Xuecheng Zhang

Assessment of marine thraustochytrid Schizochytrium limacinum OUC88 for mariculture by enriched feeds

In the present study, Schizochytrium limacinum OUC88, a thraustochytrid with high content of docosahexaenoic acid (DHA, 22∶6 n−3), was used as feed for rotifer Brachionus plicatilis and Artemia franciscana. The rotifer and Artemia were harvested at 3, 6, 9, 12, 18, and 24-h intervals, analyzed for fatty acid composition, and compared with the control which fed on yeast only. The highest DHA content resulted from an enrichment period of 12 h for both fed organisms, reached 13.4 and 10.9% of the total fatty acids (TFAs) in rotifers and Artemia nauplii, respectively, and the DHA level reduced sharply if enrichment time was longer than 12 h. The pseudoalbinism rate of turbot Scophthalmus maximus juveniles fed enriched rotifers and Artemia nauplii reduced greatly (40% lower than in control group). Thus, enrichment of rotifers and Artemia nauplii by DHA-rich Schizochytrium limacinum OUC88 may provide a practical strategy for feeding fish juveniles in aquaculture.

Production of High Docosahexaenoic Acid by Schizochytrium sp. Using Low-cost Raw Materials from Food Industry

The low-cost substrates from food industry, including maize starch hydrolysate and soybean meal hydrolysate, were used to produce docosahexaenoic acid (DHA) by Schizochytrium limacinum OUC88. Glucose derived from maize starch hydrolysate was used as the carbon source and soybean meal hydrolysate as the nitrogen sources. In 10L bioreactor fermentation, by using the soybean meal hydrolysate as the main nitrogen source, the biomass of Schizochytrium limacinum OUC88 reached 85.27 g L(-1), and the yields of DHA was 20.7g L(-1). As a comparison, when yeast extract was used as the main nitrogen source, the yields of biomass and DHA were 68.93 g L(-1) and 13.3 g L(-1), respectively. From the results of this study, these hydrolysates can provide all the nutrients required for high-density cultivation of S. limacinum OUC88 and DHA production, that will improve the economical and competitive efficiency of commercial DHA production.

Molecular cloning and expression analysis of glutathione peroxidase and glutathione reductase from Gracilaria lemaneiformis under heat stress

Glutathione peroxidase (GPx) and glutathione reductase (GR) are key enzymes in the antioxidant defense systems of living organisms and protect organisms from environmental stresses. To investigate the mechanism of resistance to heat stress in the red alga Gracilaria lemaneiformis, the cDNA sequences encoding glutathione peroxidase and glutathione reductase of two strains (wild type and heat-tolerant cultivar 981) of G. lemaneiformis were successfully cloned using reverse transcription PCR and rapid amplification cDNA ends techniques. cDNA encoding GPx has 930 nucleotides with an open reading frame (ORF) of 750 nucleotides, encoding 249 amino acid residues. cDNA encoding GR was 1,572 nucleotides with a 1,416-nucleotide ORF encoding 471 amino acids. No introns existed in genomic DNA of GPx and GR of G. lemaneiformis. Southern blotting indicated that there was only one copy of GPx and GR in both strains. Under heat stress, bioactivity and mRNA transcription levels of both GPx and GR were upregulated in cultivar 981 compared to those in the wild type. These results indicate that both GPx and GR may play important roles in the ability of G. lemaneiformis to resist high temperatures.

Gene Cloning, Expression and Activity Analysis of Manganese Superoxide Dismutase from Two Strains of Gracilaria lemaneiformis (Gracilariaceae, Rhodophyta) under Heat Stress

Manganese superoxide dismutase (Mn-SOD) plays a crucial role in antioxidant responses to environmental stress. To determine whether Mn-SOD affects heat resistance of Gracilaria lemaneiformis, we cloned Mn-SOD cDNA sequences of two strains of this red alga, wild type and cultivar 981. Both cDNA sequences contained an ORF of 675 bp encoding 224 amino acid residues. The cDNA sequences and the deduced amino acid sequences of the two strains shared relatively high identity (more than 99%). No intron existed in genomic DNA of Mn-SOD in G. lemaneiformis. Southern blotting indicated that there were multiple copies, possibly four, of Mn-SOD in both strains. Both in the wild type and cultivar 981, SOD mRNA transcription and SOD activity increased under high temperature stress, while cultivar 981 was more heat resistant based on its SOD activity. This research suggests that there may be a direct relationship between SOD activity and the heat resistance of G. lemaneiformis.

