insights on the molecular events in the ovarian development in the shrimp.

Spectral changes of C-phycocyanin with different molar ratios of SPDP

Pure C-phycocyanin was prepared from Spirulina platensis using one-step anion-exchange chromatography. The C-PC obtained was with an absorption maximum at 620 nm and a fluorescence emission maximum at 640 nm when excited by 580 nm. SPDP is an excellent heterobifunctional crosslinker for thiolating amines. Different molar ratios of SPDP have remarkable influence on the absorption and fluorescence spectra of C-phycocyanin. The absorption maximum and fluorescence emission maximum both decreased and blue-shifted from 640 run to 630 nm as the molar ratios of SPDP increased. It was found that the molar ratios of SPDP to C-phycocyanin was not more than 100 was appropriate to being conjugated with other biomolecules from the absorption and fluorescence spectra of C-phycocyanin.

Proteomic analysis of differentially expressed proteins in lymphoid organ of Fenneropenaeus chinensis response to Vibrio anguillarum stimulation

To gain an insight into the function of shrimp lymphoid organ at protein level, we analyzed the proteome of lymphoid organ in healthy Chinese shrimp Fenneropenaeus chinensis (F. chinensis) through two-dimensional gel electrophoresis (2-DE) based proteomic approach. A total of 95 spots representing 75 protein entries were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS) with both online and in-house database. According to Gene Ontology (GO) annotation of biological process, the identified proteins were classified into 13 categories. Among them, approximately 36% of proteins related to cytoskeleton are noticeable. Then, a comparative proteomic approach was employed to investigate the differentially expressed proteins in lymphoid organ of Vibrio anguillarum-challenged F. chinensis. At 24 h post-injection (hpi), 17 differentially expressed protein spots were successfully identified, including 4 up-regulated protein spots (represent 4 proteins: cathepsin L protein similar to squid CG16901-PC, protein kinase C and protein similar to T-complex Chaperonin 5 CG8439-PA), and 13 down-regulated protein spots (represent 9 proteins: actin, beta-actin, cytoplasmic actin CyII, alpha tubulin, beta tubulin, protein similar to proteasome delta, vacuolar ATP synthase subunit B, elongation factor 2, carboxypeptidase B). These data may help us to understand the function of lymphoid organ and the molecular immune mechanism of shrimp responsive to pathogen infection. (C) 2010 Elsevier Ltd. All rights reserved.

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。