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凡纳滨对虾水孔蛋白-4的cDNA克隆及盐度胁迫对其肝胰腺mRNA表达水平的影响
No full text为了探索水孔蛋白(Aquaporin,AQP)在凡纳滨对虾(Litopenaeus vannamei)渗透压调节过程中作用,本研究通过RACE方法获得克隆的凡纳滨对虾AQP(命名为LvAQP4)的c DNA全长序列,并分析了盐度胁迫对其肝胰腺m RNA表达的影响。结果发现:LvAQP4 c DNA序列全长为1 048 bp,其中包括75 bp的5￠UTR,187 bp的3￠UTR和786 bp的ORF。根据ORF序列推导出LvAQP4编码261个氨基酸,预测其分子质量为27.85 k Da,等电点为8.11。推导的氨基酸序列与其他甲壳物种的AQP相似度为48.8%到97.3%。进化分析显示LvAQP4属于AQP1-like亚族、AQP4类。定量PCR(q PCR)检测到LvAQP4在不同组织中均有表达,其中鳃的表达量最高,肝胰腺、肌肉、脑和眼柄中也较高,而血淋巴、肠、胃和胸神经节中表达量则较低。在高盐(盐度40)刺激下,凡纳滨对虾的肝胰腺LvAQP4 m RNA表达量会随着时间推移而上升,到6 h达到最高,而后逐步下降。而在低盐(盐度4)刺激下,凡纳滨对虾的肝胰腺LvAQP4 m RNA表达量并无明显变化。在原代分离和培养的肝胰腺细胞中,培养液中加入额外的Na Cl会剂量依赖地提高LvAQP4 m RNA表达水平。同样,通过在培养液中额外加入蔗糖提高培养液渗透压,也会剂量依赖地提高LvAQP4 m RNA表达水平,但作用不如Na Cl明显。这些结果表明盐度与肝胰腺LvAQP4的表达量有关,LvAQP4对凡纳滨对虾的渗透压调节具有非常重要的作用
The Application of Marine Micrioial Lipase/Esterases and Thermostable Superoxide Dismutases
No full text脂肪酶、酯酶和超氧化物歧化酶均是重要的工业用酶，脂肪酶、酯酶的立体选择性常被用来制备手性药物及其中间体，耐高温超氧化物歧化酶被广泛应用于食品、化妆品和医药行业。本研究从三株海洋来源的细菌中克隆、表达和定性了三个新颖的脂肪酶/酯酶，它们都具有手性拆分功能，另外从两株海洋来源的耐高温细菌中克隆、表达和定性了两个超氧化物歧化酶。本研究从囊孢菌Dactylosporangium aurantiacum subsp. hamdenensis NRRL 18085基因组中获取了一个新颖的酯酶DAEst6。经多态性分析，DAEst6属于脂水解酶第VII家族。酯酶DAEst6能够通过催化扁桃酸甲酯不对称的水解反应生成重要的手性药物中间体——(R)-扁桃酸甲酯。通过条件优化，实验得到DAEst6催化扁桃酸甲酯不对称的水解的最佳条件：10 mM外消旋扁桃酸在50μg DAEst6纯酶作用下，于pH 7.5缓冲液中，40 °C反应5 h，可获得光学纯度为99 %的(R)-扁桃酸甲酯，转化率为49 %。本文是第一个定性囊孢菌脂肪酶/酯酶的文献，并成功地利用囊孢菌酯酶进行手性药物中间体制备
Occurrence of antibiotics in Hailing Bay Region and mechanism on algae responses to antibiotic stress
No full text近年来，水环境中残留抗生素的存在开始受到人们的广泛关注。海洋作为陆源污染物的汇，接收来自污水直接排放、地表径流输入的抗生素。进入海洋环境的抗生素，其中相当一部分仍具有生物活性，可以通过生物富集作用对水生生物造成潜在的危害,并对人体造成饮食暴露风险。目前，国内外对于抗生素在水环境中的分布和归趋已经有了一些报道，但对海洋环境中抗生素的来源及污染特征研究仍然不足。因此，本文选取广东省海陵湾为研究区域，调查了抗生素在海湾环境中的污染分布情况，对抗生素的污染来源进行解析；研究了藻类在抗生素胁迫下的生理特征响应，并考察了藻类对抗生素的去除作用。论文研究的主要结果如下： (1)研究了37种抗生素在海陵岛典型水产养殖区的污染特征，在海水样品中，磺胺甲恶唑、盐霉素和甲氧苄胺嘧啶的检出率较高，浓度范围为0.4-36.9 ng/L，氧四环素是幼虾虾塘养殖水体中主要的抗生素。在饲料样品中恩诺沙星广泛检出，浓度范围为16.6-31.8 ng/g。在沉积物样品中红霉素检出率最高，浓度范围为0.8-4.8 ng/g。在长毛明对虾成虾肌肉组织中红霉素含量最高，浓度范围为2498-15090 ng/g。此外，甲氧苄胺嘧啶在勒氏笛鲷体内有生物富集性，其生物富集因子为6488 L/kg。根据每日摄入量进行估算，长毛明对虾成虾体内的红霉素可能对人体健康造成风险。 (2)研究了38种抗生素在海陵湾区域表层水体和沉积物中的污染特征。在海水样品、码头水样和沉积物中分别检出21、16和15种抗生素，浓度范围分别为＜0.08(克拉霉素)-15163 ng/L(氧四环素)、2.1(甲烯土霉素)-1318 ng/L(红霉素)和＜2.0(环丙沙星)-184 ng/g(氯四环素)。水相中抗生素浓度与环境变量化学需氧量和硝酸盐相关。对海陵湾抗生素来源分析表明，未经处理的生活污水是抗生素污染的主要来源。喹诺酮类抗生素吸附沉积物的能力较强。风险评价表明氧四环素、诺氟沙星和红霉素对水生生物造成高风险。 (3)考察了眼点拟微球藻和近头状伪蹄型藻在红霉素胁迫下的生理特征响应，并研究了两种藻类对诺氟沙星和磺胺嘧啶去除作用。在红霉素胁迫条件下，眼点拟微球藻和近头状伪蹄型藻的EC50值分别为6.13 mg/L和27.7 μg/L。随着红霉素浓度的增加，藻细胞的光合色素显著降低，表明藻类的细胞结构遭到破坏，细胞功能受到损伤。藻细胞过氧化氢酶和超氧化物歧化酶的活性随着红霉素浓度的增加呈现先升高后降低的趋势，酶活性增强表明藻细胞受到氧化胁迫压力，细胞内的H2O2进一步积累，H2O2去除速率与生产速率失衡导致酶活性急剧下降。眼点拟微球藻在144 h对诺氟沙星和磺胺嘧啶去除率分别达到57.0%和78.3%，近头状伪蹄型藻在120 h对诺氟沙星的去除率达到51.2%。诺氟沙星和磺胺嘧啶对所选抗生素的去除动力学均符合一级动力学方程
