Effects of polyamines on the early development of cystocarps,spore release,survival,and germination rate of Gracilariopsis lemaneiformis

Three polyamines(PAs)(spermidine(Spd),spermine(Spm),and putrescine(Put))were used as growth regulators to the marine macroalgae Gracilariopsis lemaneiformis and to female gametophyte and reproductive tissues(cystocarps were constructed),which led to early maturation of cystocarps and spore release.Laboratory cultivation revealed that exogenous PA application accelerated the development of cystocarps,and the mean sporeling growth rate was 20%–50%/d during the initial week of cultivation.The spore count(SC),survival rate(SR),and germination rate(GR)of G.lemaneiformis showed different responses to PA(10-6 mol/L and 10-3 mol/L)treatments under different temperatures(18,26,and 34℃),light intensities(30,60,and 90μmol photons/(m^(2)·s)),salinities(25,30,and 35),and nutrient levels(25꞉2.5,50꞉5.0,and 75꞉7.5μmol/L NH4Cl꞉K2HPO4),and the optimal conditions were 26℃,light intensity of 60μmol/(m^(2)·s),salinity of 35,and nutrient level of 50꞉5.0μmol/L NH4Cl꞉K2HPO4.Under the optimum conditions,the SC per cystocarp increased by 82.38%,and the spore SR and GR increased by 33.07%and 46.44%,respectively.Each experiment lasted for 7 days,with the highest SC on Day 4 and plateauing on Day 7.The results indicate that PAs could promote the early maturation,spore release,survival,and germination of G.lemaneiformis under laboratory conditions.

Transcriptome Analysis During Tetrasporogenesis of Gracilariopsis lemaneiformis and Preliminary Study of the Expressions of Its Meiotic Genes

The transcriptomes of three different parts of the fertile tetrasporophyte of Gracilariopsis lemaneiformis,including tip(T),middle(M),and subjacent(S)parts,with a gradual tetrasporangium maturity were analyzed and compared to identify the genes involved in the process of tetrasporogenesis.The number of differentially expressed genes(DEGs)for the Gple-S versus Gple-T comparison was 10296,and the numbers of DEGs for the Gple-S versus Gple-M and Gple-T versus Gple-M comparisons were 7435 and 1337,respectively.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were performed,and the results showed the enrichment of 132 KEGG pathways(corrected P<0.05).A total of 58 DEGs related to meiosis were screened and blasted against 18 meiosis-related genes(dmc1,mlh1,mnd1,msh4,msh2,msh6,mre11,pds5,pms1,rad21,rad50,rad51,smc1,smc2,smc4,smc5,smc6,and spo11),including four meiosis-specific genes.The transcriptome comparison indicated that in the T part,the meiosis,ribosome,and RNA transport-related genes were mostly up-regulated compared with those in the other two groups.In the M part,the genes related to ribosomes and the endoplasmic reticulum were also up-regulated compared with those in the lower part.Finally,in the S part,the genes associated with photosynthesis were mostly up-regulated,which might be helpful to the recovery from spore formation and release.

Seaweed Fiber Fabricated with Agar Alkali-Free Extracted from Gracilaria lemaneiformis

The sulfate groups in agar structure played a good role in the formation of fiber.However,commercially available agar is usually extracted from red algae by alkali treatment to decrease the content of sulfate group for the purpose of high gel strength.In this paper,an alkali-free method of agar extraction from Gracilaria lemaneiformis was proposed for the wet-spinning purpose.This method is environmentally friendly,reduces the extraction steps,saves energy,and reduces the production cost of agar fiber.The improved agar preparation process not only has higher agar yield,but also has higher molecular weight and sulfate group content,which is beneficial to the preparation and forming of fiber and makes the fiber have higher mechanical strength and elongation.Therefore,this extraction technology has broad application prospect in the textile field.

