Key genes expression of reductive tricarboxylic acid cycle from deep-sea hydrothermal chemolithoautotrophic Caminibacter profundus in response to salinity, pH and O2

CO2 fixation pathway of Caminibacter profundus, a chemolithoautotrophic -Proteobacteria from deep-sea hydrothermal vent, was determined and characterized by genetic and enzymatic analyses. Gene expression of key enzymes for CO2 fixation in response to salinity, pH and O2 in Medium 829 were also investigated. The results demonstrate that C. profundus contained aclB, porA and oorA, the genes encoding key enzymes of reductive tricarboxylic acid (rTCA) cycle. However, genes fragments of cbbL and cbbM encoding key enzyme of Calvin cycle were not recovered. Key enzymatic activities of ATP citrate lyase (ACL), pyruvate: ferredoxin oxidoreductase (POR) and 2-oxoglutarate: ferredoxin oxidoreductase (OOR) were also present in C. profundus. The combination of genetic and enzymatic analyses confirm that C. profundus adopted rTCA cycle for carbon assimilation. The results of aclB and oorA relative expressions of C. profundus demonstrate that the ranges of environmental factors for high genes expression were sea salt 3.0%-5.0% (optimum 3.0%), pH 5.0-6.5(optimum pH 6.5), anaerobic to microaerobic conditions (optimum 1.0% O2 ). Gene expression patterns under different conditions show similar patterns with bacterial growth, revealing that key rTCA cycle genes provided molecular basis for bacterial growth and propagation. Our results suggest that C. profundus could regulate key genes of rTCA cycle for carbon assimilation and energy metabolism in response to environmental fluctuations in hydrothermal vent.     

Enzyme variation in marine and estuarine populations of a mud crab,Macrophthalmus hirtipes (Ocypodidae)

Protein and enzyme variation in 2 populations of the mud crab Macrophthalmus hirtipes (Jacquinot, 1853) (Ocypodidae) from 1 marine and 1 estuarine habitat were investigated by poly‐acrylamide gel electrophoresis. A survey of 24 loci revealed that 22 were common to both populations; 20 were monomorphic and 2 were highly polymorphic. Two alleles were detected for each of the polymorphic loci, esterase‐2 (EST‐2) and ester‐ase‐3 (EST‐3). The frequencies of the EST‐3 alleles were similar in the 2 populations. However, the frequencies of the 2 EST‐2 alleles in the estuarine population were significantly different from those for the marine population. Expression of 1 locus, esterase‐4 (EST‐4) was confined to the estuarine population. Two alkaline phosphatase loci (AKPH‐1 and AKPH‐2) were detected in the estuarine population, but only AKPH‐1 was found in the marine population. EST‐4 and AKPH‐2 were neither sex nor age specific. These interpopulational genetic differences may reflect differences in environmental conditions measured between the 2 habitats.     

Involvement of anti-oxidative enzymes, photosynthetic pigments and flavonoid metabolism in the adaptation of Reaumuria soongorica to salt stress

Reaumuria soongorica is a short woody shrub widely found in semi-arid areas of China. It can survive severe environ- mental stress including high salinity in its natural habitat. Thus, we investigated the involvement of anti-oxidative enzymes, photosynthetic pigments and flavonoid metabolism in the adaptation of R. soongorica to saline environments. R. soon- gorica was treated with 0, 100, 200 and 400 mM NaC1 solutions for 14 days. Soil salt content increased significantly by watering with high content of NaC1 solution, and no variation between 8 and 14 days during treatment. The levels ofpe- roxidation of lipid membranes （measured by malondialdehyde content） and the activities of three antioxidant enzymes （superoxide dismutase （SOD）, peroxidase （POD） and ascorbate peroxidase （APX）） increased under salt stress. Chlorophyll and carotenoid content decreased with increasing salt content. The ratio of Chl a/Chl b and carotenoid/Chl exhibited sig- nificant increase under 400 mM NaC1. However, total flavonoid and anthocyanin contents and key enzyme activities in the flavonoid pathway including phenylalanine ammonialyase （PAL） and Chalcone isomerase （CHI） decreased under salt stress. These findings possibly suggest that R. soongorica has an adaptation protection mechanism against salt-induced oxidative damage by inducin~ the activity of antioxidant enzymes and maintaining a steady level of carotenoid/Chl.     

