饲料盐藻水平对卵形鲳鲹体色和生长的影响

以初始体质量为(13.88±0.28)g的卵形鲳鲹为试验对象,研究不同盐藻添加量(0.0%、0.1%、0.2%、0.4%、0.8%、1.6%)饲料对其体色和生长的影响。每组设3个平行,每个平行饲养20尾鱼,每日饱食投喂2次(8:00;17:00),试验周期为56d。结果显示,盐藻添加量为0.1%~1.6%时,随着饲料中盐藻水平的提高,卵形鲳鲹背部和腹部黄色值(b*)呈显著上升趋势(P0.05),饲料中添加盐藻可明显改善卵形鲳鲹体色;与此同时,卵形鲳鲹质量增加率和特定生长率随饲料盐藻水平的上升呈下降趋势(P0.05),未添加盐藻的对照组具有最高蛋白质效率和最低饲料系数,且与盐藻添加量为0.4%的试验组差异显著(P0.05),饲料中添加盐藻对卵形鲳鲹生长性能和饲料利用没有明显的促进作用;此外饲料盐藻水平对卵形鲳鲹体成分也没有显著影响(P0.05)。     

Effects of Iron Deficiency on Thylakoid Membrane Structure and Composition in the Alga Dunaliella salina

Abstract

The unicellular green alga Dunaliella was previously proposed as a model photosynthetic organism for adaptation to iron deficiency. The purpose of this study was to find out how iron limitation affects the structure and composition of the photosynthetic system of Dunaliella salina. Iron deprivation did not retard proliferation of D. salina cells, but was associated with a decrease in cell volume and chlorophyll content, and with a four‐fold reduction in iron content and a two‐fold increase in Cu content. Electron microscopic analysis revealed shrinkage of the chloroplast and decrease in stacked thylakoid membranes. Measurements of chlorophyll fluorescence induction in the presence of DCMU and of 77 K chlorophyll fluorescence emission spectra indicated gross changes in the photosynthetic efficiency of reaction centers and in the organization of their associated light harvesting antenna. Differential analysis of protein composition led to the identification of a major thylakoid membrane protein (Tidi), that was specifically induced under iron deprivation. Partial sequencing suggests that Tidi is a novel type of a chlorophyll a/b binding protein. These results clearly show that iron limitation is associated with extensive reorganization of the photosynthetic system in Dunaliella.     

巴夫杜氏藻β-肌动蛋白基因的克隆和分析

[目的]获得巴夫杜氏藻β-肌动蛋白基因cDNA全长序列。[方法]以巴夫杜氏藻cDNA为模板,采用简并引物进行PCR扩增,获得533 bp特异cDNA片段。在此基础上,设计特异引物,采用5′-GenomeWalking和3′-RACE的方法,获得基因的5′-端DNA序列和3′-端cDNA序列,进而获得β-肌动蛋白基因cDNA全长序列。[结果]获得了巴夫杜氏藻β-肌动蛋白基因的特异cDNA片段、5′-端DNA和3′-端cDNA片段。经拼接后,扩增出全长cDNA。β-肌动蛋白基因cDNA全长1 754 bp,包括1 137 bp的开放读码框和617 bp的3′-非翻译区序列。氨基酸序列相似性分析发现,巴夫杜氏藻β-肌动蛋白氨基酸序列与杜氏盐藻、莱茵衣藻等的同源性较高。系统发育分析表明,巴夫杜氏藻β-肌动蛋白与杜氏盐藻的相似性最高。[结论]首次获得了巴夫杜氏藻β-肌动蛋白基因cDNA全长序列并发现巴夫杜氏藻β-肌动蛋白基因非常保守。     

Assessment of the Antimicrobial Activity of Algae Extracts on Bacteria Responsible of External Otitis

External otitis is a diffuse inflammation around the external auditory canal and auricle, which is often occurred by microbial infection. This disease is generally treated using antibiotics, but the frequent occurrence of antibiotic resistance requires the development of new antibiotic agents. In this context, unexplored bioactive natural candidates could be a chance for the production of targeted drugs provided with antimicrobial activity. In this paper, microbial pathogens were isolated from patients with external otitis using ear swabs for over one year, and the antimicrobial activity of the two methanol extracts from selected marine (Dunaliella salina) and freshwater (Pseudokirchneriella subcapitata) microalgae was tested on the isolated pathogens. Totally, 114 bacterial and 11 fungal strains were isolated, of which Staphylococcus spp. (28.8%) and Pseudomonas aeruginosa (P. aeruginosa) (24.8%) were the major pathogens. Only three Staphylococcus aureus (S. aureus) strains and 11 coagulase-negative Staphylococci showed resistance to methicillin. The two algal extracts showed interesting antimicrobial properties, which mostly inhibited the growth of isolated S. aureus, P. aeruginosa, Escherichia coli, and Klebsiella spp. with MICs range of 1.4 × 10⁹ to 2.2 × 10¹⁰ cells/mL. These results suggest that the two algae have potential as resources for the development of antimicrobial agents.     

