•Fusion proteins of streptavidin and allophycocyanin alpha subunit were expressed in E. coli.•The fusion proteins had strong fluorescence and the ability to bind biotin.•The fusion proteins were used as fluorescent labels in immunofluorescence assay.Allophycocyanin is generally used as fluorescent label. In this study, the fusion protein (SLA) of core streptavidin from Streptomyces avidinii and allophycocyanin alpha subunit from thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, together with lyase (cpcS) and phycoerythrobilin (PEB) synthesizing enzymes (Ho1 and PebS) or phycocyanobilin (PCB) synthesizing enzymes (Ho1 and PcyA), were co-expressed in Escherichia coli. The fusion proteins were purified by metal affinity chromatography. Zinc staining analysis and spectral analysis showed that the recombinant fusion proteins covalently bound PEB (denoted as SLA-PEB) or PCB (denoted as SLA-PCB). The two proteins were highly fluorescent and were able to binding biotin. α-fetoprotein (AFP), a tumor marker, was used as analyte to evaluate the application of these two fluorescent proteins in sandwich immunofluorescence assay. The measurable ranges of AFP by these proteins were 0.02–50 ng/ml with an average coefficient of variation below 15%. The detection limit for AFP detection by SLA-PEB and SLA-PCB were 0.11 and 0.35 ng/ml, respectively. The detection sensitivities were comparable to that by streptavidin-R-phycoerythrin (SA-PE). Thus, these fusion proteins would be useful fluorescent labels in immunofluorescence assay.

•Combinational biosynthesis of two new SA-PBPs has been achieved using genetic E. coli.•These SA-PBPs were proved with desired fluorescence and efficient targeting for biotinylated substances.•The SA-PBPs were demonstrated as useful tools in immunoassay for detection of two biomarkers of liver cancers.The development of fluorescent tools with desired fluorescence and efficient targeting is of great importance in the high-throughput immunoassay. Here we report combinational biosynthesis of new dual-functional streptavidin-phycobiliproteins (SA-PBPs) in Escherichia coli by fusing streptavidin with phycobiliproteins. These recombinant proteins can achieve a purity over 95% after one-step purification, and their maximum absorption and fluorescence emission wavelength are at 556 nm and 568 nm for SA-PCA-PEB (streptavidin-phycocyanin α subunit-phycoerythrin), and 624 nm and 646 nm for SA-PCA-PCB (streptavidin-phycocyanin α subunit-phycocyanobilin), respectively. The potential application of these dual-functional SA-PBPs in immunoassay was evaluated using “sandwich” ELISA method for detection of two biomarkers of liver cancers, i.e. α-fetoprotein (AFP) and carcinoembryonic antigen (CEA). As a result, both SA-PCA-PCB and SA-PCA-PEB showed a good linear function with AFP & CEA within 0–50 ng/ml concentrations. Their limits of detection (LODs) for AFP and CEA were 0.25 ng/mL and 0.28 ng/ml using SA-PCA-PEB, and 1.01 ng/ml and 1.12 ng/ml using SA-PCA-PCB respectively. These results indicate that these novel dual-functional SA-PBPs are useful tools for a wide variety of immunoassay, and may have the advantages in their higher expandability and compatibility with existing and future immunoassay technologies.Download high-res image (70KB)Download full-size image

White spot disease is the most frequent and harmful disease affecting the shell-boring conchocelis stage of the economically important red alga Porphyra yezoensis (= Pyropia yezoensis (Ueda) Hwang & Choi) (Bangiales, Rhodophyta). To date, its potential pathogens and pathogenesis remain poorly understood. In this study, we isolated 7 bacteria, 26 fungi, and 10 actinomycetes from sick conchocelis. Re-infection experiments revealed that only one fungus, GF014, caused disease. Morphological characteristics of the colony, mycelium, sporangium, and spore of GF014 suggested that the strain belongs to the order Pleosporales and the genus Phoma. Classification based on internal transcribed spacer (ITS) sequences supported the results of morphological identification. These results indicate that GF014 is a species of Phoma and a specific pathogen for Porphyra. GF014 preferred organic nitrogen sources, and 20–28 °C and low salinity water were its optimal living conditions. Our findings will play an active role in preventing and treating white spot disease in P. yezoensis.

