Elementary study of differential expression of total soluble proteins in different life phases of Porphyra yezoensis

Total soluble proteins of different life stages, filamentous sporophytes cultivated in high temperatures, and blade gametophytes harvested in different seasons, were identified by SDS-PAGE. The types and amounts of expressed proteins also varied amongst the samples. The fewest soluble proteins were present in filamentous sporophytes. There were more types and amounts of soluble protein in conchospores than in filamentous sporophytes, but fewer than in bulgy sporophytes. More types of protein were detected in filamentous sporophytes cultivated in high temperatures than in those growing in normal situations. The most types and amounts of protein were found in blade gametophytes in all samples. Blade gametophytes harvested last year and stored at -20 ℃ showed only minor differences in expression of proteins when compared with those harvested in different seasons.

Researches on Extraction,Purification Technique and Properties of Lectin from Porphyra yezoensis

[Objective] The aim was to explore the extraction technique and purification method of lectin from Porpya yezoensis,as well as to investigate the properties of lectin. [Method] The effects of four factors such as buffer system,solid-liquid ratio,temperature,and extracting time on the lectin extraction result from P. yezoensis were investigated by the use of specific activity as the indicator. Then the lectin was purified by DEAE-Sepharose FF chromatography. The hemagglutination activity was used as the indicator,and the properties of the lectin such as carbohydrate specificity,divalent cations dependence,temperature,acidic and alkaline stability and others were detected. [Result] The optimal extraction conditions were：extraction ageat Tris-HCl buffer （25 mmol/L,pH=7.5 ）,solid-liquid ratio of 1：5,extracting temperature of 40 ℃,and extracting time of 16 h. The activity of the lectin of P. yezoensis didn＇t change after 30 min of being heated at 50 ℃,which showed a certain extent of thermal stability,and the suitable pH value was 7-9. The lectin of P. yezoensis could combine with maltose,D-galactose,D-xylose and L-Arabinose,in which the binding force with the maltose was the strongest. The lectin activity was depended on the addition of Ca2＋ and Mg2＋. [Conclusion] The research had provided theoretical basis for the clarification of immune mechanism of P. yezoensis,as well as its high effective utilization.

条斑紫菜(Porphyra yezoensis)耐高温品系的筛选及特性分析

采用60Co-γ射线诱变和高温胁迫处理条斑紫菜野生品系(WT),进行了耐高温品系的选育研究。结果表明,在18℃和22℃组,所选育的耐高温品系(TM-18)和WT品系的壳孢子存活率无显著差异,但在24℃和25℃组,TM-18与WT相比,壳孢子存活率分别提高了274%和296%,畸形苗率分别下降了286%和161%,假根发生率分别提高了151%和429%。将18℃培养50d的苗再分别在18℃、22℃和24℃下再培养30d,TM-18苗的绝对生长率分别为WT苗的3.82、3.16和4.95倍,特定生长率为WT苗的1.53、1.49、2.10倍。在24℃下培养15d或25℃下培养10d,WT苗停止生长,出现了卷曲和严重腐烂,而TM-18苗仍然生长良好,不腐烂。另外,TM-18的三种主要光合色素(Chl.a、PE和PC)含量明显比WT高。上述结果说明,TM-18是一个颜色好、生长快和耐高温的优良品系。

条斑紫菜(Porphyra yezoensis)选育品系壳孢子的放散量与耐高温性研究

模拟生产培育条件,对条斑紫菜选育品系(T-17)和野生型品系(WT)的贝壳丝状体在不同温度下的壳孢子放散量及壳孢子萌发体的生长发育进行了研究。结果表明,在18、23、24和25℃下,连续放散16d的平均每壳的壳孢子量,T-17分别为8.6×106、3.4×106、5.4×105和0个,WT分别为8.0×106、2.8×106、4.9×105和0个,两个品系之间无显著差异;18℃组的壳孢子萌发和生长均正常。将23℃下放散的壳孢子分别在三种高温条件下(23℃、24℃下培养3d再转入25℃、25℃)培养16d,壳孢子成活率,T-17分别为84.6%、59.7%和45.5%,而WT分别为33.0%、20.9%和17.1%,差异极显著。在三种高温条件下,WT的壳孢子萌发体均出现了畸形和烂苗,而T-17的壳孢子萌发体,23℃组发育正常,其余二组的发育也不正常,出现畸形。上述结果说明,T-17品系不仅具有与WT一样的壳孢子放散量,而且壳孢子和萌发体的耐高温能力均比WT强,有望在生产上得到应用。

