Ying‐Fang Niu

To obtain fast growing oil-rich microalgal strains has been urgently demanded for microalgal biofuel. Malic enzyme (ME), which is involved in pyruvate metabolism and carbon fixation, was first characterized in microalgae here. Overexpression of Phaeodactylum tricornutum ME (PtME) significantly enhanced the expression of PtME and its enzymatic activity in transgenic P. tricornutum. The total lipid content in transgenic cells markedly increased by 2.5-fold and reached a record 57.8% of dry cell weight with a similar growth rate to wild type, thus keeping a high biomass. The neutral lipid content was further increased by 31% under nitrogen-deprivation treatment, still 66% higher than that of wild type. Transgenic microalgae cells exhibited obvious morphological changes, as the cells were shorter and thicker and contained larger oil bodies. Immuno-electron microscopy targeted PtME to the mitochondrion. This study markedly increased the oil content in microalgae, suggesting a new route for developing ideal microalgal strains for industrial biodiesel production.

Microalgae have been emerging as an important source for the production of bioactive compounds. Marine diatoms can store high amounts of lipid and grow quite quickly. However, the genetic and biochemical characteristics of fatty acid biosynthesis in diatoms remain unclear. Glycerophospholipids are integral as structural and functional components of cellular membranes, as well as precursors of various lipid mediators. In addition, diacylglycerol acyltransferase (DGAT) is a key enzyme that catalyzes the last step of triacylglyceride (TAG) biosynthesis. However, a comprehensive sequence-structure and functional analysis of DGAT in diatoms is lacking. In this study, an isoform of diacylglycerol acyltransferase type 2 of the marine diatom Phaeodactylum tricornutum was characterized. Surprisingly, DGAT2 overexpression in P. tricornutum stimulated more oil bodies, and the neutral lipid content increased by 35%. The fatty acid composition showed a significant increase in the proportion of polyunsaturated fatty acids; in particular, EPA was increased by 76.2%. Moreover, the growth rate of transgenic microalgae remained similar, thereby maintaining a high biomass. Our results suggest that increased DGAT2 expression could alter fatty acid profile in the diatom, and the results thus represent a valuable strategy for polyunsaturated fatty acid production by genetic manipulation.

The marine diatom, Phaeodactylum tricornutum, has become a model for studying lipid metabolism and its triacylglycerol (TAG) synthesis pathway makes it an ideal target for metabolic engineering to improve lipid productivity. However, the genetic background and metabolic networks of fatty acid biosynthesis in diatoms are not well understood. Glycerol-3-phosphate acyltransferase (GPAT) is the critical enzyme that catalyzes the first step of TAG formation. So far, characterization of GPAT in marine microalgae has not been reported, especially at the level of comprehensive sequence-structure and functional analysis.A GPAT was cloned from P. tricornutum and overexpressed in P. tricornutum. Volumes of oil bodies were produced and the neutral lipid content was increased by twofold determined by Nile red fluorescence staining. Fatty acid composition was analyzed by GC-MS, which showed significantly higher proportion of unsaturated fatty acids compared to wild type.These results suggested that the identified GPAT could upregulate TAG biosynthesis in P. tricornutum. Moreover, this study offers insight into the lipid metabolism of diatoms and supports the role of microalgal strains for biofuels production.

The purpose of this study was to investigate the changes in mitochondria in porcine MII-stage oocytes after open pulled straw (OPS) vitrification and to determine their roles in apoptosis and in vitro developmental ability. The mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS) level, adenosine-5'-triphosphate (ATP) concentration, mitochondrial distribution, mitochondrial ultrastructure, early-stage apoptosis with Annexin V-FITC staining, survival rate, parthenogenetic developmental ability and related gene expression were measured in the present experiments. The results showed that: (1) the mitochondrial ΔΨm of vitrified-thawed oocytes (1.05) was lower than that of fresh oocytes 1.24 (P<0.05). (2) ROS level in the OPS vitrification group was much higher than that of the fresh group, while the ATP concentration was much lower than that of fresh group (P<0.05). (3) Early-stage apoptosis rate from the OPS vitrification group (57.6%) was much higher than that of fresh group (8.53%) (P<0.05), and the survival rate and parthenogenetic cleavage rate of OPS vitrified oocytes were much lower than those from fresh ones (P<0.05). (4) Vitrification not only disrupted the mitochondrial distribution of porcine MII-stage oocytes, but also damaged the mitochondrial ultrastructure. (5) After vitrification, the gene expression level of Dnm1 was up-regulated, and other four genes (SOD1, Mfn2, BAX and Bcl2) were down-regulated. The present study suggested that not only the morphology and function of mitochondria were damaged greatly during the vitrification process, but also early-stage apoptosis was observed after vitrification. Intrinsic mitochondrial pathway could be in involved in the occurrence of apoptosis in vitrified-thawed porcine oocytes.

