雨生红球藻中虾青素含量快速测定方法

雨生红球藻中虾青素以虾青素单酯、二酯以及少量游离虾青素的形式存在,为了准确测定虾青素含量,通常需要将提取的虾青素酯水解转化为游离虾青素,再利用HPLC进行定量,操作耗时,不利于生产过程的快速监测。基于系统研究分光光度法直接测定细胞提取物中的混合虾青素含量和提取-酶解-HPLC法测定的关系,发现分光光度法估算的虾青素含量与HPLC法测定的准确含量之间具有良好的线性关系(r2=0. 997)。基于此建立了雨生红球藻虾青素快速测定方法,并对提取条件进行了优化。雨生红球藻粉(约5 mg)利用1 m L二甲基亚砜和6 m L丙酮进行1次提取,准确定容后,测定474 nm处的吸光度,根据吸光度与HPLC法虾青素含量间线性关系计算雨生红球藻中虾青素的含量。该方法操作简单,仅需10～20 min,测定准确,适于生产和流通环节的所需要的快速测定领域。

南海小轴海绵的组织结构和细胞特征

海绵动物是天然活性产物重要的生物来源。近年来针对海绵的研究主要集中在其化合物的分离鉴定和活性检测,而对其本身组织结构和细胞生物学的探索较少。文章采用组织学方法和透射电镜对小轴海绵的结构和原位细胞形态进行了观察和鉴定;通过细胞分离手段,将4种主要类型细胞富集,并进一步对其密度分布、光学特性、内含物、培养特性等进行了全面的检测。结果表明,小轴海绵水沟系为复沟型;具有外扁平细胞层、皮层和髓层结构;主要的细胞类型为小球细胞(A、B)、原细胞和领细胞;两种小球细胞具有较高密度、特殊的荧光和细胞内含物;原细胞和领细胞内含有糖类和脂类能量物质,提取物在可见光区有类胡萝卜素的吸收峰;除小球细胞B外,其余细胞均可富集后培养;领细胞与小球细胞可能存在分化衍生的关系。研究结果为进一步分析小轴海绵细胞分化、活性化合物的代谢通路和细胞培养模式提供了基础依据。

类火星环境的低气压和水对发状念珠藻复苏的影响

为了考察发状念珠藻(发菜)在类火星表面半开放体系复苏的可行性,搭建了在线监测模拟类火星环境密闭体系内发菜复苏时放氧速率的试验装置,并考察了水对发菜光合放氧复苏的影响。结果显示,在气压超过5℃、15℃、25℃所对应的水饱和蒸气压时,发菜放氧最高速率分别为5、7和10μmol O_2·h^(-1)·g^(-1)(DW),分别是发菜最大放氧速率的1%、2%和3%;而在气压低于相应的水饱和蒸气压时,发菜放氧平均速率分别下降为1、2和3μmol O_2·h^(-1)·g^(-1)(DW),且随着反应时间的延长,发菜在5℃放氧速率下降为0;然而,在5℃,气压为1.7~1.9 k Pa,加入10 mL BG11时,发菜放氧速率最高上调至12μmol O_2·h^(-1)·g^(-1)(DW)。以上结果显示,水对发菜在类火星表面半开放体系复苏的影响大于气压。