^(60)Co-γ射线对条斑紫菜(Porphyra yezoensis)的诱变效果与色素突变体分离

用60Co-γ射线辐照处理野生型条斑紫菜的壳孢子萌发体以获得色素变异细胞,随后用酶解法获得单离的变异细胞进行个体再生培养,从中分离出不同的条斑紫菜色素突变体。结果表明,叶状体经剂量分别为100、300、500、700和900Gy的γ射线辐照后再培养1周,均出现了多种色素变异细胞,随后,它们逐渐分裂成为变异细胞块。在0—500Gy辐照剂量范围内,色素变异细胞块的百分率随着辐照剂量的增加而增加,但增至700Gy时,变异细胞块的百分率反而下降,说明500Gy是最合适的剂量。从单离变异细胞的再生叶状体中分离出桔红、紫红、桔黄和墨绿等颜色的单色变异体,并获得了它们的丝状体纯系。各变异品系的F1叶状体均为单色,其颜色与各自的母本叶状体相同,表明它们是稳定突变体。各色素突变品系的叶状体活体吸收光谱不仅与野生型品系有明显的差异,而且各突变品系之间也存在较大的差异。

条斑紫菜(Porphyra yezoensis)丝状体的超微结构观察

本文报道了条斑紫菜自由丝状体的超微结构。观察到丝状体的细胞主要由细胞壁、细胞膜、色素体、细胞核、线粒体、内质网、淀粉粒和液泡等构成。细胞之间有栓子状的孔状联系存在。并对色素体的存在部位做了探讨。

条斑紫菜(Porphyra yezoensis)dbEST中筛选微卫星位点及引物种间转移扩增

采用电子查询的方法,借助Blast软件从条斑紫菜表达序列标签数据库的20979条序列中筛选微卫星位点,共发现非冗余微卫星位点211个,占整个条斑紫菜EST数据库的1.01%。其中双碱基重复微卫星位点共有35个,占查找微卫星序列总数的16.6%;三碱基微卫星有176个,占83.4%。双碱基重复微卫星中AG/CT形式最多,而三碱基中GGC/GCC最多。从筛选出的微卫星位点中选取序列设计合成引物,用在坛紫菜上进行引物种间转移扩增研究。在总共15对引物中有13对引物在两个种间都有扩增产物。结果表明,微卫星位点在这两种紫菜之间具有很高的同源性。获得的微卫星引物可用来进行种群遗传多样性研究、谱系鉴定和遗传图谱的构建。而微卫星标记的共显性可用来进行紫菜二倍体丝状体纯合性检验,为纯系紫菜品系的鉴定提供分子生物学鉴定方法。

Community structure and controlled factor of attached green algae on the Porphyra yezoensis aquaculture rafts in the Subei Shoal, China

This study analyzes the community structure, the quantity changes of the algae and the effect of important environmental factors and estimates the total biomass of the attached green algae in the survey areas. The study uses data from surveys of the attached green algae on the Porphyra yezoensis aquaculture rafts and data regarding the environmental factors from October 2010 to April 2011 in the Subei Shoal. The attached green algae on the rafts included Ulva prolifera, Capsosiphon groenlandicus, U. linza, U. intestinalis, U. clathrata, and U. cornpressa. The biomass changes of the attached green algae exhibited an inverted parabola： the biomass was the highest （14 898 t） in April, and was the second highest （2 034 t） in November; it was lowest in February （only 729 t） and increased sharply from March to April. The species diversity differed significantly among the seasons. In September and October, when the P. yezoensis aquaculture rafts were initially set up, the attached green algae had a high biodiversity, while from December to the next February, a variety of green algae species coexisted on the rafts, although the biomass was low, and from March to April, as the biomass increased sharply, the species diversity dropped to the minimum. During this time, C. groenlandicus was apparently dominant with the maximum biomass proportion up to 80%, while the U. prolifera proportion increased exponentially to 20% to 40%. The water temperature had a direct regulating effect on the biomass and the species succession of the attached green algae. The estimation of the community dynamics and the biomass of the green algae provided the evidence needed to track the origin of the large-scale green tide in the southern Yellow Sea.