Transcriptome Analysis and Physiological Responses of Economic Macroalga Gracilariopsis lemaneiformis under Sulfate Deficiency

Sulfur is an essential macronutrient for the growth of all photosynthetic organisms and plays important roles in different metabolic pathways.However,sulfur metabolism and its related research on macroalgae with important ecological value is rather limited.In this study,marine ecological valued macroalga Gracilariopsis lemaneiformis was used to study the general physiological responses and transcriptome profiling under the sulfate deficiency.The relative growth rate of algae under sulfate deficiency was statistically significantly lower than that of control after 6 days.However,no significant differences were observed in the pigments content and Fv/Fm value,indicating that the photosynthesis was not obviously affected under the sulfate deficiency.Furthermore,the significantly increased soluble protein and carbohydrate contents,and indistinctively differentially expressed sulfate transporter/permease(ST)and ATP sulfurylase(ATPS)genes suggesting that G.lemaneiformis cells did not use sulfur from internal protein and carbohydrate pools but utilize stored sulfur from vacuole to satisfy their nutritional requirements during the sulfate deficiency.Transcriptome data showed that most annotated differentially expressed genes(DEGs)were down-regulated under the sulfate deficiency,including a large proportion of genes involved in the pathway of sulfur metabolism.Therefore,it was concluded that the pathway of sulfur metabolism was suppressed under the sulfate deficiency in G.lemaneiformis.The results and analysis in this study provide a comprehensive understanding for the physiological and molecular response of G.lemaneiformis to sulfate deficiency.

CLONING AND ANALYSIS OF PHYCOERYTHRIN GENES IN GRACILARIA LEMANEIFORMIS (RHODOPHYCEAE)

Cloning and sequencing of the genes coding for the α and β subunit of phycoerythrin (PE) of a red alga- Gracilaria lemaneiformis (GL) are reported. Alignment of 1084 nucleotides sequenced with three known red algal PE genes, Rhodella violacea (RV), Polysiphonia boldii (PB) and Aglaothamnion neglectum (AN), showed high level of conservation, and similarities of 77.6% (between GL and RV), 77.9% (GL and AN) and 79.0% (GL and PB). The similarities of amino acids were 84.8% (between GL and RV), 85.7% (GL and PB), and 80.6% (AN and GL), higher than those among nucleotides.

Genome Size of Alexandrium catenella and Gracilariopsis lemaneiformis Estimated by Flow Cytometry

Flow cytometry(FCM) technique has been widely applied to estimating the genome size of various higher plants. However, there is few report about its application in algae. In this study, an optimized procedure of FCM was exploited to estimate the genome size of two eukaryotic algae. For analyzing Alexandrium catenella, an important red tide species, the whole cell instead of isolated nucleus was studied, and chicken erythrocytes were used as an internal reference. The genome size of A. catenella was estimated to be 56.48 ± 4.14 Gb(1C), approximately nineteen times larger than that of human genome. For analyzing Gracilariopsis lemaneiformis, an important economical red alga, the purified nucleus was employed, and Arabidopsis thaliana and Chondrus crispus were used as internal references, respectively. The genome size of Gp. lemaneiformis was 97.35 ± 2.58 Mb(1C) and 112.73 ± 14.00 Mb(1C), respectively, depending on the different internal references. The results of this research will promote the related studies on the genomics and evolution of these two species.

Changes in the Photosynthetic Pigment Contents and Transcription Levels of Phycoerythrin-Related Genes in Three Gracilariopsis lemaneiformis Strains Under Different Light Intensities

