大叶藻(Zostera marina L.)PSⅠ和PSⅡ复合物的分离鉴定

采用蔗糖密度梯度离心法分离纯化大叶藻类囊体膜 ,经 1 0 %SDS增溶后 ,用蔗糖密度梯度超速离心分离其色素蛋白质复合物。经稳态光谱分析、DCIP光还原活性测定及P680 、P70 0 差示光谱检测结果表明 ,2 0 %蔗糖层的CP3和 40 %蔗糖层 (上 )的CP4为PSⅡ复合物 ,具有光化学活性 ;40 %蔗糖层 (下 )的CP5为PSⅠ复合物 ,其P70 0 特征吸收峰位于 695nm处。CP3和CP4的DCIP光还原活性 :CP3为 34.2 7微电子当量 /(mgchl·h) ,CP4为 7.2 9微电子当量 /(mgchl·h)。

Artificial seed germination and seedling production of Zostera marina L.by salinity manipulation

Vast declines in Zostera marina seagrass beds demand effective methods of rehabilitation. In this study, we developed a practical method by reducing salinity to induce seed germination followed with recovering salinity to facilitate seedling production of Z. marina. The results showed that Z. marina seeds collected from natural seawater（salinity 30） were induced to germinate at reduced salinities. Percent germination（GR） was higher and mean-time-to-germinate（MTG） was shorter at lower salinities. The highest GR and shortest MTG occurred at salinity 0（deionized freshwater）. After germination in freshwater, seeds could develop into seedlings at salinities5–30 and continue the growth. Viability or development of germinated seeds was not significantly different during the 40 d of post-germination incubation at salinities 5–15 after 1–20 d of germination in freshwater. However,during the process of translating germinated seeds from salinity 0 and 5 to salinity 30, reducing the gradients of post-germination acclimation facilitated more seeds forming seedlings in less time. On average, after 60 d of static incubation, including 20 d in freshwater for germination followed with immediate shift to salinity 5 and increasing to salinity 30 at increment of 5 every two days until cultivation at constant salinity 30, 33% of Z. marina seeds produced healthy seedlings. The results indicate that the salinity-manipulation based method of artificial germination and seedling production is practical and effective in supporting rehabilitation of Z. marina bed.

Influence of Temperature and Salinity on Germination of Eelgrass (Zostera marina L.) Seeds

Seagrass restoration as part of ocean ecosystem protection has been launched for many years all over the world, but intensive research on this subject in China has just begun in recent years. Seed broadcasting has been widely accepted as the most potentially useful method for seagrass restoration over large areas. We examined the influence of key environmental factors on seed germination to help promote eelgrass bed restoration. Under anoxic conditions, the influence of temperature and salinity on the germination rate of eelgrass (Zostera marina L.) seeds was examined at different combinations of four temperatures (4, 9, 14, and 24℃) and nine salinities (5 to 45, increment of 5). The effect of significant interaction of temperature and salinity on germination rate was observed (ANOVA) (P<0.001). The highest germination rate (83.3 ± 3.5)% was reached in 8 weeks at 14℃ and salinity 5. Higher temperature significantly increased the germination rate at salinity 5 (P<0.001) during the whole observation period except for 24℃, while lower salinity significantly increased the germination rate at 14℃ (P<0.001). Although significant interaction was found between temperature and salinity (P<0.001), the influence of salinity was stronger than that of temperature for the germination of eelgrass seeds. These results provide useful information for the propagation of artificial seedlings for seagrass restoration in China.

Habitat suitability analysis of eelgrass Zostera marina L. in the subtidal zone of Xiaoheishan Island

We present a GIS-based habitat suitability index(HSI) model to identify suitable areas for Zostera marina L. restoration in the subtidal zone of Xiaoheishan Island. The controlling factors in the model,in order of importance,are Secchi depth,sediment composition,water temperature,salinity,current velocity,water depth and nutrient quality. Specific factor piecewise functions have been used to transform parameter values into normalized quality indexes. The weight of each factor was defined using expert knowledge and the analytic hierarchy process(AHP) method. All of the data thus obtained were interpolated using the inverse distance weighted(IDW) interpolation method to create maps for the entire region. In this study,the analysis of habitat suitability in the subtidal zone of Xiaoheishan Island was conducted for four seasons. According to the GIS-based HSI model,the optimal habitat of Z ostera marina L. appears in spring,although habitat remains suitable all year round. On the whole,the optimum site for eelgrass restoration is located in the eastern region,followed by the western and southern regions. We believe that the GIS-based HSI model could be a promising tool to select sites for Z ostera marina L. restoration and could also be applicable in other types of habitat evaluation.