Ecotoxicological evaluation of Harbour sediments using marine organisms from different trophic levels

Background, Aim and Scope  The toxicity of contaminated sediments should be evaluated considering the direct exposure of laboratory organisms to whole sediments and the indirect exposure to elutriates or extracts (Tay et al. 1992, Byrne and Halloran 1999, Nendza 2002). The alga Dunaliella tertiolecta is indicated for the use in toxicity bioassays because it is highly sensitive to several xenobiotics. Harpacticoid copepods have been already used for toxicity testing and Tigriopus fulvus is a promising Mediterranean target-species in ecotoxicology (Todaro et al. 2001, Faraponova et al. 2003, Pane et al. 2005a). In this study, the toxicity of sediments collected in harbour sites of the Northeastern Adriatic Sea was evaluated by growth inhibition test with free living and alginate-immobilized Dunaliella tertiolecta and acute toxicity test with nauplii and adult Tigriopus fulvus with the aim of pointing out the importance to utilize model organisms from different trophic levels in sediment ecotoxicology. Methodology  Elutriates and whole sediments were tested on free living and immobilized (Pane et al. 1998) algal cells, and on laboratory reared copepods. Free-living D. tertiolecta were exposed to diluted elutriates in a static, multi-well plate system. Naalginate immobilized D. tertiolecta were placed in polystyrene inserts fitted with polyester mesh bottoms and exposed to a thin layer (2 mm) of whole sediments in multi-well plates (EPS 1992, Pane and Bertino 1999). Toxicity tests with copepods were carried out on Tigriopus fulvus nauplii (elutriates) and adults (whole sediments and elutriates). Same-aged nauplii useful for toxicity tests were obtained by egg sac detaching and consequent hatching stimulation (Pane et al. 2006). Newborn nauplii (I–II stage) were exposed to elutriates in multi-well plates provided with polystyrene inserts. Adult T. fulvus maintained in polystyrene inserts fitted with polyester mesh bottoms were placed in contact with a thin layer (2 mm) of whole sediment placed on multi-well plate bottoms. All end-points were evaluated after 96 h. Results  In general, the effects increased with the increasing of elutriate concentration up to 50%; the stimulation or inhibition of algal growth was statistically significant in comparison to the control. The inhibiting elutriates induced EC₅₀ variations of algal growth ranging from 66.9% to 74.3%. The mortality of T. fulvus nauplii was always < 25% after treatment with 100% elutriates and < 10% after treatment with 50% dilution; no effect was shown up with 25% dilution; therefore LC₅₀ was not calculable. The effect of elutriates was negligible on adult copepods and LC₅₀ values were never calculable; percent mortality always resulted in < 10% after treatment with whole sediments. Discussion  Both experimental systems gave substantially similar results after exposition to whole sediments and elutriates. During the experiment with algal cells, the immobilization in Na-alginate and the employment of inserts which allowed the contact of organisms with sediments and their easy counting were particularly useful. Likewise, the employment of inserts of adequate mesh size in the tests with copepods allowed the contact of organisms with the sediment and made organism handling and counting easy, as well as the evaluation of mortality. The methodology here described and the utilization of the proposed test-species could have an importance also considering that the current trend in ecotoxicological research is towards finding the most appropriate organism for specific areas of concern by using indigenous species (Mariani et al. 2006) and towards the major significance of chronic and reproductive end-points. Conclusions  Based on the above results, it can be stated that the bioassay with Dunaliella tertiolecta could be a good estimation tool for the ecotoxicological assessment of marine sediments. The immobilization of algae in Na-alginate was seen to be useful to evaluate the toxicity of whole sediments; the employment of polystyrene inserts allowed an improvement of the procedures. T. fulvus nauplii and adults, as other harpacticoids such as Tigriopus japonicus (Yoon et al. 2006), satisfy the basic criteria for the employment of a standard species in marine bioassays. To date only pelagic Acartia tonsa are utilized in the standardized procedure to evaluate the risk assessment of chemicals or wastewaters (ISO 1999). As, on the contrary, the exposure of copepods to solid-phase contaminants it is not yet standardized, the employment of polystyrene inserts improved the procedures for T. fulvus too. So, the rapidity and the possibility to solve practical problems could be the main attractive features of this technique (Pane et al. 2005a) when applied to whole sediments. Recommendations and Perspectives  The methodology here developed being also applicable to long term and reproduction tests should be recommended because it provides relevant information in comparison with other frequently applied, standardized biotests with crustaceans (ISO 1999). The procedure has been shown to be easily applicable to selected marine organisms. ESS-Submission Editor: Prof. Dr. Henner Hollert (henner.hollert@bio5.rwth-aachen.de)     