条斑紫菜(Porphyra yezoensis)游离氨基酸组成分析

采用邻苯二甲醛/9-氯甲酸芴甲酯(OPA/FMOC)柱前衍生反相高效液相色谱法(RP-HPLC),对江苏南通海区栽培的12个条斑紫菜(Porphyra yezoensis)样品游离氨基酸组成进行分析。结果显示:供试样品游离氨基酸总量在19.82～38.91mg.g-1之间,共检出16种游离氨基酸,其中Ala、Arg、Glu和Asp在所有测试样品中含量显著,占游离氨基酸总量的80%以上,Glu与Ala质量分数与游离氨基酸总量呈极显著正相关性(P<0.01);样品游离氨基酸中必需氨基酸质量分数在9.48～13.06 mg.g-1之间,占总量的33.44～52.75%;呈味氨基酸以Ala、Asp、Glu和Gly等鲜甜味氨基酸为主,质量分数在10.23～25.86 mg.g-1之间,占游离氨基酸总量的47.53～66.88%。

In Silico Screening for Microsatellite Markers from Expressed Sequence Tags of Porphyra yezoensis (Bangiales, Rhodophyta)

The genomic resources of Porphyra yezoensis expressed sequence tags (ESTs) were utilized to identify simple sequence repeats (SSRs), or microsatellites. This method took the advantage of using ESTs and microsatellites either for the establishment of gene identities or for the acquisition of high polymorphism. The microsatellites can be used as gene markers when microsatellites are tagged to genes. Revealed by bioinformatics analysis, 1 162 out of 21 954 ESTs contained microsatellites and cluster analysis indi- cated that 984 of these ESTs fell into 112 contigs, while the other 178 ESTs were singletons. A total of 290 unique SSR-containing genes were identified. The AAC SSRs were the most populous type of microsatellites. GC-rich microsatellites were predominant among all the microsatellites.

Genetic variation of Porphyra yezoensis by using AFLP1

Genetic variation of 11 lines of Porphyra yezoensis from the coastline of Kagoshima of Japan, Qingdao, Nantong, Putuo and Nanji Islands of China were studied by using amplified fragment length polymorphism (AFLP). 778 bands were obtained with AFLP analysis of 16 primer combinations, among which 15 were unique, about 98.07% were polymorphic. The AFLP data showed that the closest genetic distance was 0 - 180 between two Kagoshima samples, and the farthest one was 0.397 between Kagoshima No. 1 and Nantong No - 9 line. The genetic distance showed that the variation was within the inner species scope. Neighbor-joining cluster and UPGMA cluster indicated that samples from Kagoshima and Qingdao were with high similarity and either with the samples of Nantong, Putuo and Nanji Islands. P. yezoensis in China shared high genetic diversity, and the genetic distance showed positive correlation with the geographic distance.

Efficient gusA Transient Expression in Porphyra yezoensis Protoplasts Mediated by Endogenous Beta-tubulin Flanking Sequences

Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are unknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were con-structed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5’- and 3’-flanking regions (Tub5’ and Tub3’) up- and down-stream of β-glucuronidase (GUS) gene (gusA), respectively, into pA, a derivative of pCAT?3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3’. These con-structs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5’-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when com-pared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35 S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.