大叶藻(Zostera marina L.)胚的结构和种子萌发过程的研究

以采自山东荣成褚岛桑沟湾南岸成熟的大叶藻种子为材料,研究了种子的外部形态特征、胚乳成分及胚的解剖结构,并初步了解了大叶藻种子的萌发过程。结果表明,大叶藻种子是单子叶有胚乳的种子,呈暗褐色,椭圆形或长圆形,种皮革质且有17～20条纵肋,胚乳丰厚坚硬,胚乳细胞被淀粉粒充满;胚弯曲,由胚根、胚轴、胚芽和子叶组成,子叶U形棒状,其弯曲部有一裂缝,胚芽位于裂缝处,胚轴较短,其下是两个对生的胚根,有中央维管束贯穿胚根、胚轴、胚芽和子叶。在子叶出土萌发的植物类型中,大叶藻种子萌发过程是罕见的。萌发初期,子叶优先生长突破种皮而长出地面,此后胚根才开始生长并产生不定根。对大叶藻种子结构和萌发过程的研究,为探讨大叶藻种子萌发和幼苗更新的限制因素提供了一定的理论依据。

Identification of Light-Harvesting Chlorophyll a/b-Binding Protein Genes of Zostera marina L.and Their Expression Under Different Environmental Conditions

Photosynthesis includes the collection of light and the transfer of solar energy using light-harvesting chlorophyll a/b-binding(LHC) proteins.In high plants,the LHC gene family includes LHCA and LHCB sub-families,which encode proteins constituting the light-harvesting complex of photosystems I and II.Zostera marina L.is a monocotyledonous angiosperm and inhabits submerged marine environments rather than land environments.We characterized the Lhca and Lhcb gene families of Z.marina from the expressed sequence tags(EST) database.In total,13 unigenes were annotated as Zm Lhc,6 in Lhca family and 7 in Zm Lhcb family.Zm LHCA and Zm LHCB contained the conservative LHC motifs and amino acid residues binding chlorophyll.The average similarity among mature Zm LHCA and Zm LHCB was 48.91% and 48.66%,respectively,which indicated a high degree of divergence within Zm LHChc gene family.The reconstructed phylogenetic tree showed that the tree topology and phylogenetic relationship were similar to those reported in other high plants,suggesting that the Lhc genes were highly conservative and the classification of Zm Lhc genes was consistent with the evolutionary position of Z.marina.Real-time reverse transcription(RT) PCR analysis showed that different members of Zm Lhca and Zm Lhcb responded to a stress in different expression patterns.Salinity,temperature,light intensity and light quality may affect the expression of most Zm Lhca and Zm Lhcb genes.Inorganic carbon concentration and acidity had no obvious effect on Zm Lhca and Zm Lhcb gene expression,except for Zm Lhca6.

Generation and analysis of expressed sequence tags from the salt-tolerant eelgrass species, Zostera marina