基于海绵共附生放线菌的抗结核活性物质筛选

目的:从海绵相关放线菌中高通量筛选具有拮抗分枝杆菌活性的菌株及其代谢物,评估已建立的海洋相关放线菌菌种库中抗分枝杆菌的活性比例,为发现新的拮抗结核病的先导化合物奠定基础.方法:对已建立的海绵相关放线菌菌种库中461株进行发酵,分别制备菌体丙酮提取物和发酵液乙酸乙酯萃取物,以抗耻垢分枝杆菌(Mycobacterium smegmatis,Msm)活性为导向,刃天青为筛选指示剂,于96孔板中测定放线菌菌体与发酵液萃取物的抗菌活性.通过测定供试菌株的16S rDNA序列,对抗Msm菌株进行聚类分析,获得具Msm抗性菌株种群特性.结果:筛选的461株放线菌中394株具有抗Msm活性,阳性率为85.5%,中等活性以上菌株占25.6%,32.2%的菌株菌体与发酵液均有活性,表明该菌种库所选择的评价体系阳性率高,具有抗结核药物及先导化合物的较好开发潜力.对23株具有高抑菌活性菌株16S rDNA分析表明,这些菌株均为链霉菌属.培养液乙酸乙酯萃取物中,菌株编号772,596与DL238抑菌活性最高,MIC为0.24μg/mL,低于目前广泛应用的乙胺丁醇(ethambutol)、链霉素(streptomycin)和吡嗪酰胺(pyrazinamide)等抗结核药物;菌体丙酮提物中,菌株D98活性最高为4.9μg/mL,与卷曲霉素(capreomycin)和卡那霉素(kanamycin)抗结核活性水平相当.结论:本研究结果表明海绵相关放线菌种库有望成为开发新型拮抗结核药物先导物的资源库.

湛江等鞭金藻藻际细菌群落与其耐高温关系分析

为解析湛江等鞭金藻(Isochrysis zhangjiangensis)野生藻株(IZ)与常压室温等离子体技术诱变获得的耐高温诱变藻株(IM)藻际细菌群落与耐高温能力的关联性,本研究采用16S rDNA扩增子测序方法对两株藻在25℃和35℃下细菌群落多样性进行分析。ACE和Chao1指数分析表明,25℃下培养两株藻的细菌群落组成无显著差异,但35℃下IM组(IM35L)中细菌群落多样性显著高于IZ组(IZ35L)。无度量多维标定法(NMDS)分析表明,25℃下IM组(IM25L)和IZ组(IZ25L)的藻际细菌群落较为相似,而35℃下两者的藻际细菌群落差异明显。另外,IM25L、IZ25L和IZ35L组中丰度前三的属均一致,但IM35L组中噬冷菌属(Algoriphagus)细菌相对丰度明显提升。MetaStat分析发现,变形菌门红螺菌目细菌阿尔法变形菌DG1252(Alpha proteobacterium DG1252)在25℃下IZ组和IM组的丰度无明显差异,但在35℃下IM组的丰度显著高于IZ组。因此,湛江等鞭金藻耐高温能力与其藻际细菌多样性存在关联,其中噬冷菌属细菌和阿尔法变形菌DG1252可能与IM的耐高温性状密切相关。本研究为深入探究湛江等鞭金藻高温抗逆性与藻际细菌之间的联系奠定了基础,有助于夏季高温条件下贝类饵料微藻的培育和扩繁。

Purification and Characterization of 2-Haloacid Dehalogenase from Marine Bacterium Paracoccus sp. DEH99, Isolated from Marine Sponge Hymeniacidon perlevis

2-haloacid dehalogenases constitute a group of dehalogenases which are capable of dehalogenating the halogenated organic compounds. So far, the 2-haloacid dehalogenases have been found in many bacteria, but not in Paracoccus genus. In the present study, one enzyme 2-haloacid dehalogenase(designated as Deh99), induced by DL-2-chloropropionate(DL-2-CPA), was purified from the marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis. The enzyme of Deh99 was purified to homogeneity by ammonium sulfate precipitation, ion exchange chromatography(Q-Sepharose HP), and Superdex 200 gel filtration chromatography. The molecular weight of Deh99 was estimated to be 25.0 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), and 50.0 kDa natively by gel filtration chromatography. The enzyme of Deh99 stereospecifically dehalogenated L-2-CPA to produce D-lactate, with an apparent Michaelis-Menten constant(Km) value of 0.21 mmol L-1 for L-2-CPA. The optimal pH and temperature for Deh99 activity were 10.0 and 40℃, respectively. The enzyme of Deh99 acted on short-carbon-chain 2-haloacids, with the highest activity towards monochloroacetate. The activity of Deh99 was slightly affected by DTT and EDTA, but strongly inhibited by Cu2+ and Zn2+. The enzyme of Deh99 shows unique substrate specificity and inhibitor sensitivities compared to previously characterized 2-haloacid dehalogenases and is the reported one about purified 2-haloacid dehalogenase isolated from the bacteria of Paracoccus genus.