Green tides have occurred in the Yellow Sea successively from 2007 to 2011. Genetic analysis of the 5-year green-tide-forming algae needs to be performed to determine the source of the biomass and understand the mechanism of the green tide blooms. In this study, free-floating green algae were collected at different sites in the Yellow Sea in 2010 and 2011. Data on 182 free-floating samples and 155 attached Ulva samples from previous studies on the Yellow Sea green tides from 2007 to 2009 were also taken into consideration. Morphology observation and molecular phylogenetic analyses indicated that the Yellow Sea green tides were dominated by a single species, Ulva prolifera, from 2007 to 2011. Considering that at least five Ulva species inhabit the north coast of China, the unialgal composition of the green tides implied that (1) there may be some special physiology and propagation pathways of U. prolifera for its rapid expansion, (2) the mechanisms of the Yellow Sea green tide formation were similar for the last five years, and (3) the intra-species genetic variation and population structure of U. prolifera need to be studied to determine the exact origin of the bloom-forming biomass.

In 2008, a massive Ulva prolifera bloom, with a 3-million-ton biomass covering an area of 1.29 × 104 km2 at its largest, suddenly appeared from May to July in South Yellow Sea. The mechanism behind the rapid growth of these seaweeds was investigated. Molecular phylogenetic analysis of free-floating algal samples from the Yellow Sea suggested that U. prolifera belong to one population, and that temporary cyclonic eddies in the Yellow Sea in late spring and early summer may help promote the proliferation of this bloom by providing seaweeds with sufficient growth time, abundant nutrition, and favorable habitats. The initial investigation on the relationship between marine cyclonic eddies and the route of free-floating algae extends our knowledge on how the emergence of free-floating macroalgal blooms in coastal areas could yield a large biomass.

Zinc-decorated silica-coated magnetic nanoparticles (ZnSiMNPs) were prepared by adsorbing zinc onto colloidal silica. These nanoparticles were used for the rapid purification of 6×His-tagged recombinant phycobiliprotein. The surface changes in the magnetic nanoparticles after zinc adsorption were characterized by transmission electron microscopy. The adsorption of the 6× His-tagged phycobiliprotein onto ZnSiMNPs in 10 mM PBS at 25 °C was found to follow the Langmuir isotherm. ZnSiMNPs could be used to extract 6×His-tagged phycobiliprotein from lysate to single-band purity, as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. No spectral variation was observed in the purified phycobiliprotein. Thus, ZnSiMNPs served as a useful tool for the magnetic separation and delivery of the 6×His-tagged phycobiliprotein.

Ulva (Enteromorpha) prolifera, widely distributed from the intertidal to the upper subtidal zones around the world, was the dominant species of the massive green tides in the Yellow Sea in the summers of 2007, 2008, and 2009. However, little is known about its intra-species genetic diversity. In this study, six attached and seven floating U. prolifera samples collected from different sites distributed from the north of the Yellow Sea to the south of the East China Sea were taken in inter-simple sequence repeat (ISSR) analysis. Based on the results of the 90 polymorphic bands from four ISSR primers, the genetic diversity level of the floating samples (H = 0.1663, I = 0.2608) was found to be lower than that of the attached samples (H = 0.2105, I = 0.3346). Unweighted pair-group mean analysis (UPGMA) and principal coordinate analysis (PCoA) suggested that floating U. prolifera samples in the Yellow Sea from 2007 to 2009 had a close genetic relationship, and the floating samples were separated from the attached samples. Genetic differentiation and limited gene flow among attached U. prolifera populations were indicated by analysis of molecular variance.

This study investigated the delivery of a SV40 promoter driving lacZ gene into cells of Kappaphycus alvarezii using particle bombardment. Thallus pieces 0.5–0.8 mm in diameter and 1 cm in length were prepared as gene recipients. Bombardment parameters of 450 psi (rupture pressures) × 6 cm (particle travel distances), 650 psi × 6 cm, 1,100 psi × 6 cm and 1,100 psi × 9 cm were used. A significant increase in transformation efficiency from about 33% under the rupture pressure of 450 psi to 87% at 650 psi was observed in transformed thalli. Most of the positive cells appeared in epidermal cells bombarded at 450 psi, whereas positive signals were seen in both epidermal and medullary cells at 650 psi. No positive transient expression was detected at a bombardment of 1,100 psi, or in negative or blank controls. For the conditions tested, the best parameter was obtained at 650 psi at a distance of 6 cm. Thus, the strategy of taking vegetative thalli as recipients, using particle bombardment, and combining this with micro-propagation, together with developing an in vivo selectable marker, is a viable way to produce stable transformants, to eliminate chimeric expression, and to achieve transgenic breeding in K. alvarezii.