Zostera marina, a monocotyledonous angiosperm, is one of the most important seagrass species. To inves- tigate the salt-tolerance mechanism and discover salt-tolerant genes in Z. marina, a cDNA library was con- structed. Single-pass sequencing of the 5＇ ends of 4 081 clones yielded 4 002 high quality expressed sequence tags （ESTs）, which were assembled into 241 contigs and 1 673 singletons, representing 1 914 unigenes. The average length of the ESTs was 582 bp, with sizes ranging from 100-1 500 bp. Basic Local Alignment Search Tool （BLASTX） analysis revealed that 1 664 unigenes had significant homology to known genes in the Na- tional Center for Biotechnology Information （NCBI） non-redundant （nr） database （E-value≤5-10）. Among them, the two most abundant genes encoded metallothionein （157 ESTs） and chlorophyll a/b-binding pro- tein （38 ESTs）, accounting for 7.1% and 1.7% of the total ESTs, respectively. Using Kyoto Encyclopedia of Genes and Genomes （KEGG）, 1 462 unigenes were assigned to 1 161 pathways （E-value≤5-10）. A total of 938 unigenes were assigned Gene Ontology （GO） terms based on the GO hierarchy analysis, and InterProScan searches recognized 1 003 InterPro families. Three genes for metallothionein in Z. marina that belonged to Class II was identified. Results of this study will improve understanding of the molecular mechanisms of saline tolerance in Z. marina.

Biotic and Abiotic Conditions Can Change the Reproductive Allocation of Zostera marina Inhabiting the Coastal Areas of North China

The relationships between reproductive allocation and different factors have been widely recognized for terrestrial plants, but scarcely for marine angiosperms. Focusing on the seagrass Zostera marina, we examined the change in biomass when it was partitioned to growth, vegetative propagation and reproductive allocation of Z. marina. We also evaluated the relationships between reproductive allocation and biotic and abiotic conditions in the coastal areas of north China. The results indicated that the reproductive allocation of Z. marina at the levels of genet, shoot and spathe was positively correlated with shoot characteristics such as height and biomass(P < 0.01), but was negatively correlated with branch number and leaf number per genet(P < 0.01). In addition, the allocation was also affected by other abiotic factors such as water column phosphate concentration(P < 0.05) and sediment total petroleum(P < 0.01). These findings indicated that both biotic and abiotic conditions significantly influenced the reproductive allocation of Z. marina, which may affect further the population dynamics.

Cloning, characterization and expression analysis of a microsomal glutathione S-transferase gene from the seagrass Zostera marina

The response of glutathione S-transferase(GST)in Zostera marina to temperature variation was analyzed at molecular level by cloning the microsomal GST gene and texting the microsomal GST expression regularity under different temperature.Specific speaking,express ZmGST in Escherichia coli,then purify the recombinant protein and make the thermal stability analysis.Therefore,the experiments were carried out to provide a theoretical basis for the further elaboration to the population degradation mechanisms of Z.marina.In conclusion,the thermostability and the response of ZmGST gene to temperature changes can determine its temperature tolerance range,and affect its resilience in turn.

Influences of diel and tidal cycles on fish assemblage in eelgrass(Zostera marina) bed of southern Korea during autumn

Short-term tidal and diel variations of autumn fish assemblage in a Zostera marina bed were investigated using 3 h interval samplings for 24 h in both spring and neap tide using a small beam trawl.A total of 1 346 fishes belonging to 19 species were collected at spring tide,whereas 1 115 fishes belonging to 17 species were at neap tide.The common fish species were Nuchequula nuchalis,Acanthogobius flavimanus,Takifugu niphobles,Acentrogobius pflaumii,and Pholis nebulosa with the former three species dominating at spring tide,while the latter two species being abundant at neap tide.Diel variation in abundance was significant with higher abundance at night than day,but there were no significant differences between spring and neap tides,and between ebb and flood tides（three-way ANOVAs）.Diel variation in the abundance of fishes may be influenced by tidal range and cycle,and day-night differences of food availability and behaviors of fishes indirectly.Non-metric multidimensional scaling（n MDS） ordination and analysis of similarity（ANOSIM） results revealed significant differences in species compositions both between day and night,and between spring and neap tide.Eelgrass beds are highly productive marine ecosystem,and thus,our results will contribute to conservation of seagrass ecosystem in the study area.