The Light Regime Effect on Triacylglycerol Accumulation of Isochrysis zhangjiangensis

Stress state of microalgal cells is caused under unfavorable conditions such as disordered light regime and depleted nitrogen. The stress state can impair photosynthetic efficiency, inhibit cell growth and result in the accumulation of triacylglycerol(TAG) from protective mechanisms. Continuous light or nitrogen starvation was applied on microalgae and performed effectively on inducing TAG production. To evaluate the light regime effect on inducing TAG production, the effect of different light regimes on nitrogen-starved Isochrysis zhangjiangensis was investigated in this work. The continuous light and nitrogen starvation elevated TAG content of biomass by 73% and 193%, respectively. Furthermore, the TAG accumulation of I. zhangjiangensis cell under nitrogen starvation decreased under aggravated stress from continuous illumination. Our results demonstrated that culturing the cells with 14 L: 10 D light regime under nitrogen starvation is the optimal mode to achieve maximal accumulation of TAG. A recovery in light regime was necessary for I. zhangjiangensis cultivation.

Determination of Internal Controls for Quantitative Gene Expression of Isochrysis zhangjiangensis at Nitrogen Stress Condition

Isochrysis zhangjiangensis is a potential marine microalga for biodiesel production,which accumulates lipid under nitrogen limitation conditions,but the mechanism on molecular level is veiled.Quantitative real-time polymerase chain reaction(q PCR) provides the possibility to investigate the gene expression levels,and a valid reference for data normalization is an essential prerequisite for firing up the analysis.In this study,five housekeeping genes,actin(ACT),α-tubulin(TUA),β-tubulin(TUB),ubiquitin(UBI),18 S r RNA(18S) and one target gene,diacylglycerol acyltransferase(DGAT),were used for determining the reference.By analyzing the stabilities based on calculation of the stability index and on operating the two types of software,ge Norm and bestkeeper,it showed that the reference genes widely used in higher plant and microalgae,such as UBI,TUA and 18 S,were not the most stable ones in nitrogen-stressed I.zhangjiangensis,and thus are not suitable for exploring the m RNA expression levels under these experimental conditions.Our results show that ACT together with TUB is the most feasible internal control for investigating gene expression under nitrogen-stressed conditions.Our findings will contribute not only to future q PCR studies of I.zhangjiangensis,but also to verification of comparative transcriptomics studies of the microalgae under similar conditions.

Positive Effects of Isopropanol as a Co-Precipitant in Glycerol-3-Phosphate Acyltransferase Crystallization

Glycerol-3-phosphate acyltransferase(GPAT) is considered as the rate-limiting enzyme of glycerolipid synthesis pathway and the core element in lysophosphatidic acid(LPA) synthesis. For understanding its catalytic mechanism, the structural biology study is expected, but is always hindered by obtaining crystals for X-ray diffraction analysis. In this study, a progressive strategy to optimize the crystal of microalgae plastidial GPAT was presented. After the expression and purification of GPAT, the crystals were screened by hanging-drop and only clusters were obtained. The crystals were optimized by adjusting temperature, pH, protein concentration, or precipitant, but little improvement. To improve the interaction between protein and precipitant, the isopropanol was applied as co-precipitant. The qualified crystals formed. It's suggested that isopropanol is critical to affect protein crystallization by altering polyethylene glycol(PEG)-water-protein interaction when PEG serves as precipitant. The resulting crystal diffracted to a resolution of 2.75 ? and belonged to space group P1, with unit-cell parameters a = 50.79, b = 80.09, c = 88.21 ?, and α = 62.85, β = 73.04, γ = 80.53?. This work introduced a new strategy to optimize the crystal of the heterogeneous catalysis enzymes like GPAT and provided the fundamental structural information for the oriented synthesis of marine microalgae glycerolipid.