Flavor Characteristics of Different Crops of Laver(Porphyra yezoensis)During One Harvest Cycle

The taste and odor analysis of the first,second,fourth and sixth crops of laver(Porphyra yezoensis)was obtained by electronic tongue,taste-related compound,electronic nose and gas chromatography-ion mobility spectrometer(GC-IMS).Through the detection of the electronic tongue,the pleasant tastes such as umami and richness are more intense in the early crops,which is consistent with the analysis results of free amino acids and flavored nucleotides.The electronic nose can clearly distinguish between different laver crops.GC-IMS separated and identified 36 volatile components from lavers,mainly including aldehydes,ketones,alcohols,esters,acids and aromatic substances.For the early harvested crops,nonanal,octanal,hexanal and benzaldehyde have a significant contribution to odor because of the high concentration and low threshold of these compounds.For the later harvested crops,the concentration of most ketones and some aldehydes increased.1-Octen-3-ol,malondialdehyde and heptanal constituted the main odor characteristics.

Cloning and Analysis of Calmodulin Gene from Porphyra yezoensis Ueda (Bangiales, Rhodophyta)

In order to understand the mechanisms of signal transduction and anti-desiccation mechanisms of Porphyra yezoensis, cDNA and its genomic sequence of Calmodulin gene (CaM) was cloned by the technique of polymerase chain reaction (PCR) based on the analysis of P. yezoensis ESTs from dbEST database. The result shows that the full-length cDNA of CaM consists of 603 bps including an ORF encoding for 151 amino acids and a terminate codon UGA, while the length of genomic sequence is 1231 bps including 2 exons and 1 intron. The average GC content of the coding region is 58.77%, while the GC content of the third position of this gene is as high as 82.23%. Four Ca2+ binding sites (EF-hand) are found in this gene. The predicted molecular mass of the deduced peptide is 16688.72 Da and the pI is 4.222. By aligning with known CaM genes, the similarity of CaM gene sequence with homologous genes in Chlamydomonas incerta and Chlamydomonas reinhardtii is 72.7% and 72.2% respectively, and the similarity of the deduced amino acid sequence of CaM gene with homologous genes in C. incerta and C. reinhardtii are both 71.5%. This is the first report on CaM from a species of Rhodophyta.

Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

The wild Porphyra yezoensis collected from the Qingdao coast was used to prepare protoplasts by enzyme digestion. The pure line was constructed by cultivating the protoplasts. The 18S rDNA of the P. yezoensis pure line was cloned and sequenced. Sequence analysis was executed for this sequence and other 22 sequences retrieved from GenBank. A phylogenetic tree was constructed using the neighbor joining method. The results revealed a high diversity of 18S rDNA sequences in genus Porphyra and the considerable variation of 18S rDNA sequences in different strains of the same species P. yezoensis and P. tenera. Significant difference of 18S rDNA sequence was observed between P. yezoensis from Qingdao, China, and the two strains of P. yezoensis from Japan, but the three strains of P. yezoensis formed a stable clade in the phylogenetic tree. These results indicate the possibility of interspecies and intraspecies discrimination of Porphyra using the 18S rDNA sequences.

Exploring valid internal-control genes in Porphyra yezoensis(Bangiaceae) during stress response conditions

To screen the stable expression genes related to the stress （strong light, dehydration and temperature shock） we applied Absolute real-time PCR technology to determine the transcription numbers of the selected test genes in Porphyra yezoensis, which has been regarded as a potential model species responding the stress conditions in the intertidal. Absolute real-time PCR technology was applied to determine the transcription numbers of the selected test genes in Porphyra yezoensis, which has been regarded as a potential model species in stress responding. According to the results of photosynthesis parameters, we observed that Y（II） and Fv/Fm were significantly affected when stress was imposed on the thalli of Porphyra yezoensis, but underwent almost completely recovered under normal conditions, which were collected for the following experiments. Then three samples, which were treated with different grade stresses combined with salinity, irradiation and temperature, were collected. The transcription numbers of seven constitutive expression genes in above samples were determined after RNA extraction and cDNA synthesis. Finally, a general insight into the selection of internal control genes during stress response was obtained. We found that there were no obvious effects in terms of salinity stress （at salinity 90） on transcription of most genes used in the study. The 18S ribosomal RNA gene had the highest expression level, varying remarkably among different tested groups. RPS8 expression showed a high irregular variance between samples. GAPDH presented comparatively stable expression and could thus be selected as the internal control. EF-la showed stable expression during the series of multiple-stress tests. Our research provided available references for the selection of internal control genes for transcripts determination of P. yezoensis.