Functional Studies of Toxin-antitoxin System and H-NS on the Native Plasmid of Pseudoalteromonas rubra
No full text假交替单胞菌作为一类典型的海洋细菌，在海洋环境中分布广泛。本研究对两株不同海域来源的，产灵菌红素的假交替单胞菌SCSIO 6842和DSM 6842进行了亲缘关系鉴定，发现这两株菌株属于同属同种中的不同亚种；对其表型特征进行分析，发现其表型特征如生物被膜形成等存在明显的差异；同时，通过全基因组序列比对分析，发现其基因组序列存在着区域性差异，而且这些差异主要为可遗传元件如转座酶和基因岛；尤其SCSIO 6842携带了一个内源质粒pMBL6842，而DSM 6842中不存在质粒。利用我们实验室建立的适合假交替单胞菌的遗传操作体系，成功得到了质粒pMBL6842的缺失突变株；表型分析发现质粒的存在能够增加宿主对外源质粒的抗性。进一步分析发现，质粒为潜在的接合转移型质粒，并携带一对潜在的毒素-抗毒素系统和一个DNA结合蛋白H-NS。因此，对于质粒pMBL6842的研究我们开展了如下的工作
Study of oyster peptide separation and fishy odor substance removal
No full text香港巨牡蛎（Crassostrea hongkongensis）是我国南方沿海主要经济贝类和养殖优良品种，经酶解后获得的小分子肽具有多种生物活性，具有广阔的开发利用前景。但其酶解液中的腥味影响并限制了其在生物医药、保健品、化妆品中的应用。本论文通过体外细胞模型研究了牡蛎小分子肽的对肠黏膜细胞的生物活性，并利用RP-HPLC、ESI-MS、MALDI-TOF-MS和Edman氮端测序等手段分析纯化活性肽的组成、分子量、蛋白次序等结构信息；采用HS-SPME-GC-MS 分析了牡蛎酶解液及不同相、不同分子量段腥味物质，确定海洋经济贝类共性腥味物质；构建活性炭吸附三甲胺模型对吸附机理进行探讨；以感官评价和氨基酸回收率为指标，采用吸附法、包埋法、掩蔽法、美拉德反应、柠檬酸反应法、抗氧化法等对牡蛎酶解液进行脱腥工艺优化研究。以上研究获得了以下主要结论
Antiviral function of Tachyplesin I against iridovirus and nodavirus
No full textAntimicrobial peptides (AMPs) are ubiquitously found in living organisms and are an important component in innate immune response. Tachyplesin I is a potent antimicrobial peptide isolated from the hemocytes of the horseshoe crab, Tachypleus tridentatus. Previous studies have shown that the 17-residue peptide exhibits a wide spectrum of antimicrobial activity against Gram-negative and Grampositive bacteria, fungi, protozoa, and viruses. However, the efficiencies and defense mechanisms of the Tachyplesin I against fish viruses are still unknown. In this study, Tachyplesin I showed a key role in inhibiting the infection and replication of two kinds of newly emerging marine fish viruses, an enveloped DNA virus of Singapore grouper iridovirus (SGIV), and a non-enveloped RNA virus of viral nervous necrosis virus (RGNNV). Synthetic peptides of Tachyplesin I incubated with virus or cells before infection reduced the viral infectivity. Synthetic peptides of Tachyplesin I drastically decreased SGIV and RGNNV titers and viral gene expression. Grouper spleen (GS) and brain (GB) cells over-expressing Tachyplesin I (GS/pcDNA3.1-flag-Tac I and GB/pcDNA3.1-flag-Tac I) support the inhibition of viral infection. Tachyplesin I activated type I IFN and Interferon-sensitive response element (ISRE) in vitro. The promoter activity of IFN-13 and ISRE were significantly up-regulated in cells transfected with pcDNA3.1-flag-Tac I after infection with SGIV and VNNV. These results suggest that Tachyplesin I is importantly involved in host immune responses to invasion of viral pathogens. (C) 2016 Elsevier Ltd. All rights reserved