Lysobacter hymeniacidonis sp. nov., Isolated from a Crude Oil-Contaminated Marine Sponge

An aerobic, Gram-negative bacterium, strain 2-5~T, was isolated from a crude oil-contaminated marine sponge collected near Dalian Bay, China, and subjected to a polyphasic taxonomic investigation. Cells of strain 2-5~T were non-spore forming, non-motile, rods 0.2–0.3 μm wide and 1.1–1.2μm long. Strain 2-5~T grew well on nutrient agar, ~TSA, R2 A agar and LB agar. Colonies of strain 2-5~T on LB agar were circular, smooth with entire margins, non-transparent and pale yellow after 3 d of incubation at 30℃. Growth of strain 2-5~T occurred in LN medium with 0–6% Na Cl; no growth occurred in the presence of 8.0% NaCl. Strain 2-5~T grew at 15–42℃ and at pH 6.0–8.0. Comparative 16 S r RNA gene sequence analysis showed that strain 2-5~T clustered with the species of the genus Lysobacter. Its closet neighbors were the type strains of Lysobacter concretionis KC^TC 12205~T(97% similarity), Lysobacter arseniciresistens ZS79~T(96%), and Lysobacter defluii APB-9 ~T(96%). ~The value for DNA-DNA relatedness between strain 2-5~~T and L. concretionis KC^TC 12205 ~T was 23%. Branched fatty acids iso-C16: 0, iso-C15: 0, iso-C11: 0 3-OH, iso-C17: 1ω9c and iso-C11: 0 were found to be predominant. ~The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Strain 2-5~T had a DNA G+C content of 63.8 mol%. On the basis of the phenotypic, chemotaxonomic, DNA-DNA hybridization and phylogenetic data, strain 2-5~T represents a novel species of the genus Lysobacter, for which the name Lysobacter hymeniacidonis sp. nov. is proposed. ~The type strain is 2-5~T(=CGMCC 1.12190 ~T = JCM 18137~T).

A Novel Material for Selective Separation of Mono-galactosyldiacylglycerols from Microalgae

Monogalactosyldiacylglycerols(MGDGs)have potential applications in food products,cosmetics and pharmaceuticals.MGDGs from microalgae with high amounts of polyunsaturated fatty acids(PUFAs)have potential functions,which arise the interest of the researchers.MGDGs were prepared by silica gel column chromatography with the appropriate mobile phase,while due to the similarity to molecular structure of MGDGs,digalactosyldiacylglycerols(DGDGs)were the most difficult impurities to separate during the extraction process of MGDGs from Nannochloropsis oceanica IMET1 and Arthrospira platensis.In order to obtain MGDGs from microalgae using low toxic solvent system,a novel material Click thiol-ene cysteine(Click TE-Cys)was employed to achieve its selective separation by differentiation of the hydrophilic interaction.The mixture of MGDGs and DGDGs standards were separated from each other by Click TE-Cys solid phase extraction(SPE),which was further confirmed by the result of LC-MS.The molecular interaction of MGDGs and DGDGs with Click TE-Cys demonstrated that DGDGs had more hydrogen bonds with Click TE-Cys material,which might cause a higher hydrophilic interaction.In this study,the Click TE-Cys material exhibited higher hydrophilicity with DGDGs and effectively separated MGDGs from 4 species microalgae by‘flow-through'mode using ethanol as mobile phase.

Discussion on Spatial Optimization Modeling of Water Resource Conservation Forests and Management Practice of Forest Functions

According to the unification principle of system structure and system function of the Water Resource Conservation Forests,seven factors were selected from stand spatial structure,trees structure and healthy of woods,the multifunction management optimization model target was confirmed by using nonlinearity multi-objective programming approach, and the target function-stand spatial structure homogeneity index was defined to establish spatial optimization models with restraining conditions set up in diversity of stand structure and spatial structure.The spatial structure of available typical stand in the wet land area in southern Donting Lake was optimized by means of selective cutting and reinforcement planting.The results showed that the spatial structure was improved obviously and trees diversity and stand health were not weakened.It’s suggested that the established optimization model can effectively guide multifunction management,stand structure optimization and the steadiness of ecological ecosystem and it is convenient and operable in practice,and it’s very important to protect and recover ecosystem of Water Resource Conservation Forests.