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.

条斑紫菜(Porphyra Yezoensis Ueda)和孔石莼(Ulve Pertusa Kjellm)对酸耐受力的研究——Ⅰ.pH对条斑紫菜和孔石莼呼吸强度和致死酸度试验

不同pH海水和不同时间处理紫菜、石莼对呼吸强度和致死酸度试验结果表明,紫菜比石莼耐酸力强。在海水温度为18℃条件下,分别用pH_2、pH_3、pH_4、pH_5、pH_6、pH_7、pH_8处理,处理时间20分钟,紫菜除pH_2处理对呼吸强度有抑制作用和培养5天后有20％死亡外,pH_3、pH_4、pH_5、pH_6、pH_7、pH_8各组处理对呼吸强度都有促进作用,培养5天后没有死亡现象,而石莼经pH_2、pH_3处理,对呼吸强度都有抑制作用,培养5天后,pH_2处理组全部死亡,pH_3处理有50％死亡。pH_3和pH_4在海水温度为18℃条件下,处理时间为40分钟时,对紫菜呼吸强度有促进作用,培养5天报,pH_3处理组仅有20％死亡。而pH_3对石莼呼吸强度有抑制作用,培养5天后,全部灭亡。在生产上是否可以考虑用pH_3的海水处理紫菜网帘以达到消除石莼(杂藻)的目的,将是一项具有生产意义的待试验课题。

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)遗传多样性的AFLP分析

利用扩增片段长度多态性技术(AFLP)分析了13个条斑紫菜品系,在80对引物中,有11对能得到重复性好的多态性扩增条带,共扩增出619条谱带,每对引物扩增带数为40—77条,并且各对引物扩增结果均显示样品间存在差异。共得到609条多态性条带,多态位点的比例高达98.38%。根据AFLP图谱计算了不同样品间的遗传距离(GD)和相似性系数(GS)。聚类结果显示,品系海安与二室间的遗传相似系数较高而聚合在一起,不同样品间的相似性系数介于0.595—0.845之间。结果表明,条斑紫菜的遗传多样性极为丰富,而部分材料间的遗传距离有随地理间距增大而逐渐增大的趋势。聚类分析结果反映了目前条斑紫菜养殖品种间存在着相互混杂。

GAMMA-RAYS INDUCTION OF MUTATION IN CONCHOCELIS OF PORPHYRA YEZOENSIS

Free living conchocelis of Porphyra yezoensis Ueda (Bangiales, Rhodophyta) were treated with 6 0Co γ rays of different doses (ranging from 100 Gy to 1000 Gy) to induce mutation. Most of the conchocelis maintained the capability of penetrating into shells, growing and forming colonies in shells, but their vitality was seriously impaired by the irradiation of γ rays. A few conchocelis pigments were mutagenized directly into different color pigment mutants whose progeny conchospores and foliose thalli had the same colors. However, some irradiated conchocelis did not show the change in color at the conchocelis stage. The pigment mutation could be observed only after the conchospores of these conchocelis had germinated into young foliose thalli. Irradiation of low dose (100 Gy) promoted the growth of thallus and many with altered morphology were observed. Conchospores of the irradiated conchocelis attached to the culture nets were cultured in the sea, and growth of these progenies was observed and measured.

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.

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.

Isolated Cells of Porphyra yezoensis Cultured on Solid Medium

Vegetative cells of Porphyra yezoensis are isolated with sea snail enzyme and cultured on the solidified agar medium. The results of experiments show that the isolated cells can survive,divide and regenerate well on the medium solidified with agar. The first division on the solid medium starts after 7 days' culture, 4 days later than the liquid culture. The survival rate of isolated cells is 71.3% on the solid medium, lower than the 86.2% of that in seawater.Thalli, thalloids,conchocelis, spermatangia and multicellular masses are developed on the solid/medium in the first month, slowly but normally. Spermatangia sacs disappear within 4 weeks. Without adding nutrient liquid onto the surface of solid medium or injecting seawater under the agar layer in order to keep moisture, the thalli and cell groups release monospores to form new thalli instead of enlarging their areas after 5 weeks' culturing. Some monospores regenerate new thalli. Other monospores lose their pigments and minimize their volume and divide quickly to form light pink calli. After 16 weeks, numerous calli can be seen on the solid medium and after 24 weeks' culturing, almost only calli and conchocelis can be seen. If the calli are immersed in seawater, the monospores are released and may develop into young thallus.