Selection of reference gene from Gracilaria lemaneiformis under temperature stress

Selection of a reliable reference gene for the normalization of RT-qPCR data is important to obtain accurate results in the study of gene expression. To elucidate the effects of temperature on the gene expression in Gracilaria lemaneiformis, this study aimed to find an appropriate reference gene from ten candidate genes, including actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1 (EF1), eukaryotic translation initiation factor (eIF), histone (H2B), 18S ribosomal rRNA (18S rRNA), internal transcribed spacer 2 (ITS2), cytochrome c oxidase (COX), cytochrome b-5 reductase (CR) and phycoerythrin (PEB) of G. lemaneiformis under temperature stress. Programs geNorm and NormFinder were used to analyze the data of RT-qPCR. The results showed that the best reference gene combination was eIF, ACT for 32°C different treatments, GAPDH, ITS2, CR, and 18SrRNA for 8xa0°C different treatments, GAPDH, EF1 and ACT for different temperature treatments, and GAPDH and ACT for different life generations. This study provides the useful reference genes for future gene expression studies of G. lemaneiformis under temperature stress.

Cloning and transcription analysis of six members of the calmodulin family in Gracilaria lemaneiformis under heat shock

Calmodulin (CaM) is essential in plants for signal transduction under environmental stress. To determine the function of CaMs in the thermal response of the red alga Gracilaria lemaneiformis, six cams were cloned from three strains, wild type and heat-tolerant cultivar 981 and 07-2. In wild type, w-cam1, w-cam2, and w-cam3 were cloned; in cultivar 981, 981-cam1 and 981-cam2 were obtained; and in cultivar 07-2, only 072-cam1 was identified. The cam1s in the three strains were identical. They contain 832 nucleotides, including four exons and three introns. The ORFs of w-cam2 and 981-cam2 were the same length, having 450 nucleotides without introns, and they shared 78xa0% similarity in nucleotides and 87xa0% in amino acids. In addition, w-cam3, a gene of 809 nucleotides, consisting of three exons and two introns, was cloned from the genomic DNA of wild type. The transcription levels of the cams under heat shock were tested by real-time quantitative PCR, except for w-cam2 and w-cam3, because these two cams could not be amplified effectively by RT-qPCR. Among all the cam1s, 981-cam1 expressed more highly under heat shock. Though w-cam1 and 072-cam1 were down-regulated under heat, the suppression level of 072-cam1 was slightly less than the wild type. For 981-cam2, the expression level was lower than that of 981-cam1 at 28xa0°C, while at 32xa0°C, the two cams of 981 had similar expression profiles. These results indicate that the cams may play an important role in G. lemaneiformis and that cam1 responds the most to heat shock.

Expression of a chimeric human/salmon calcitonin gene integrated into the Saccharomyces cerevisiae genome using rDNA sequences as recombination sites