Antibacterial Activity of Extracts from Zostera marina Against Pathogens of Apostichopus japonicus Skin Ulceration Disease

The purpose of this study was to investigate the antibacterial activity of extracts from Zostera marina against the pathogens of Apostichopus japonicus skin ulceration disease.When 95% ethanol (v/v) solvent was used to extract Zostera marina at 50℃,aqueous extract (ZA) showed obvious bacteriostatic effects on the tested bacterial strains (inhibition halo diameters between 8.23 mm and 13.62 mm),whereas the ethyl acetate extract (ZE) was almost inactive.The minimal inhibitory concentration (MIC) of ZA against four pathogens were homogeneous at 12.8 g L-1.ZA components were analyzed by thin layer chromatography (TLC) assay and six fractions were obtained.In another study,the six fractions showed inhibitory effects against the tested bacteria while their functions seemed to counteract the ZA activity.

A genome-wide analysis of the chloroplast NADH dehydrogenase - like genes in Zostera marina

The chloroplast NADH dehydrogenase-like(NDH)complex,homologous to respiratory complex I,participates in photosystem I cyclic electron flow(PSI-CEF)and chlororespiration in photosynthesis.Phylogenetic analyses indicated that Zostera marina,a widely distributed seagrass,has a complete NDH complex,which is rarely observed in marine macrophytes.We identified all 31 ndh genes necessary for the functional NDH complex,of which ndhB and pnsb3 occurred as duplication events.Secondary structural analyses of antiporter-like subunits showed that the long amphipathic helix of NdhF was lost in Z.marina,which could exhibit an alternative mode in the generation of trans-thylakoid proton gradient.The splicing pattern of ndh exhibited tissue-specific patterns and responded to light stress.RNA editing in Z.marina presented the ancestral pattern with many of the primitive editing sites and types.The partial editing in ndhF reflected the link between light stress and RNA editing.Moreover,the predominant expression in leaves of most ndh genes suggested that their major function is in photosynthesis.The quantitative real time-PCR results show that the expression of ndh was signifi cantly upregulated in response to light stress.Nevertheless,there were two diverse responsive mechanisms of the NDH complex in PSI-CEF and chlororespiration.Overall,the presence of a complete structure,upregulated gene expression level,and multiple post-transcriptional regulations could provide a molecular basis for the powerful NDH complex and enable Z.marina to maintain eff ective photosynthetic performance.

Isolation and Identification of Flavonoids Found in Zostera marina Collected in Norwegian Coastal Waters

In extracts of the seagrass Zostera marina, collected in coastal waters of West-Norway, fourteen different flavones and high amounts of rosmarinic acid were identified. Five of the flavones were found to be sulphated, among these were luteolin 7,3'-disulphate and chrysoeriol 7-sulphate structures previously not published with complete NMR assignments. Luteolin 7-O-β-(6''-malonyl) glucoside, and two other malonylated flavone compounds occurring in trace amounts, were identified for the first time in Z. marina. The sulphated flavones were fairly stable in slightly acidified (0.1% trifluoroacetic acid) extracts stored for months, however, under more acidic conditions (0.5% trifluoroacetic acid in the extracts) they were susceptible to undergo hydrolyses. When the solvents of purified fractions were removed by rotary evaporation, the sulphated flavones quickly decomposed to their corresponding aglycones due to the increased acid concentrations.

Recent Trends of Zostera marina (Eelgrass) in a Highly Eutrophic Coastal Lagoon in the Mid-Atlantic Region (USA)

Spatial and temporal characteristics of Zostera marina (eelgrass) in the Barnegat Bay-Little Egg Harbor Estuary are compared before (2004-2011) and after (2012-2013) major fertilizer legislation (Fertilizer Law A2290) was enacted in New Jersey (USA) to reduce nutrient inputs from fertilizers to water bodies in the state. A significant decrease of Z. marina biomass and areal cover occurred in this eutrophic estuary between 2004 and 2011 concomitantly with increasing nitrogen and phosphorus loading from the watershed. The rate of decline in aboveground and belowground biomass was significantly sharper during 2004-2006 than during 2008-2010. In 2010, Z. marina biomass dropped to a very low level (mean aboveground biomass = 7.7 g dry wt mDŽ;mean belowground biomass = 27.0 g dry wt mDŽ), persisting through the last sampling period (October-November) in 2013. Biomass and areal cover of Z. marina decreased even further after Fertilizer Law A2290 was enacted in January 2012, with the lowest values recorded from August to November each year. These low values are the result of ongoing eutrophication of the system. More seagrass monitoring and research are necessary in future years to determine if the fertilizer law will have a positive effect on Z. marina condition in the estuary over the long term.

