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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Observation of female gamete (Carpogonia) germination of Porphyra yezoensis and P. oligospermatangia

Several papers have reported that part or whole leafy thallus seemingly consisting of zygotospores can give rise to both blades and conchocelis in the same culture of Porphyra. Study on samples of wild and cul- tivated Porphyra yezoensis and P. oligospermatangia were conducted to clarify the origination of the young blades in the culture. It is confirmed that single cells on the blade of both species, which normally intermixed with zygotospores, germinated into young blades. TEM and SEM observation has shown that the single cells of Porphyra yezoensis had typical features of female gamete (carpogonia) but archeospore. Therefore, the female gametes are responsible in developing leafy thalli.

乳酸菌分离鉴定及其条斑紫菜乳酸菌混合发酵理化因子的变化

对泡菜老坛水中的乳酸菌进行分离鉴定,获得3株乳酸菌,确定为短乳杆菌(Lactobacillus brevis)、植物乳杆菌(Lactobacillus plantarum)、干酪乳杆菌(Lactobacillus casei)。以条斑紫菜(Porphyra yezoensis)为原料,以4种不同配比的上述乳酸菌对条斑紫菜进行直投式发酵,探讨发酵过程中发酵液部分理化因子的变化规律。结果显示,4种发酵液的pH下降趋势相似,从开始的4.51下降到2.90;1∶1∶2(体积比)混合乳酸菌发酵液的产酸能力最强,发酵终点总酸含量为13.75 g/L,为发酵开始的6倍;1∶1∶2混合乳酸菌发酵液的总糖含量由开始的29.95 g/L下降为发酵终点的18.97 g/L;1∶1∶2混合乳酸菌发酵液亚硝酸盐含量的峰值最小,为3.75 mg/kg;1∶1∶1混合乳酸菌发酵液氨基酸含量较高,最大值为58.16μmol/mL;蛋白质含量下降幅度最大的是1∶1∶3混合乳酸菌发酵液。

条斑紫菜叶绿体基因密码子偏爱性分析

条斑紫菜是一种具有重要经济价值的藻类植物,广泛应用于医学、纺织和食品等领域。本研究通过Python、OGDRAW、CodonW、CUSP、DNAstar及SPSS20等软件进行多元统计和密码子偏爱性分析,并获得了56个条斑紫菜叶绿体基因序列的GC_(1)、GC_(2)、GC_(3)、ENC值和RSCU值,同时获得了条斑紫菜叶绿体基因组结构图和密码子使用热图。结果显示,条斑紫菜叶绿体基因的AT碱基含量高于GC碱基含量,说明其密码子偏爱于A/T碱基结尾。中性绘图分析、ENC绘图分析和PR2-plot分析结果表明影响条斑紫菜叶绿体基因密码子偏爱性的主要影响因素是自然选择。将高频密码子和高表达优越密码子结合,最终获得了9个条斑紫菜叶绿体基因的最优密码子,如UUU、UCA和CCU,均偏爱于A/U碱基结尾。研究结果为条斑紫菜的资源开发和培育利用,以及外源基因的稳定表达提供了重要的科学依据。