Calcitonin participates in controlling homeostasis of calcium and phosphorus and plays an important role in bone metabolism. The aim of this study was to endow an industrial strain of Saccharomyces cerevisiae with the ability to express chimeric human/salmon calcitonin (hsCT) without the use of antibiotics. To do so, a homologous recombination plasmid pUC18-rDNA2-ura3-Ppgk-5hsCT-rDNA1 was constructed, which contains two segments of ribosomal DNA of 1.1xa0kb (rDNA1) and 1.4xa0kb (rDNA2), to integrate the heterologous gene into host rDNA. A DNA fragment containing five copies of a chimeric human/salmon calcitonin gene (5hsCT) under the control of the promoter for phosphoglycerate kinase (Ppgk) was constructed to express 5hsCT in S. cerevisiae using ura3 as a selectable auxotrophic marker gene. After digestion by restriction endonuclease HpaI, a linear fragment, rDNA2-ura3-Ppgk-5hsCT-rDNA1, was obtained and transformed into the △ura3 mutant of S. cerevisiae by the lithium acetate method. The ura3-Ppgk-5hsCT sequence was introduced into the genome at rDNA sites by homologous recombination, and the recombinant strain YS-5hsCT was obtained. Southern blot analysis revealed that the 5hsCT had been integrated successfully into the genome of S. cerevisiae. The results of Western blot and ELISA confirmed that the 5hsCT protein had been expressed in the recombinant strain YS-5hsCT. The expression level reached 2.04xa0% of total proteins. S. cerevisiae YS-5hsCT decreased serum calcium in mice by oral administration and even 0.01xa0g lyophilized S. cerevisiae YS-5hsCT/kg decreased serum calcium by 0.498xa0mM. This work has produced a commercial yeast strain potentially useful for the treatment of osteoporosis.

Stable cell-surface expression of Japanese flounder growth hormone in yeast Saccharomyces cerevisiae and growth-promoting effect on juvenile fish by oral administration

A cost-effective method for producing and using recombinant growth hormone (GH) has been developed. A cell-surface protein display system was constructed to produce recombinant Japanese flounder growth hormone (r-JfGH) in yeast Saccharomyces cerevisiae and the biological activity of expressed r-JfGH was evaluated by oral delivery to juvenile Japanese flounder. The expression vector pYD-E2, containing the JfGH gene, S. cerevisiae GAL1 promoter, and the S.cerevisiae URA3 gene downstream and upstream sequences, was designed and constructed to integrate the GH gene into the yeast genome and secrete r-JfGH stably on the surface of the yeast cell. Expression of r-JfGH was confirmed by flow cytometry until the 50th continuous generation of transgenic yeast strain y-E2, and over 0.98xa0μg r-JfGH per 1xa0mg total protein of y-E2 was estimated by an ELISA–RA assay. Growth of Japanese flounder was promoted significantly by the direct supplementation of freeze-dried transgenic yeast into the Japanese flounder juvenile feed at levels of 0.5 and 1%. The weight-specific growth rate and feed conversion ratio of the treated groups were 23.07 and 21.57% (0.5% feeding level) and 28.85 and 41.18% (1% feeding level), respectively, higher than that of the control group in a 7-week bioactivity study.

Analysis on the factors affecting start-up intensity in the upstream sequence of phycocyanin β subunit gene from Arthrospira platensis by site-directed mutagenesis

Six promoters in the 419xa0bp upstream sequence of the phycocyanin β subunit gene of Arthrospira platensis FACHB341 have been previously cloned. Site-directed mutagenesis has now been used to introduce mutations in the -10 and -35xa0boxes of promoter 3, -10 box of promoter 4, and -35xa0box of promoter 6. The expression level of green fluorescent protein gene was measured by flow cytometry. Results showed that the effects of site-directed mutagenesis in different promoters were dissimilar: some increased and some declined.

Molecular Cloning of cpcU and Heterodimeric Bilin Lyase Activity Analysis of CpcU and CpcS for Attachment of Phycocyanobilin to Cys-82 on the β-Subunit of Phycocyanin in Arthrospira platensis FACHB314

A new bilin lyase gene cpcU was cloned from Arthrospira platensis FACHB314 to study the assembly of the phycocyanin β-Subunit. Two recombinant plasmids, one contained the phycocyanobilin (PCB) producing genes (hoxI and pcyA), while the other contained the gene of the β-Subunit of phycobiliprotein (cpcB) and the lyase gene (cpcU, cpcS, or cpcU/S) were constructed and separately transferred into Escherichia coli in order to test the activities of relevant lyases for catalyzing PCB addition to CpcB during synthesizing fluorescent β-PC of A. platensis FACHB314. The fluorescence intensity examination showed that Cys-82 maybe the active site for the β-Subunit binding to PCBs and the attachment could be carried out by CpcU, CpcS, or co-expressed cpcU/S in A. platensis FACHB314.