Effect of cadmium, copper and mercury on antioxidant enzyme activities and lipid peroxidation in the gills of the hydrothermal vent mussel Bathymodiolus azoricus

Metals are known to influence lipid peroxidation and oxidative status of marine organisms. Hydrothermal vent mussels Bathymodiolus azoricus live in deep-sea environments with anomalous conditions, including high metal concentrations. Although B. azoricus are aerobic organisms they possess abundant methano and thioautotrophic symbiotic bacteria in the gills. The enzymatic defences (superoxide dismutase (SOD), catalase (CAT), total glutathione peroxidase (Total GPx) and selenium-dependent glutathione peroxidase (Se–GPx)) and lipid peroxidation were determined in the gills of B. azoricus exposed to Cd (0.9 μM), Cu (0.4 μM) and Hg (0.1 μM) with different times of exposure. The experiments were performed in pressurized containers at 9 ± 1 °C and 85 bars.Results show that vent mussels possess antioxidant enzymatic protection in the gills. Cd and Cu had an inhibitory effect in the enzymatic defence system, contrarily to Hg. These enzymatic systems are not completely understood in the B. azoricus, since reactive oxygen species might be produced through other processes than natural redox cycling, due to hydrogen sulphide and oxygen content present. Also the symbiotic bacteria may play an important contribution in the antioxidant protection of the gills.     

Determinations of dimethylsulphoniopropionate (DMSP) lyase activity using headspace analysis of dimethylsulphide (DMS)

The osmolyte dimethylsulphoniopropionate (DMSP) can be enzymatically cleaved to dimethylsulphide (DMS), acrylate and a proton. The enzyme involved in this reaction is dimethylpropiothetin dethiomethylase (DMSP lyase; enzyme classification number 4.4.1.3.). Although the importance of this reaction for the global sulphur cycle, the influence of DMS on atmospheric acidity and the possible effect on climate regulation have been widely recognised, our knowledge of DMSP lyases is limited to just a few studies. Activity measurements of DMSP lyases offer an important step towards a better understanding of the conditions under which DMS is produced. In the available published data somewhat similar methods have been used before, but a critical examination of the method limitations has not been reported. To encourage further research on this enzyme, we suggest and detail two protocols for measurements of DMSP lyase activity: An in vitro assay for crude cell extracts or purified enzyme and an in vivo method for whole cells, which we recently started to use. After addition of DMSP, samples incubated in a gas tight vial may produce DMS from enzymatic cleavage under suitable conditions, and a DMS production rate can be estimated from time-series measurements of DMS in the headspace of the vial. Headspace analysis of DMS is a useful and rapid technique to estimate and compare DMSP lyase activities from different sources. The relative rates of DMS production in the liquid and of the gas transfer between liquid and headspace, determine the rate of DMS production measured via headspace analysis. If DMS production in the liquid is higher than the rate of transfer, headspace measurements will not reflect the actual amount of DMS produced in the liquid. In this case, extracts have to be diluted to a level that ensures linearity between dilution factor and reduction of enzyme activity. Additionally, incubation volumes and vials should be selected to provide a high surface-to-volume ratio to ensure maximum flux of DMS from the aqueous phase into the headspace. The methods can be adapted to further investigate species- and strain-specific activities, biogeographical distribution, cellular location and biochemical properties of various DMSP lyases.     

降雨量对科尔沁沙地3种沙生植物生长和生理的影响

对降水格局变化的响应是植物适应环境的重要方面。通过野外增减雨试验,研究了降水变化对科尔沁沙地3种沙生植物生长特性和生理特征的影响。结果表明：（1）6月植被平均密度最大,7月平均盖度最大,降雨量增加60%时,植被盖度最大,为58.0%。（2）增雨区的主要植物为雾冰藜（Bassia dasyphylla）和猪毛菜（Salsola collina）,减雨区主要植物为蒺藜（Tribulus terrestris）,降雨量减少60%时,蒺藜在6、7、8月密度分别为70%、80%、79%,显著高于其他植物。（3）降雨量减少时,3种沙生植物的相对含水量（RWC）减少,而细胞膜透性增加;蒺藜RWC高于雾冰藜和猪毛菜,但是丙二醛（MDA）正好相反;蒺藜的耐脱水能力和细胞膜的耐伤害程度强于雾冰藜和猪毛菜。（4）随着降雨量的增加,3种植物的光能转化效率（F_v/F_m、ΦPSⅡ）逐渐增加,但随干旱天数的增加而减小。     