大叶藻(Zostera marina)的热值和元素含量(碳、氮、磷)及其相关性

选取辽宁獐子岛近岸海域大叶藻样品,对其须根、根状茎和不同生长阶段叶片的灰分含量、热值和元素含量(碳、氮、磷)及其相关性进行研究,结果表明:在不同生长阶段,叶片长度随着生长顺序先增大后减小;灰分含量随着叶片生长逐渐升高,且极显著小于根状茎和须根(P<0.01);不同部位干质量热值差异极显著(P<0.01),以初生叶最高,为(14.89±0.040)k J/g,后逐渐减小,在须根中最小,为(8.15±0.079)k J/g;不同生长阶段叶片的去灰分热值差异不大,但极显著大于须根和根状茎(P<0.01);碳、氮、磷含量均呈同一趋势:初生叶>幼叶>成熟叶1>成熟叶2>衰老叶>根状茎>须根;N/P比值为27.08,说明大叶藻存在P限制;N/P比值在根状茎中最大,须根中最小,这说明大叶藻存在能量的转移和存贮;大叶藻不同部位灰分含量与干质量热值、去灰分热值、碳含量、氮含量、磷含量之间呈极显著的负相关(P<0.01),干质量热值与碳含量、氮含量、磷含量之间呈极显著的正相关(P<0.01);大叶藻不同部位的热值与碳、氮、磷元素含量之间均呈显著的线性关系(P<0.05)。

荣成天鹅湖大叶藻(Zostera marina)间2 种小型腹足类的分类学研究

小型腹足类生物在我国种类繁多,但其相关研究在国内基本属于空白。本文对荣成天鹅湖大叶藻间的2种小型腹足类生物进行了形态学及分子生物学(16S和28SrRNA基因片段)研究。结果表明,根据形态学所鉴定的短剑螺属未定种Machae roplax sp.,应为畦螺属未定种Lirularia sp.;分子生物学研究结果支持形态学对刺绣翼螺Alabapicta的鉴定。同时,与A.picta有一定形态学差异的其它4种相近形态的小型腹足类,在基因序列及遗传距离上与刺绣翼螺非常接近,亦应为刺绣翼螺Alaba picta。

不同水体浊度对鳗草存活、生长和生理的影响

海水浊度升高是导致海草床退化的主要因素之一。通过3个月的室内实验,研究了不同水体浊度[2(对照)、10、20、30和40 NTU]对鳗草存活、生长和生理的影响。结果显示,3个月后,鳗草植株的存活率随水体浊度升高呈现逐渐降低趋势,10~40 NTU处理组植株存活均显著低于对照组(P<0.05),至40 NTU处理组,植株存活率仅为对照组的56.1%;随水体浊度升高,植株的生长速率和生产力亦呈现下降趋势,至40 NTU处理组,植株茎节延伸速率和叶片延伸速率达到最小值,分别下降至对照组的48.9%和61.6%,地上生产力和地下生产力与对照组相比下降了64.6%和78.8%;相关性分析表明,长期浊度胁迫主要是通过影响植株非结构性碳水化合物含量,进而对植株生长存活产生负面影响。植株非结构性碳水化合物含量随水体浊度增加而逐渐下降,10~40 NTU处理组植株碳水化合物含量均显著低于对照组(P<0.05),各浊度升高处理组植株地上组织可溶性糖含量相比对照组降低了20.2%~74.7%。研究表明,水体浊度长期升高导致鳗草植株非结构性碳水化合物显著下降,对鳗草的生长存活不利。本研究为阐明鳗草海草床退化机理和选划适宜修复区等提供了理论依据。