Genomic structure and expression pattern of MHC II alpha and II beta genes reveal an unusual immune trait in lined seahorse Hippocampus erectus
No full textThe major histocompatibility complex (MHC) genes are crucial in the adaptive immune system, and the gene duplication of MHC in animals can generally result in immune flexibility. In this study, we found that the lined seahorse (Hippocampus erectus) has only one gene copy number (GCN) of MHC II alpha and II beta, which is different from that in other teleosts. Together with the lack of spleen and gut-associated lymphatic tissue (GALT), the seahorse may be referred to as having a partial but natural "immunodeficiency". Highly variable amino acid residues were found in the II alpha and 1113 domains, especially in the alpha 1 and beta 1 domains with 9.62% and 8.43% allelic variation, respectively. Site models revealed seven and ten positively selected positions in the alpha 1 and beta 1 domains, respectively. Real-time PCR experiments showed high expression levels of the MHC II genes in intestine (In), gill (Gi) and trunk kidney (TK) and medium in muscle (Mu) and brood pouch (BP), and the expression levels were significantly up-regulated after bacterial infection. Specially, relative higher expression level of both MHC II alpha and II beta was found in Mu and BP when compared with other fish species, in which MHC II is expressed negligibly in Mu. These results indicate that apart from TIC, Gi and In, MU and BP play an important role in the immune response against pathogens in the seahorse. In conclusion, high allelic variation and strong positive selection in PBR and relative higher expression in MU and BP are speculated to partly compensate for the immunodeficiency. (C) 2016 Elsevier Ltd. All rights reserved
Changes in microbial communities, photosynthesis and calcification of the coral Acropora gemmifera in response to ocean acidification
No full textWith the increasing anthropogenic CO2 concentration, ocean acidification (OA) can have dramatic effects on coral reefs. However, the effects of OA on coral physiology and the associated microbes remain largely unknown. In the present study, reef-building coral Acropora gemmifera collected from a reef flat with highly fluctuating environmental condition in the South China Sea were exposed to three levels of partial pressure of carbon dioxide (pCO(2)) (i.e., 421, 923, and 2070 mu atm) for four weeks. The microbial community structures associated with A. gemmifera under