Gene cloning and expression profile of a novel carotenoid hydroxylase (CYP97C) from the green alga Haematococcus pluvialis

A full-length complementary DNA (cDNA) sequence of epsilon-ring CHY (designated Haecyp97c) was cloned from the green alga Haematococcus pluvialis by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. The Haecyp97c cDNA sequence was 1,995 base pairs (bp) in length, which contained a 1,620-bp open reading frame, a 46-bp 5'-untranslated region (UTR), and a 329-bp 3'-UTR with the characteristic of the poly (A) tail. The deduced protein had a calculated molecular mass of 58.71 kDa with an estimated isoelectric point of 7.94. Multiple alignment analysis revealed that the deduced amino acid sequence of HaeCYP97C shared high identity of 72-85 % with corresponding CYP97Cs from other eukaryotes. The catalytic motifs of cytochrome P450s were detected in the amino acid sequence of HaeCYP97C. The transcriptional levels of Haecyp97c and xanthophylls accumulation under high light (HL) stress have been examined. The results revealed that Haecyp97c transcript was strongly increased after 13-28 h under HL stress. Meanwhile, the concentrations of chlorophylls, carotenes, and lutein were decreased, and zeaxanthin and astaxanthin concentrations were increased rapidly, respectively. These facts indicated that HaeCYP97C was perhaps involved in xanthophyll biosynthesis, which plays an important role in adaption to HL for H. pluvialis.A full-length complementary DNA (cDNA) sequence of epsilon-ring CHY (designated Haecyp97c) was cloned from the green alga Haematococcus pluvialis by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. The Haecyp97c cDNA sequence was 1,995 base pairs (bp) in length, which contained a 1,620-bp open reading frame, a 46-bp 5'-untranslated region (UTR), and a 329-bp 3'-UTR with the characteristic of the poly (A) tail. The deduced protein had a calculated molecular mass of 58.71 kDa with an estimated isoelectric point of 7.94. Multiple alignment analysis revealed that the deduced amino acid sequence of HaeCYP97C shared high identity of 72-85 % with corresponding CYP97Cs from other eukaryotes. The catalytic motifs of cytochrome P450s were detected in the amino acid sequence of HaeCYP97C. The transcriptional levels of Haecyp97c and xanthophylls accumulation under high light (HL) stress have been examined. The results revealed that Haecyp97c transcript was strongly increased after 13-28 h under HL stress. Meanwhile, the concentrations of chlorophylls, carotenes, and lutein were decreased, and zeaxanthin and astaxanthin concentrations were increased rapidly, respectively. These facts indicated that HaeCYP97C was perhaps involved in xanthophyll biosynthesis, which plays an important role in adaption to HL for H. pluvialis

Characteristics of Sediment Bacterial Community in Response to Environmental Impacts in a Sewage Polluted River

The Jiaolai River is the main source of industrial and irrigation water for its catchment of 3900 km(2). Anthropogenic activities have caused heavy pollution of this river, but their impacts on biota have never been evaluated. In this study, molecular techniques were applied to investigate the impacts of environmental pollution on the river. Quantitative PCR revealed that total bacterial abundance ranged from 2.90x10(7) to 2.12x10(8) copies/g, with no significant differences among sampling sites or seasons. Bacterial abundance and pore water ammonium concentration were negatively correlated. Cluster analysis revealed that bacterial communities were mainly distributed into groups corresponding to nitrate concentration. Two clone libraries were constructed to compare the bacterial composition of samples with high (J308) and moderate (J304) nitrate impact. Sample J308 was characterized by more members in Clostridia and disappearance of Betaproteobacteria members, which are the primary contributors to nitrogen biogeochemical cycling. Bacterial communities in the sediment were clearly differentiated by environmental nitrogen pollution, suggesting that nitrogen eutrophication was the main environmental problem influencing the Jiaolai River

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-trnl (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

Enhanced green fluorescent protein (egfp) gene expression in Tetraselmis subcordiformis chloroplast with endogenous regulators

On the basis of fundamental genetic transformation technologies, the goal of this study was to optimize Tetraselmis subcordiformis chloroplast transformation through the use of endogenous regulators. The genes rrn16S, rbcL, psbA, and psbC are commonly highly expressed in chloroplasts, and the regulators of these genes are often used in chloroplast transformation. For lack of a known chloroplast genome sequence, the genome-walking method was used here to obtain full sequences of T. subcordiformis endogenous regulators. The resulting regulators, including three promoters, two terminators, and a ribosome combination sequence, were inserted into the previously constructed plasmid pPSC-R, with the egfp gene included as a reporter gene, and five chloroplast expression vectors prepared. These vectors were successfully transformed into T. subcordiformis by particle bombardment and the efficiency of each vector tested by assessing EGFP fluorescence via microscopy. The results showed that these vectors exhibited higher efficiency than the former vector pPSC-G carrying exogenous regulators, and the vector pRFA with Prrn, psbA-5'RE, and TpsbA showed the highest efficiency. This research provides a set of effective endogenous regulators for T. subcordiformis and will facilitate future fundamental studies of this alga