The influence of Fe^(2+) on growth and development of cells enzy-matically isolated from Porphyra yezoensis blades

Fe2+ acted as an accessorial factor for many cellular enzymatic reactions is very important for seaweed growth and development, but the Fe2+ requirement in nori had not been seen. Porphyra yezoensis cells were separated enzymatically and cultured in a series of sterilized seawater media containing various concentra- tions of Fe2+. The growth development and cell were investigated in this work. Through this experiment, two biologically-meant concentration scales were found, one is low concentrations, 12.1–102.1 μg/L, 10–100 times than that in seawater, favoring the development of isolated cells of Porphyra and the other was high concentra- tions, more than 10mg/L inhibiting the cell growth, leading to the deformity and shrinkage of the cells. At the concentration of 50 mg/L, the cells stopped growing and died eventually.

Purification and Photodynamic Bioactivity of Phycoerythrin and Phycocyanin from Porphyra yezoensis Ueda

Phycoerythrin and phycocyanin were purified from Porphyra yezoensis Ueda with their bioactivity determined in this study. Continuous precipitation with ammonium sulfate at different concentrations(10%, 20%, 40% and 50%) increased the purity(A564:A280) of phycoerythrin to 1.49, 3.92 fold of the raw extract(0.38) and the purity(A615:A280) of phycocyanin to 0.70, 3.33 fold of the raw extract(0.21). Two more times of chromatography with hydroxylapatites finally made the purity of phycoerythrin and phycocyanin reach 5.50, 14.47 fold of the raw extract, and 5.10, 24.29 fold of the raw extract, respectviely. The yield of high purity phycoerythrin and phycocyanin were 0.21% and 0.09% of dried P. yezoensis blade, respectively. The photodynamic cytotoxic experiment showed that both phycoerythrin and phycocyanin inhibited the growth of liver tumor cells significantly. It was found that 250 mg L-1 purified phycoerythrin and phycocyanin inhibited the growth of hepatocellular carcinoma cells 24 h after laser-irradiation by 80% and 59%, respectively, and 100 mg L-1 purified phycoerythrin and phycocyanin induced the apoptosis of 31.54% and 32.54% of the cells, respectively, 8 h after photodynamic therapy. Oue findings demonstrated that P. yezoensis can serve as photosensitizer(phycoerythrin and phycocyanin) producer.

Extraction of plasmid-like DNA and high-quality total DNA from Porphyra yezoensis

Somatic cells were prepared from sea snail enzyme digests of Porphyra yezoensis thalli. Us ing SDS - Proteinase K as extraction solution, total DNA was isolated from the somatic cells. The crude extracts of total DNA were purified with glassmilk, and the resulting DNA was of sufficient quality for digestion of restriction endonuclease. DNA bands were clearly observed in the restriction patterns of EcoRI, PstI and HaeIII respectively. The presence of DNA hands in the restriction pattern of total DNA indicated that the genome of Porphyra yezoensis may be small. Unexpectedly, using guanidinium isoth iocyanate and sarcosyl as extraction solution, a plasmid-like DNA band (2.3 Kb) was directly found in the isolated total DNA of Porphyra yezoensis. A very simple and convenient method for plasmid-like DNA isolation has been established.

Ultrastructure of Single Cells, Callus-like and Monospore-like Cells in Porphyra yezoensis Ueda on Semi-solid Culture Medium

It had been demonstrated that individual cells or protoplasts isolated fromPorphyrathallus by enzyme could develop into normal leafy thalli in the same way as monospores, and that isolated cells develop in different way in liquid and on semi-solid media. The authors observed the ultrastructure of isolated vegetative cells cultured on semi-solid media and compared them with those of monospores and isolated cells cultured in liquid media. The results showed that subcellular structures were quite different among cells in different conditions. In their development, isolated cells on semi-solid media did not show the characteristic subcellular feature of monospore formation, such as production of fibrous vesicles. Callus-like cells formed on semi-solid media underwent a distinctive modification in cellular organization. They developed characteristic cell inclusions and a special 2-layer cell covering. Golgi bodies, ER, starch grains, mitochondria. Vacuoles were not commonly found in them.