杜氏盐藻MAPKK激酶基因DsMAPKKK的克隆、原核表达与纯化

为了进一步研究MAPKK激酶的生理功能,利用RT-PCR技术扩增了DsMAPKKK的开放阅读框序列,并利用T4连接酶与质粒pGS21a连接,构建了原核表达载体pGS21a-DsMAPKKK。将该重组质粒导入大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导成功表达了融合蛋白。经SDS-PAGE检测,融合蛋白在上清和包涵体中均存在。可溶性蛋白经过GST柱纯化,电泳分析结果表明,在68kD左右有单一的蛋白条带,说明融合蛋白得到有效纯化。蛋白印迹结果显示在68kD左右有明显的杂交条带,初步证明纯化的蛋白就是带有GST标签的MAPKK激酶。DsMAPKKK的成功表达与纯化,为深入探讨DsMAPKK激酶的性质及功能打下了坚实的基础。     

硼铜组合对盐藻生长与物质积累的影响

为了探索培养盐藻的微量元素条件,设计了室内模拟海洋环境下微量元素硼、铜的不同浓度组合,研究其对盐藻细胞生长与物质积累的影响。结果表明,培养液中适当的硼铜浓度组合对盐藻细胞的生长和物质积累有促进作用,而硼铜浓度过高或过低则对其不利。在试验的9种硼铜浓度组合中,以培养液中4 mg/L的硼和125 μg/L的铜浓度组合对盐藻细胞的生长和物质积累促进作用最好,它可使培养液中的盐藻细胞密度、蛋白质积累量和β-萝卜素积累量都达到最高。培养液中6 mg/L的硼和175 μg/L的铜浓度组合可使单个盐藻细胞中β-萝卜素和蛋白质积累量都最高,但可能仅是在铜、硼过多的逆境条件下,盐藻细胞的适应性反应。     

铁对盐藻生长与物质积累的调控作用研究

实验研究了不同浓度的铁对盐藻细胞生长与物质积累的调控作用。结果表明,培养液中供给铁过多或过少都不利于盐藻细胞的生长与物质积累。以培养基中30mg/L的铁浓度对盐藻细胞生长、蛋白质合成与β-胡萝卜素积累的促进作用最大。这一铁浓度可用于盐藻的生产性培养。     

杜氏盐藻和亚心型扁藻混合培养生长的初步研究

在实验室条件下研究了杜氏盐藻和亚心型扁藻在单独培养和相同接种比例混合培养下的生长情况。结果显示,单独培养盐藻的生长经历了3个明显的阶段,生长曲线呈现"S"型;单独培养扁藻与混合培养藻在18 d内还未到达稳定期,仍保持一定的生长态势。混合培养、单独培养盐藻以及单独培养扁藻的最大光密度值(OD680)分别为0.784、0.702和0.765。混合培养藻的生物量(0.841 mg/ml)也稍高于单独培养盐藻(0.582 mg/ml)和单独培养扁藻的生物量(0.819 mg/ml)。试验结果表明,混合培养盐藻和扁藻具有一定的促进藻生长和提高生物量产出的潜力。     

盐藻运动特性及其影响因素

为了揭示盐藻的运动特性,在不同条件下观察了玻璃U形管两端盐藻的密度变化。结果表明,盐藻运动受光照、ATP、丙二酸钠和粘度的影响。盐藻具有向光源方向聚集的运动特性;供给ATP时盐藻运动加速,加入丙二酸钠时盐藻运动减慢,盐藻的运动是一个消耗能量的过程;增加介质粘稠度会使其运动速度减慢。