Yulin Cui

Genetic variation of Ulva (Enteromorpha) prolifera (Ulvales, Chlorophyta)—the causative species of the green tides in the Yellow Sea, China

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.

Chloroplast Transformation of Platymonas (Tetraselmis) subcordiformis with the bar Gene as Selectable Marker

The objective of this research was to establish a chloroplast transformation technique for Platymonas (Tetraselmis) subcordiformis. Employing the gfp gene as a reporter and the bar gene as a selectable marker, transformation vectors of P. subcordiformis chloroplast were constructed with endogenous fragments rrn16S–trnI (left) and trnA–rrn23S (right) as a recombination site of the chloroplast genome. The plasmids were transferred into P. subcordiformis via particle bombardment. Confocal laser scanning microscopy indicated that the green fluorescence protein was localized in the chloroplast of P. subcordiformis, confirming the activity of the Chlamydomonas reinhardtii promoter. Cells transformed with the bar gene were selected using the herbicide Basta. Resistant colonies were analyzed by PCR and Southern blotting, and the results indicated that the bar gene was successfully integrated into the chloroplast genome via homologous recombination. The technique will improve genetic engineering of this alga.

Gene transfer into conchospores of Porphyra haitanensis (Bangiales, Rhodophyta) by glass bead agitation

Wang J., Jiang P., Cui Y., Guan X. and Qin S. 2010. Gene transfer into conchospores of Porphyra haitanensis (Bangiales, Rhodophyta) by glass bead agitation. Phycologia 49: 355–360. DOI: 10.2216/09-91.1 Genetic transformation by electroporation of protoplasts is a standard procedure for many plants. However, for the genus Porphyra, the method is not effective because of low viability of protoplasts and is a time-consuming and expensive procedure. Based on the life history of Porphyra, a spore-targeted strategy of genetic transformation was developed, that is, using fresh conchospores of Porphyra haitanensis Chang & Zheng transformed by agitation with glass beads. A SV40 promoter-driven lacZ reporter gene was expressed in conchospores 48 h after the agitation. More transformants were obtained by increasing the agitation time from 10 to 25 s. The maximum number of transformants was more than six out of 1 million agitated conchospores. Transfer of a SV40 promoter-driven egfp gene into conchospores resulted in significant green GFP fluorescence. The expression of lacZ and egfp revealed that this strategy of spore-targeted transformation using glass bead agitation is feasible in P. haitanensis and that the SV40 promoter is effective for monitoring expression of foreign genes in this red algal species.

Evolutionary origins, molecular cloning and expression of carotenoid hydroxylases in eukaryotic photosynthetic algae

BackgroundXanthophylls, oxygenated derivatives of carotenes, play critical roles in photosynthetic apparatus of cyanobacteria, algae, and higher plants. Although the xanthophylls biosynthetic pathway of algae is largely unknown, it is of particular interest because they have a very complicated evolutionary history. Carotenoid hydroxylase (CHY) is an important protein that plays essential roles in xanthophylls biosynthesis. With the availability of 18 sequenced algal genomes, we performed a comprehensive comparative analysis of chy genes and explored their distribution, structure, evolution, origins, and expression.ResultsOverall 60 putative chy genes were identified and classified into two major subfamilies (bch and cyp97) according to their domain structures. Genes in the bch subfamily were found in 10 green algae and 1 red alga, but absent in other algae. In the phylogenetic tree, bch genes of green algae and higher plants share a common ancestor and are of non-cyanobacterial origin, whereas that of red algae is of cyanobacteria. The homologs of cyp97a/c genes were widespread only in green algae, while cyp97b paralogs were seen in most of algae. Phylogenetic analysis on cyp97 genes supported the hypothesis that cyp97b is an ancient gene originated before the formation of extant algal groups. The cyp97a gene is more closely related to cyp97c in evolution than to cyp97b. The two cyp97 genes were isolated from the green alga Haematococcus pluvialis, and transcriptional expression profiles of chy genes were observed under high light stress of different wavelength.ConclusionsGreen algae received a β-xanthophylls biosynthetic pathway from host organisms. Although red algae inherited the pathway from cyanobacteria during primary endosymbiosis, it remains unclear in Chromalveolates. The α-xanthophylls biosynthetic pathway is a common feature in green algae and higher plants. The origination of cyp97a/c is most likely due to gene duplication before divergence of green algae and higher plants. Protein domain structures and expression analyses in green alga H. pluvialis indicate that various chy genes are in different manners response to light. The knowledge of evolution of chy genes in photosynthetic eukaryotes provided information of gene cloning and functional investigation of chy genes in algae in the future.