Protective effects of C-phycocyanin on alcohol-induced subacute liver injury in mice

Excessive and long-term alcohol consumption leads to liver disease and low immunity. Extensive evidence suggests that C-phycocyanin (C-PC), a chromophore phycocyanobilin derived from Arthrospira (Spirulina) platensis, exerts protective effects against chemical-induced organ damage and improves immunity. In this study, we investigated whether C-PC could protect against ethanol-induced subacute liver injury and improve immunity. KM mice with ethanol-induced liver injury were established, and animals were divided into three groups that were treated with high, medium, and low doses of C-PC. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (CHOL), low-density lipoprotein (LDL), total bilirubin (TBIL), liver homogenate malondialdehyde (MDA), and superoxide dismutase (SOD) levels were measured. In addition, the number of thymus T cell subsets was assessed, and liver sections were examined pathologically. C-PC exhibited obvious inhibitory effects on serum ALT, AST, TG, CHOL, LDL, and MDA levels and increased SOD content significantly in the liver. C-PC also increased serum CD3+ and CD4+ cell activation and T cell proliferation significantly compared with the model group. The structure of the hepatic lobules was clear, the liver sinus returned to normal, and the liver cell cords were arranged in neat rows. Therefore, C-PC could protect against ethanol-induced subacute liver injury significantly

Spatial Diversity of Bacterioplankton Communities in Surface Water of Northern South China Sea

The South China Sea is one of the largest marginal seas, with relatively frequent passage of eddies and featuring distinct spatial variation in the western tropical Pacific Ocean. Here, we report a phylogenetic study of bacterial community structures in surface seawater of the northern South China Sea (nSCS). Samples collected from 31 sites across large environmental gradients were used to construct clone libraries and yielded 2,443 sequences grouped into 170 OTUs. Phylogenetic analysis revealed 23 bacterial classes with major components alpha-, beta- and gamma-Proteobacteria, as well as Cyanobacteria. At class and genus taxon levels, community structure of coastal waters was distinctively different from that of deep-sea waters and displayed a higher diversity index. Redundancy analyses revealed that bacterial community structures displayed a significant correlation with the water depth of individual sampling sites. Members of alpha-Proteobacteria were the principal component contributing to the differences of the clone libraries. Furthermore, the bacterial communities exhibited heterogeneity within zones of upwelling and anticyclonic eddies. Our results suggested that surface bacterial communities in nSCS had two-level patterns of spatial distribution structured by ecological types (coastal VS. oceanic zones) and mesoscale physical processes, and also provided evidence for bacterial phylogenetic phyla shaped by ecological preferences.The South China Sea is one of the largest marginal seas, with relatively frequent passage of eddies and featuring distinct spatial variation in the western tropical Pacific Ocean. Here, we report a phylogenetic study of bacterial community structures in surface seawater of the northern South China Sea (nSCS). Samples collected from 31 sites across large environmental gradients were used to construct clone libraries and yielded 2,443 sequences grouped into 170 OTUs. Phylogenetic analysis revealed 23 bacterial classes with major components alpha-, beta- and gamma-Proteobacteria, as well as Cyanobacteria. At class and genus taxon levels, community structure of coastal waters was distinctively different from that of deep-sea waters and displayed a higher diversity index. Redundancy analyses revealed that bacterial community structures displayed a significant correlation with the water depth of individual sampling sites. Members of alpha-Proteobacteria were the principal component contributing to the differences of the clone libraries. Furthermore, the bacterial communities exhibited heterogeneity within zones of upwelling and anticyclonic eddies. Our results suggested that surface bacterial communities in nSCS had two-level patterns of spatial distribution structured by ecological types (coastal VS. oceanic zones) and mesoscale physical processes, and also provided evidence for bacterial phylogenetic phyla shaped by ecological preferences

Protective effects of C-phycocyanin on alcohol-induced acute liver injury in mice

Excessive alcohol consumption leads to liver disease. Extensive evidence suggests that C-phycocyanin (C-PC), a chromophore phycocyanobilin derived from Spirulina platensis, exerts protective effects against chemical-induced organ damage. In this study, we investigated whether C-PC could protect against ethanol-induced acute liver injury. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (CHOL), low-density lipoprotein (LDL), liver homogenate malondialdehyde (MDA), superoxide dismutase (SOD) content were measured, and pathological examination of liver sections were examined. C-PC showed obvious inhibitory effects on serum ALT, AST, TG, CHOL, LDL and MDA, and SOD content significantly increased in the liver. The structure of hepatic lobules was clear, liver sinus returned to normal, and liver cell cords were arranged in neat rows. Cloudiness, swelling, inflammatory cell infiltration and spotty necrosis of liver cells were significantly reduced. Therefore, C-PC can significantly protect against ethanol-induced acute liver injury