Three Gracilariopsis lemaneiformis strains,including wild type and high-temperature-resistant cultivars 981 and 2007,were studied with the changes in their photosynthetic pigment contents and related gene transcription levels under different light intensities(10,60,100,and 200μmolm^(−2)s^(−1)).The three G.lemaneiformis strains had the following photosynthetic pigments with high-to-low contents:phycoerythrin(PE),phycocyanin(PC),allophycocyanin(APC),and chlorophyll a(Chl a).Among the three strains,cultivar 981 had the highest PE content,followed by cultivar 2007.The PC and APC contents were similar among the three strains,but they were higher in cultivars 981 and 2007 than in the wild type.The Chl a contents in the three G.lemaneiformis strains were equal.A low light intensity(10μmolm^(−2)s^(−1))promoted photosynthetic pigment accumulation in G.lemaneiformis and improved the relative PE gene transcription(peA and peB)in a short period(≤6 d).A high light intensity decreased the PE content.PebA and PebB,which catalyzed phycoerythrobilin synthesis,showed no compensatory upregulation at a low light intensity among the strains except for the wild type.At a high light intensity,transcription levels of pebA and pebB in the three strains were upregulated.This study provided an experimental basis for elucidating the photosynthesis of G.lemaneiformis.As key genes of algal growth,photo-synthesis-related genes served as useful gene markers for screening elite varieties with good traits in breeding.Cultivar 2007 was superior to cultivar 981 in terms of maintaining high pigment levels in a wide range of light intensities,which is the most suitable for aquaculture.

Eff ects of temperature on photosynthetic performance and nitrate reductase activity in vivo assay in Gracilariopsis lemaneiformis (Rhodophyta)

Gracilariopsis lemaneiformis is an economically-valued species and widely cultured in China at present.After being acclimated to diff erent growth temperatures(15,20,25,and 30°C)for 7 days,the relative growth rate(RGR),nitrate reductase activity,soluble protein content and chlorophyll a fl uorescence of G.lemaneiformis were examined.Results show that RGR was markedly aff ected by temperature especially at 20°C at which G.lemaneiformis exhibited the highest eff ective quantum yield of PSII[Y(II)]and lightsaturated electron transport rate(ETR max),but the lowest non-photochemical quenching.Irrespective of growth temperature,the nitrate reductase activity increased with the incubation temperature from 15 to 30°C.In addition,the greatest nitrate reductase activity was found in the thalli grown at 20°C.The value of temperature coeffi cient Q10 of alga cultured in 15°C was the greatest among those of other temperatures tested.Results indicate that the optimum temperature for nitrate reductase synthesis was relatively lower than that for nitrate reductase activity,and the relationship among growth,photosynthesis,and nitrate reductase activity showed that the optimum temperature for activity of nitrate reductase in vivo assay should be the same to the optimal growth temperature.

Adverse Effect of TiO2 Nanoparticles on Antioxidant System and Antitumor Activities of Macroalgae Gracilaria lemaneiformis

Recently,the widespread use of TiO2 nanoparticles(nano-TiO2)has attracted the global concern regarding their adverse effects to aquatic organisms.Nanoparticles may get to the aquatic environments and harm the growth of algae.This paper presents the studies of nano-TiO2 concentrations influence on the photosynthetic characteristics,antioxidant system and antitumor activity of marine macroalgae Gracilaria lemaneiformis after exposed to 0,5,10,20 or 40 mg L^-1 of nano-TiO2.Results showed that the photosynthetic pigment content of G.lemaneiformis obviously decreased and the antioxidant defense system was significantly disturbed after exposure to nano-TiO2.Compared with control,the maximum level of malondialdehyde showed 3.3-fold increase for 15 days exposure to nano-TiO2.The activity of superoxide dismutase and catalase enhanced obviously by 1.10-2.24-fold.The aqueous extract of G.lemaneiformis(AEGL)possesses anticancer activity against HepG2 liver cancer cells proliferation and the 48 h-cell inhibition exceeds 35%.But the toxicity of nano-TiO2 would inhibit the anticancer activity of AEGL.When G.lemaneiformis was exposed to nano-TiO2,the effect of the aqueous extract of G.lemaneiformis on the cell inhibition,reactive oxygen species production,apoptosis,cycle arrest of HepG2 cells significantly lessened,and which were linearly associated with nano-TiO2 exposure concentration.Those results further confirmed that nano-TiO2 can inhibit the anticancer activity of G.lemaneiformis.The results in this work imply that macroalgae can be an effective biomarker of nano-TiO2 contaminant,and can be useful indicator to evaluate ecological effects of increasing pollutants in marine ecosystem.