Characterization and engineering of a dual-function diacylglycerol acyltransferase in the oleaginous marine diatom Phaeodactylum tricornutum

BackgroundPhotosynthetic oleaginous microalgae are promising feedstocks for biofuels. Acyl-CoA:diacylglycerol acyltransferases (DGATs) represent rich sources for engineering microalgal lipid production. The principal activity of DGATs has been defined as a single-function enzyme catalyzing the esterification of diacylglycerol with acyl-CoA.ResultsA dual-function PtWS/DGAT associated with diatom Phaeodactylum tricornutum is discovered in the current study. Distinctive to documented microalgal DGAT types, PtWS/DGAT exhibits activities of both a wax ester synthase (WS) and a DGAT. WS/DGATs are broadly distributed in microalgae, with different topology and phylogeny from those of DGAT1s, DGAT2s, and DGAT3s. In vitro and in vivo assays revealed that PtWS/DGAT, functioning as either a WS or a DGAT, exhibited a preference on saturated FA substrate. Endogenous overexpression of PtWS/DGAT demonstrated that the DGAT activity was dominant, whereas the WS activity was condition dependent and relatively minor. Compared with the wild type (WT), overexpression of PtWS/DGAT in the diatom resulted in increased levels of total lipids (TL) and triacylglycerol (TAG) regardless of nitrogen availability. The stability and scalability of the introduced traits were further investigated at a 10-L photobioreactor, where the mutant growth resembled WT, with moderately increased productivity of TL and TAG. Furthermore, the production of wax esters increased considerably (from undetectable levels to 2.83%) under nitrogen-deplete conditions.ConclusionsPtWS/DGAT is a bifunctional enzyme and may serve as a promising target for the engineering of microalga-based oils and waxes for future industrial use.

Transcriptomic analysis of Synechocystis sp PCC6803 under low-temperature stress

In this study, cDNA microarrays were developed from 3569 mRNA reads to analyze the expression profiles of the transcriptomes of Synechocystis sp. PCC6803 under low temperature (LT) stress. Among the genes on the cDNA microarrays, 899 LT-affected genes exhibited a 1.5-fold (or greater) difference in expression compared with the genes from normal unstressed Synechocystis sp. PCC6803. Of the differentially expressed genes, 353 were up-regulated and 246 were down-regulated. The results showed that genes involved in photosynthesis were activated at LT (10°C), including genes for photosystem I, photosystem II, photosynthetic electron transport, and cytochrome b6/f complex. Moreover, desB, one of four genes that encode the fatty acid desaturases, was also induced by LT. However, the LT conditions to some degree enhanced the transcription of some genes. In addition, LT (10°C) may reduce cellular motility by regulating the transcription of spkA (sll1575), a serine/threonine protein kinase. The results reported in this study may contribute to a better understanding of the responses of the Synechocystis cell to LT, including pathways involved in photosynthesis and repair.

Genetic transformation of Platymonas ( Tetraselmis ) subcordiformis (Prasinophyceae, Chlorophyta) using particle bombardment and glass-bead agitation

Platymonas (Tetraselmis) subcordiformis is a unicellular marine green alga. It was found to be very sensitive to the herbicide Basta through a sensitivity test indicating it could be employed as a selective agent. The bar gene is a practicable and selectable marker gene. The vector containing the expression cassette of the bar gene was transferred to P. subcordiformis by both particle bombardment and glass-bead agitation and transformants were then selected using Basta. Finally, Southern blotting analysis indicated that the bar gene had been successfully integrated into the nuclear genome of P. subcordiformis using both of the transgenic techniques, with the transformation efficiency of the glass-bead method being slightly higher than that of particle bombardment. This is the first report on stable transformation of P. subcordiformis, and will improve fundamental research and enlarge application of this alga.

Purification of 6×His-tagged phycobiliprotein using zinc-decorated silica-coated magnetic nanoparticles

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.

Transient expression of the enhanced green fluorescent protein (egfp) gene in Sargassum horneri

Sargassum horneri is a macroalga widespread in North Asia-Pacific region, and these years its bloom has caused huge damage to the environment and the economic in China. To make up the blank on genetic engineering research, a transient transformation system for the multicellular marine brown alga S. horneri was established in this research. The algae used in this research were collected from the Yellow Sea of China and verified as a same species S. horneri with analysis of molecular markers. The S. horneri parietal leaves were transformed with the enhanced green fluorescent gene as the reporter by micro-particle bombardment. The results show that GFP is an effective transgene reporter for S. horneri and that particle bombardment is a suitable method for transformation of S. horneri. Through selection of four different promoters for EGFP and six groups’ bombardment characters, the highest transformation efficiency approximately 1.31% was got with the vector pEGFP-N1 at bombardment characters 900 spi and 6 cm distance. This research paves a way for the further research and application of S. horneri.

The influences of phytohormones on triacylglycerol accumulation in an oleaginous marine diatom Phaeodactylum tricornutum

Environmental stresses such as nitrate deprivation and high light are effective at increasing lipid content in microalgae, but they can also slow down and even stop growth. In this study, the phytohormones methyl jasmonate, salicylic acid, gibberellin, abscisic acid, and ethephon were introduced to cultures of the oleaginous marine diatom Phaeodactylum tricornutum in an attempt to increase growth and lipid production. Single-factor experiments showed that the influences of some of the phytohormones were closely related to their concentrations. Methyl jasmonate, abscisic acid, and salicylic acid promoted P. tricornutum growth and lipid accumulation at certain concentrations. The differing effects of the three phytohormones on P. tricornutum may be related to the respective phytohormone’s responsive cis-regulatory elements in the upstream regions of the triacylglycerol (TAG) synthesis genes. Methyl jasmonate, abscisic acid, and salicylic acid were further studied in response surface experiments, through which a 141% increase in TAG production was attained for 10-L cultures of P. tricornutum grown under optimal conditions. This study suggests that some phytohormones can promote P. tricornutum lipid accumulation without hindering growth. It also provides another strategy for improving the production of microalgae for use as biodiesel.