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.

Defensive physiological characters of Pyropia yezoensis resistant lines to the red rot disease

To explore ef fective physiological indexes to distinguish the resistant lines and susceptible lines of Pyt hium porphyrae,the causal agent of red rot disease of Pyropia yezoensis,and establish the disease-resistance breeding strategy,we obtained and analyzed the candidate resistant and susceptible lines by population selection.Gametophytes of the candidate lines were cultured in seawater containing Pyt.porphyrae zoospores.Antioxidase activities,including superoxide dismutase(SOD),peroxidase(POD)and polyphenol oxidase(PPO),were measured and compared between the two lines before and after infection.In the resistant lines,SOD and POD activities increased and then decreased,but PPO activity rose with the prolongation of the infection time.The phenylalanine ammonia lyase(PAL)activities also increased and then decreased after infection,but it had significantly different expression in the two lines without pathogen attack.The synthesis rates ofβ-1,3-glucanase,and cell-wall degrading enzyme were different from each other between the two lines after infection of P yt.porphyrae.Comparison in the contents of malondialdehyde(MDA)and reactive oxygen species(ROS)in the two lines showed that,the two contents varied synchronously in response to the pathogen attack.Changes of these enzymes activities or contents demonstrated that Pyr.yezoensis could resist against the pathogen of P yt.porphyrae with the antioxidant defense capacity.In addition,β-1.3-glucanase content showed extremely significant dif ference between the two lines,and the PAL had consistent expression dif ference.Therefore,phenylalanine ammonia lyase(PAL)andβ-1,3-glucanase can be considered as an ef fective index to distinguish susceptible line and resistant line,with which the workload of the resistant breeding could be reduced in the future.

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.

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.

Photosynthesis-Dependent Extracellular Ca2+ Influx Triggers an Asexual Reproductive Cycle in the Marine Red Macroalga Porphyra yezoensis

Asexual propagation to increase the number of gametophytic clones via the growth of asexual haploid spores is a unique survival strategy found in marine multicellular algae. However, the mechanisms regulating the asexual life cycle are largely unknown. Here, factors involved in the regulation of production and discharge of asexual spores, so-called monospores, are identified in the marine red macroalga Porphyra yezoensis. First, enhanced discharge of monospores was found by incubation of gametophytes in ASPMT1, a modified version of the previously established synthetic medium ASP12. Comparison of the compositions of ASPMT1 and our standard medium, ESL, indicated that the Ca2+ concentration in ASPMT1 was three times lower than that in ESL medium. Thus, we modified ASPMT1 by increasing its Ca2+ concentration, resulting in reduction of monospore discharge. These findings demonstrate the role of reduced Ca2+ concentrations in enhancing monospore production and release. Moreover, it was also observed that initiation of asexual life cycle required illumination, was repressed by DCMU, and was induced by a Ca2+ ionophore in the dark. Taken together, these results indicate that photosynthesis-dependent Ca2+ influx triggers the asexual life cycle by promoting the production and discharge of monospores in P. yezoensis.

Large scale preparation of phycobiliproteins from <i>Porphyra yezoensis</i>using co-precipitation with ammonium sulfate

The purpose of this study was to extract phyco-biliproteins (PBP) from Porphyra yezoensis using an optimized procedure and further establish a large scale process for protein production. According to our previous experiences on the extraction of PBP, salting out methods, e.g. ammonium sulfate precipitation, worked more efficiently than isoelectric precipitation, differential centrifugation or ultrafiltration. Thus, we chose ammonium sulfate to coprecipitate PBP in crude solution. After four times of precipitation followed by one time of high speed centrifugation, the maximum purity of crude phycoerythrin and phycocyanin reached 1.94 (A565/A280) and 0.85 (A615/A280), with a yield of 0.50% and 0.37%, respectively. A total of 0.94 mg phycoerythrin and 0.54 mg phycocyanin with purity of more than 3.2 were obtained from 1 g dried P. yezoensis after additional chromatography. We further scaled up the frozen dried P. yezoensis from 20 g to 400 g, with 1295 mg phycoerythrin and 593 mg phycocyanin obtained.