APPLICATION OF RAPD IN GENETIC DIVERSITY STUDY ON GRACILARIA LEMANEIFORMIS Ⅲ.PHASE AND SEX RELATED MARKERS

Random amplification of polymorphic DNA (RAPD) was conducted by using 10 ran-dom primers (P-1 to P-10) in Gracilaria lemaneiformis. Phase and sex specific bands were amplified byprimers P-2, P-6, P-7 and P-8: for P-2 a 1 .4 kb band was found in female gametophytes and tetrasporo-phytes, for P-6, a 0.6 kb band appeared in male gametophytes and tetrasporophytes; for P-7, a 0. 76 kbband appeared in male gametophytes and tetrasporophytes; for P-7, a 0.72 kb band appeared in femalegametophytes and tetrasporophytes; for P-8, a 0. 73 kb band only appeared in male gametophytes.

Structure of reproductive apparatus of Gracilaria/Gracilariopsis lemaneiformis (Gracilariaceae,Rhodophyta)

Reproductive apparatus of Gracilaria/Gracilariopsis lemaneiformis collected from Qingdao city were studied with a light and a transmission electron microscope. The special superficial arrangement of spermatangium for this species was clearly observed, and the ultrastructure of spermatangial devel-opment revealed the similar cytodynamic pattern followed by all the Gracilariaceae members devel-oped from spermatangial mother cells to spermatangium. The female reproductive apparatus before fertilization was also observed and trichogyne was found protruding above the cortex, contrary to the earlier reports. Tetrasporangium was formed by an outer cortical cell and the tetraspores became spherical and expended after being released.

温度和氮营养盐对龙须菜生长与光合生理的影响

以栽培龙须菜(Gracilariopsis lemaneiformis)为试验材料,设置2个温度水平(LT:20℃;HT:24℃)和3个氮营养盐浓度(LN:3.1μmol/L;MN:30.1μmol/L;HN:103.1μmol/L),研究2个环境因子变化对龙须菜生长和光合生理的影响。结果表明,温度、氮营养盐及二者交互作用对龙须菜的相对生长速率(RGR)均具有显著影响(P<0.05),RGR随着温度的升高、氮营养盐浓度的增加而升高。叶绿素荧光参数分析显示,在同一氮营养盐浓度下,HT处理组的有效光合量子产量[Y(Ⅱ)]在0.18~0.24内波动,均高于LT处理组,在同一温度培养条件下,HN处理组的Y(Ⅱ)普遍高于LN处理组;温度对龙须菜的最大相对电子传递速率(rETRmax)存在显著影响(P<0.05);氮营养盐对不同处理下龙须菜的rETRmax、光能利用效率(α)、饱和光强(Ik)均存在显著影响(P<0.05),二者交互作用对α存在显著影响(P<0.05)。此外,温度、氮营养盐及二者交互作用对龙须菜的光合色素含量均具有显著影响(P<0.05);氮营养盐及二者交互作用对其可溶性蛋白质(SP)含量存在显著影响(P<0.05);在LT或HT条件下,氮营养盐浓度的增加显著促进叶绿素a(Chl a)、类胡萝卜素(Car)和SP的合成(P<0.05)。HN和HT促进了龙须菜的生长,结果为解析龙须菜应对核电站温排水区的环境变化提供了理论数据。

龙须菜在滩涂贝藻混养系统中的生态作用模拟研究

2005年5-7月,在室内采用实验生态学方法对壳长为48.35 mm±4.27 mm文蛤(Meretrix meretrix Linnaeus)成贝和龙须菜(Gracilaria lemaneiformis Weber-van Bosse)的4种配比(生物量配比分别为0∶1、1∶1、2∶1、4∶1)进行了混养实验,每周测定养殖水体中营养盐变化情况以及文蛤、龙须菜的存活和生长情况等。实验表明,加入了大型藻类的文蛤养殖系统,相对于空白对照组,其氨氮、亚硝氮和磷酸盐浓度显著降低,NH4-N从5.52μmol/L降至1.30μmol/L、NO2-N从0.31μmol/L降至0.06μmol/L,而PO4-P从0.48μmol/L降至0.006μmol/L。实验结束后,文蛤及龙须菜生长情况良好,文蛤特定生长率最高达到了0.84%,龙须菜最高则达到了1.79%。本实验条件下,在文蛤成贝养殖系统中加入大型藻类,对养殖水体中的NH4-N、NO2-N、PO4-P有明显的吸收效果,吸收率分别为86%、98%和99%,起到了良好的生态作用。