不同栽培方式对鳗草实生苗建成和生理的影响

在15℃条件下,研究了3种栽培方式(水培促萌+萌中插植、水培促萌+萌后插植以及基质栽培)对鳗草(Zosteramarina)种子萌发、实生苗建成与生长的影响,分析了鳗草实生苗的培育成本及其应对不同栽培方式的生理响应。结果显示,水培促萌+萌中插植、水培促萌+萌后插植的鳗草种子萌发率和实生苗建成率无显著差异(P>0.05),平均达到54.8%和46.5%,分别是基质栽培处理组的1.3和1.6倍(P<0.05)。水培促萌+萌中插植、水培促萌+萌后插植的鳗草实生苗的各项生长指标亦无显著差异(P>0.05),其平均值均显著高于基质栽培处理组(P<0.05),特别是根生成速率平均达到基质促萌处理组的1.6倍。主成分分析显示,水培促萌+基质插植的鳗草实生苗生理状态较优,其中,总氮和可溶性糖含量的贡献度较大,贡献度平均分别达到16.6%和15.0%,是基质栽培处理组的1.2倍(P<0.05)。培育成本分析显示,水培促萌+萌后插植栽培方式的培育成本最低(3.6元/株),是水培促萌+萌中插植栽培方式的81.9%。综合实生苗培育效果和培育成本,水培促萌+萌后插植的栽培方式是适宜的鳗草实生苗栽培方式,其主要通过提升实生苗非结构性碳水化合物的形成和提高光合色素含量,实现对实生苗建成与生长的促进作用。

赤霉素和弱酸对鳗草种子萌发和生理特性的影响

本文研究了赤霉素和弱酸对鳗草(Zostera marina)种子萌发和生理特性的影响,监测了鳗草种子的累积萌发率、幼苗建成率以及种子萌发过程中种子干重、水分、呼吸速率、可溶性糖、淀粉、赤霉素、脱落酸含量和α-淀粉酶、β-淀粉酶活性的动态变化,探究了2种外源处理对鳗草种子的促萌作用,分析了鳗草种子应对外源促萌的生理响应过程。结果显示,2种促萌处理均能有效促进鳗草种子萌发和幼苗建成,其中,赤霉素处理组促萌效果最优,其种子萌发率和幼苗建成率为对照组的1.6倍;促萌期间,2个促萌处理组种子α-淀粉酶活性呈先上升后下降的趋势,淀粉含量呈下降趋势,可溶性糖含量呈持续上升趋势;至实验结束时,赤霉素处理组种子可溶性糖含量最高,是促萌前初始值的3.3倍,亦显著高于弱酸处理组和对照组(P<0.05),弱酸处理组种子淀粉含量最低,显著低于其他2组(P<0.05),而各处理组之间种子的α-淀粉酶活性无显著差异(P>0.05);主成分分析显示,种子α-淀粉酶活性、淀粉和可溶性糖含量为种子萌发过程中的关键因子。综合分析认为,外源赤霉素处理是破除鳗草种子休眠的有效方法,其主要通过调节种子淀粉酶活性、提高淀粉分解速率和提高可溶性糖含量为种子萌发提供能量实现破除休眠的作用。本研究结果为鳗草种子快速萌发技术提供了理论依据和科学基础。

大叶藻海草床的生态恢复:根茎棉线绑石移植法及其效果

于2009年和2010年在青岛汇泉湾用根茎棉线绑石法进行大叶藻（Zostera marina L.）移植，并于2012年4月20日至11月19日对移植大叶藻的生长情况进行观察（包括形态学变化、密度、茎枝高度、地上生物量、底质粒径几个方面）。观察期间水温为7.8-26.1℃。结果显示，移植底质可定性为粉砂质；移植大叶藻的有性繁殖期为2012年4-8月；无性繁殖在秋季达到高峰；密度在6月和9月分别高达411茎枝/m2和481茎枝/m2；高度与地上生物量的最大值出现在6-7月份。与2009年青岛湾天然大叶藻进行比较后可以发现，移植大叶藻的高度、地上生物量及其季节变化与天然大叶藻基本保持一致，说明移植大叶藻的生长状况良好，同时说明根茎棉线绑石法是一种高效且实用的海草床生态恢复方法。