these treatments were analyzed using 16S rRNA gene barcode sequencing. The results revealed that the microbial community associated with A. gemmifera was highly diverse at the genus level and dominated by Alphaproteobacteria. More importantly, the microbial community structure remained rather stable under different pCO2 treatments. Photosynthesis and calcification in A. gemmifera, as indicated by enrichment of delta O-18 and increased depletion of delta C-13 in the coral skeleton, were significantly impaired only at the high pCO(2) (2070 mu atm). These results suggest that A. gemmifera can maintain a high degree of stable microbial communities despite of significant physiological changes in response to extremely high pCO(2)
Cryptophyte farming by symbiotic ciliate host detected in situ
No full textProtist-alga symbiosis is widespread in the ocean, but its characteristics and function in situ remain largely unexplored. Here we report the symbiosis of the ciliate Mesodinium rubrum with cryptophyte cells during a red-tide bloom in Long Island Sound. In contrast to the current notion that Mesodinium retains cryptophyte chloroplasts or organelles, our multiapproach analyses reveal that in this bloom the endosymbiotic Teleaulax amphioxeia cells were intact and expressing genes of membrane transporters, nucleus-to-cytoplasm RNA transporters, and all major metabolic pathways. Among the most highly expressed were ammonium transporters in both organisms, indicating cooperative acquisition of ammonium as a major N nutrient, and genes for photosynthesis and cell division in the cryptophyte, showing active population proliferation of the endosymbiont. We posit this as a "Mesodinium-farming-Teleaulax" relationship, a model of protist-alga symbiosis worth further investigation by metatranscriptomic technology
Synthesis and antibacterial evaluation of hamacanthin B analogues
No full textHamacanthins are a class of antibacterial bisindole alkaloids isolated from marine sponges. Based on structure-activity relationships and in silico MRSA PK binding analysis of these bisindole alkaloids, the authors designed new hamacanthin B derivatives and evaluated their antibacterial activities against drug-resistant pathogens. Racemates of the synthetic products were resolved into their enantiomers by chiral separation using a cellulose column, and antibacterial activities were compared. Unsaturation of the central heterocyclic ring structure and bromine substitution at the indole moiety were found to enhance the antibacterial activities of hamacanthin B analogues. (C) 2016 Elsevier Ltd. All rights reserved