光照和营养盐胁迫对龙须菜生长及生化组成的影响

以龙须菜(Gracilaria lemaneiformis)为实验材料,分别研究了光照强度、营养盐胁迫对其生长及生化组成的影响。结果表明,在800～3000lx的范围内随光强的升高,龙须菜生长率增加,但藻红素(PE)、叶绿素a、蛋白质和可溶性总糖含量却降低,即低光强更利于龙须菜生化组分的积累。饥饿处理组、饱和处理组及正常对照组的生长率分别为0.9%,5.01%,3.06%。饥饿处理组的总C含量最高,总N及C/N比值高(>15),饱和处理组则相反,C/N比值<10,即存在C高N低、N高C低的现象。表明水体营养状况对龙须菜生长、体内生化组分有显著影响。总C、总N及C/N比都能很好地反映藻体的营养状态。饱和处理组藻体呈现深红色,饥饿处理组藻体色泽为浅黄色,因此也可根据藻体色泽的变化来估计藻体的营养状态。

4种酞酸酯在龙须菜-篮子鱼食物链中的积累放大研究

在模拟受控条件下,利用气相色谱方法研究分析了4种酞酸酯类化合物在龙须菜(Gracilaria lemane-iformis)-篮子鱼(Siganus oramin)食物链中的积累、分布和放大状况。结果表明,4种酞酸酯在龙须菜组织内的积累表现为:邻苯二甲酸二异辛酯(DEHP)>邻苯二甲酸二丁酯(DBP)>邻苯二甲酸二乙酯(DEP)>邻苯二甲酸二甲酯(DMP)。侧链最长的DEHP在龙须菜藻体内的含量随暴露时间的延长呈增加的趋势,30 d体内积累量可达2.35 mg/kg(鲜质量);侧链最短的DMP在龙须菜藻体内的质量比随时间变化差异不显著,约为0.05 mg/kg;在30 d的暴露中,4种酞酸酯DMP,DEP,DBP和DEHP在篮子鱼体内总质量比分别为0.08,0.41,1.72,3.11 mg/kg。其中DBP和DEHP在鱼内脏组织中有较高的积累和分布,分别为1.14 mg/kg和1.83 mg/kg,其次为鱼残体组织,肌肉组织中含量最低。DEHP和DBP在龙须菜和篮子鱼体不同组织中均有一定程度的富集(生物富集系数FBC均大于1)。其中DEHP和DBP在内脏组织中的FBC值分别高达9.16和5.68,在龙须菜-篮子鱼这个简单食物链传递中DEHP和DBP在篮子鱼内脏组织中存在放大现象。

龙须菜琼胶的提取方法研究

采用多种方法提取了龙须菜(Gracilaria lemaneiformis)中的琼胶,并研究了碱处理、微波处理、超声波处理等方法对龙须菜的琼胶提取率、硫酸基质量分数和多糖质量分数的影响。结果表明:当碱质量分数由2.5%增加到10%时,琼胶产率由15%降低到9.8%,硫酸基质量分数由2.66%降低到2.06%,同时,多糖质量分数由98.3%增加到102.6%;微波和超声波处理在不同程度上增加了琼胶的提取率和多糖质量分数,同时降低了硫酸基质量分数。另外,还从宏观和微观两个角度考察了龙须菜碱处理前后藻体的变化。