A transient transformation system for the unicellular marine green alga, Platymonas subcordiformis, was established in this study. We introduced the pEGFP-N1 vector into P. subcordiformis with a glass bead method. P.subcordiformis was incubated in cell wall lytic enzymes (abalone acetone powder and cellulase solutions) to degrade the cell wall. The applicable conditions for production of viable protoplasts were pH 6.5, 25°C, and 3 h of enzyme treatment. The protoplast yield was 61.2% when P. subcordiformis cells were added to the enzyme solution at a concentration of 107 cell ml−1. The protoplasts were immediately transformed with the pEGFP-N1 vector using glass-bead method. The transformation frequency was about 10−5, and there was no GFP activity observed in either the negative or the blank controls. This study indicated that GFP was a sensitively transgenic reporter for P. subcordiformis, and the method of cell wall enzymolysis followed by glass bead agitation was applicable for the transformation of P. subcordiformis.

Ulva pertusa is a native species to Asia along the western coast of Pacific Ocean, with new occurrence records in the eastern coast of Pacific, the northwest coast of Atlantic and the Mediterranean Sea. However, little is known about its population genetic structure. In this study, twelve U. pertusa populations from 3 coastal areas of China: Qingdao, Yantai and Dalian, were applied to ISSR analysis. The selected 4 ISSR primers amplified 120 polymorphic bands totally. Nei’s gene diversity (H) ranged from 0.0729 to 0.1496, and Shannon’s information index (I) ranged from 0.1072 to 0.2196. Genetic diversity was greater within Qingdao populations (H = 0.2069, I = 0.3232). Analysis of molecular variance (AMOVA) showed the greatest variance within populations (68.57%), much less variance among populations (22.63%) and among areas (8.79%). Unweighted pair-group mean analysis (UPGMA) indicated that clustering of U. pertusa individuals mainly relates to their populations and geographic distances separating those populations. Genetic differentiation and limited gene flow among U. pertusa populations were indicated by ISSR analysis.

Allophycocyanin (APC) is widely used as a fluorescent tag for fluorescence detection techniques. In this study, the apcB gene from a thermophilic cyanobacterium strain was cloned and ligated into an expression vector to construct a pathway for the biosynthesis of an allophycocyanin beta subunit (holo-apcBT) in Escherichia coli. Isopropyl β-d-1-thiogalactopyranoside induction successfully reconstituted holo-apcBT in E. coli. The recombinant holo-apcB showed spectroscopic properties similar to native APC. The stability and spectroscopic properties of this protein were then compared with another recombinant allophycocyanin beta subunit (holo-apcBM) whose apcB gene was cloned from mesophilic cyanobacterium. At high temperatures and during the course of storage, holo-apcBT was significantly more stable than holo-apcBM. In addition, holo-apcBT had an unexpectedly higher extinction coefficient and fluorescence quantum yield than holo-apcBM, suggesting that holo-apcBT would be a promising fluorescent tag and serve as a substitute for native APC.

Allophycocyanin (APC) is generally used as fluorescent labels. In this study, apcA genes from a mesophilic cyanobacterium Synechocystis sp. PCC 6803 and a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 were cloned into expression vectors to construct pathway for biosynthesis of allophycocyanin holo-α subunits (named as holo-ApcAS for Synechocystis sp. PCC 6803 and holo-ApcAT for T. elongatus BP-1) in Escherichia coli. The two holo-ApcAs were successfully reconstituted in E. coli and purified by metal affinity chromatography. Spectral analysis showed that the two proteins have similar spectroscopic properties, with absorbance maximum both at 614 nm and emission maximum at 639 nm for holo-ApcAS and 638 nm for holo-ApcAT. At high temperature, the recombinant holo-ApcAT was much more stable than the recombinant holo-ApcAS. Holo-ApcAS was most fluorescent at pH 8.5 and stable in pH range of 6.0–9.0 while holo-ApcAT was most fluorescent at pH 6.0 and stable in pH range of 5.0–7.0, with residual fluorescence intensity no less than 90% of the maximum fluorescence. These findings will pave the way for further protein engineering to achieve high stable APC from extremophiles.