皱纹盘鲍群体选育第四代成鲍4种多糖裂解酶基因表达的初步研究

根据已发表c DNA序列设计引物扩增了2龄皱纹盘鲍(Haliotis discus hannai Ino)的褐藻酸酶、海带淀粉酶、α-淀粉酶和纤维素酶基因片段并测定其序列,同时用半定量反转录PCR和实时荧光定量PCR技术分析了它们在不同组织中的表达。结果表明:以cDNA为模板的PCR产物序列与已发表的相应基因序列一致;以DNA为模板扩增的a-淀粉酶和纤维素酶基因片段分别含438 bp和667 bp的内含子;在纤维素酶基因片段的外显子和内含子区各检测到2个SNP;4种多糖裂解酶基因均主要在消化腺中表达,褐藻酸酶基因的表达最强,其次为纤维素酶,表明在摄食海带的条件下,褐藻酸是成体皱纹盘鲍可利用的最重要多糖类物质之一。本文的结果为进一步开展皱纹盘鲍"97"选育群体在不同发育期、摄食不同藻类及不同培育环境下的多糖水解酶基因表达研究奠定了基础。     

微泡菌QZHA1褐藻胶裂解酶MAAL1的酶学性质研究

为探究Microbulbifer sp. QZHA1褐藻胶裂解(Escherichia coli)酶MAAL1的酶学性质,将褐藻胶裂解酶基因maal1构建至pET-28a表达载体并利用大肠杆菌BL21(DE3)进行异源表达。研究发现：重组酶MAAL1与来源于Microbulbifer sp. ALW1菌株的褐藻胶裂解酶(WP＿23625014.1)同源性最高,为93.69%,且与PL7家族蛋白聚为一支;重组酶MAAL1最适温度为35℃,最适pH为7.5,在pH为5.5～10.5范围内保存24 h仍能保持60%以上的酶活力;MAAL1具备良好的耐有机溶剂特性,在测试的9种有机溶剂中,除异丙醇外,其他有机溶剂在添加量达到30%(体积分数)后,酶活力依然保持在59%以上;重组酶MAAL1最适条件下酶活力为4.3 U/mg,米氏常数(K_m)值为1.08 mg/mL,最大反应速率(V_max)为4.75 mg/(mL·min),催化常数(K_cat)值为4.52 s^-1;重组酶MAAL1对聚β-D-甘露糖醛酸(p...     

3种致病弧菌感染对大黄鱼7种酶活性的影响

大黄鱼溃疡病主要由3种致病弧菌引起:溶藻弧菌Vibrio alginolyticus、哈维弧菌V. harveyi和副溶血弧菌V. parahaemolyticus。为了解大黄鱼抗病原菌感染的免疫反应机理,对其疾病防御提供重要的理论基础,本实验将健康的大黄鱼随机分为3个实验组和1个对照组,分别注射0.2 mL的哈维弧菌、溶藻弧菌、副溶血弧菌及灭菌生理盐水。在人工感染后的第0,1,2,4,7,10,13,16,20天分别采样,通过测定每组大黄鱼血清中7种免疫相关酶活性,比较这3种致病弧菌对大黄鱼影响的差异性。结果表明:在感染后的1～10 d,实验组大黄鱼血清中血清溶菌酶(LSZ)、酸性磷酸酶(ACP)、过氧化物酶(POD)、髓过氧化物酶(MPO)的活性变化,总体表现为先升高、后降低再逐渐升高最后趋于-致的现象,且不同病原菌之间存在-定的时效差异。 碱性磷酸酶(AKP)对入侵体内的哈氏弧菌反应较敏感,ACP、POD和MPO对溶藻弧菌反应较敏感,LSZ和PO对副溶血弧菌反应较敏感。