4种经济海藻脂肪酸组成分析

采用改进的Bligh-Dyer法提取脂溶性成分,气相色谱-质谱联用法(GC-MS)进行分离和鉴定,C19:0内标确定总脂及各组分含量,研究了鼠尾藻(Sargassum thunbergii)、浒苔(Enteromorpha prolifera)、龙须菜(Gracilaria lemaneiformis)和红毛菜(Bangia sp.)4种经济海藻的脂肪酸组成及含量。结果表明,4种海藻都检测出C14-C22脂肪酸,总脂含量在12～19 mg/g之间,不饱和脂肪酸为主要组成成分,含量均超过60%。不饱和脂肪酸中以多不饱和脂肪酸(PUFAs)为主,富含n-3和n-6系列PUFAs,n-6与n-3系列PUFAs之比均低于2。比较4种海藻脂肪酸组成特点表明,鼠尾藻以C16、C18和C20为主要组成成分,具褐藻类脂肪酸组成特征;浒苔以C16和C18为主要组成成分,具绿藻类脂肪酸组成特征;龙须菜和红毛菜以C16和C20类脂肪酸为主,具典型红藻类脂肪酸组成特征,同时二者又有不同之处,分别显示真红藻与原始红藻脂肪酸组成的特点。

酞酸酯对龙须菜的生态毒理研究

研究了酞酸酯类化合物对龙须菜（Gracilaria lemaneiformis）生长的影响以及酞酸酯在龙须菜体内的积累状况。结果表明，在邻苯二甲酸二甲酯（DMP）、邻苯二甲酸二乙酯（DEP）、邻苯二甲酸二丁酯（DBP）、邻苯二甲酯二异辛酯（DEHP）4种酞酸酯混合质量浓度为0．05，0．10，0．20和0．40mg／L的暴露条件下，龙须菜的相对生长速率和叶绿素a含量均受到了明显影响。与对照组比较，在高质量浓度处理组（0．1～0．4mg／L）中，两指标分别下降了18．4％～21.3％和10．4％～15．3％，致毒效应与酞酸酯暴露浓度呈正相关。在受控水体的暴露试验中，利用气相色谱（GC—FID）分析表明，4种酞酸酯类（DMP，DEP，DBP和DEHP）在龙须菜体内均有一定程度的积累，其残留量分别为0～0．14mg／kg，0～0．83mg／kg，0．11～1189mg／kg，0．01～10．4mg／kg，其积累程度与酞酸酯分子结构相关，侧链较长的酞酸酯比侧链较短的积累量要大，并随处理浓度的增加和时间推移呈增加趋势。

厦门筼筜湖龙须菜生长的主要影响因素

通过在高度富营养化的海水泻湖——厦门筼筜湖开展龙须菜(Gracilaria lemaneiformis)的延绳夹苗挂养与网笼挂养实验和室内端足类啃食海藻实验,探讨了影响筼筜湖龙须菜生长的主要因素。结果显示,筼筜湖龙须菜的表观生长率为0.42%/d,实际生长率为4.80%/d,强壮藻钩虾(Ampithoe valida)的摄食量占龙须菜总生长量的91.4%。而且,筼筜湖的水流缓慢,使藻体附着最高可达2.32g/g FW的颗粒物,明显抑制了龙须菜的生长。以上研究表明,端足类的啃食作用和不良的水动力条件是导致筼筜湖龙须菜表观生长率低的主要原因。

不同N、P浓度条件下龙须菜对镉胁迫的生理响应

通过室内生态学对比实验,研究了龙须菜(Gracilaria lemaneiformis)在不同N、P营养盐浓度条件下对重金属Cd2+胁迫的生理响应。结果表明,环境中加入适当浓度的N、P营养盐(N280μmol/L、P56μmol/L)有利于提高龙须菜光合色素的含量和生长率,同时藻体内超氧化物歧化酶(SOD)和过氧化物酶(POD)酶活性增高,丙二醛(MDA)含量降低,从而增强了龙须菜对重金属Cd2+胁迫的耐受性;而N、P饥饿(N、P均为0μmol/L)或N、P营养盐浓度过量(N、P分别为216μmol/L、1620μmol/L)状态下,龙须菜抗氧化能力下降、脂质过氧化和膜系统受伤害程度加剧,光合色素合成和生长率受到抑制,不利于龙须菜对重金属Cd2+胁迫的生理适应。