Microalgae are ideal raw materials for biodiesel and bioactive compounds. Glycerol-3-phosphate is formed from dihydroxyacetone phosphate (DHAP) through the glycolytic pathway catalyzed by glycerol-3-phosphate dehydrogenase (GPDH). GPDH was characterized in the marine diatom Phaeodactylum tricornutum. In the GPDH-overexpressing P. tricornutum cells, the glycerol concentration per cell in the transformed diatom increased by 6.8-fold compared with the wild type, indicating that the overexpression of GPDH promoted the conversion of DHAP to glycerol-3-phosphate. There was a 60% increase in neutral lipid content, reaching 39.7% of dry cell weight in transgenic cells in the stationary phase, despite a 20% decrease in cell concentration. Fatty acid profiling showed that the levels of 16- and 18-carbon monounsaturated fatty acids significantly increased. GPDH had a significant impact on numerous metabolic processes in diatom cells, including the biosynthesis of glycerol and neutral lipids. These findings are instructive for the metabolic engineering of microalgae for biofuel production.

Summary Phosphorus is an important macronutrient. To understand the molecular and cellular responses to phosphorus stress better, transcriptome profiling in combination with biochemical investigations was conducted in the model diatom P haeodactylum tricornutum . Out of 10 402 predicted genes, 2491 and 405 genes were significantly upregulated or downregulated respectively. Unsurprisingly, genes associated with phosphate uptake were upregulated, such as the phosphate transporters and alkaline phosphatases. Genes encoding stress‐shock proteins were accordingly upregulated, including genes associated with stress‐responsive proteins, signal transduction and secondary metabolism. Additionally, genes related to protein translation, carbon fixation, glycolysis and the citric acid cycle were also upregulated. Genes associated with gene transcription were downregulated, thereby resulting in the upregulation of translation to compensate for the limited supply of messenger RNA . The downregulation of genes related to β ‐oxidation could contribute to the accumulation of fatty acids. Accordingly, triacylglycerols, which are important for energy storage, were determined to increase by 1.65‐fold. Intracellular membranes, other than chloroplast membranes, tended to be dispersed; this finding was in accordance with the increased transcription of a total of 11 genes encoding putative phospholipases. Taken together, this work revealed the coordination of multiple metabolic pathways and certain key genes in the adaptation of P . tricornutum to phosphorus stress.

Diatoms are important primary producers in the marine ecosystem. Currently it is difficult to genetically transform diatoms due to the technical limitations of existing methods. The promoter/terminator of the nitrate reductase gene of the model diatom Phaeodactylum tricornutum was cloned and used to drive chloramphenicol acetyltransferase (CAT) reporter gene expression. The construct was transferred by electroporation into P. tricornutum grown in medium lacking silicon. CAT expression was induced in transformed diatoms in the presence of nitrate, enabling growth in selective medium, and was repressed when ammonium was the only nitrogen source. Expression of CAT transcript and protein were demonstrated by RT-PCR and Western blot analysis, respectively. Our study is the first to report a successful genetic transformation of diatom by electroporation in an economical and efficient manner and provides a tightly regulated inducible gene expression system for diatom.

Microalgae have been an emerging biofuel resource; however, the germplasm improvement has been slow due to the lack of molecular tools. Pyruvate dehydrogenase kinase (PDK) deactivates the pyruvate dehydrogenase complex (PDC) which catalyzes the oxidative decarboxylation of pyruvate. Acetyl-CoA production via PDC is important in plant tissues that are active in fatty acid synthesis.A 1261-bp cDNA of a putative PDK gene (PtPDK) was cloned from a diatom Phaeodactylum tricornutum, and PtPDK antisense knockdown transgenic diatoms were generated. Both PtPDK transcript abundance and enzyme activity were reduced significantly due to antisense knockdown of PtPDK. Neutral lipid content of transgenic diatom cells increased up to 82% as determined by Nile red staining, and fatty acid composition was not altered. Transgenic cells showed slightly lower growth rate but similar cell size with the wild type, hence retaining similar biomass productivity.This work first obtained a successful engineered diatom regulating a key gene involved in lipid metabolism. Our findings also provide powerful indications in enhancing microalgal lipid production by metabolic engineering for biofuel industry.

Genetic transformation is useful for basic research and applied biotechnology. However, genetic transformation of microalgae is usually quite difficult due to the technical limitations of existing methods. We cloned the promoter and terminator of the nitrate reductase gene from the microalga Phaeodactylum tricornutum and used them for optimization of a transformation system of the microalga Chlorella vulgaris. This species has been used for food production and is a promising candidate as a bioreactor for large-scale production of value-added proteins. A construct was made containing the CAT (chloramphenicol acetyltransferase) reporter gene driven by the nitrate reductase promoter. This construct was transferred into the C. vulgaris genome by electroporation. Expression of CAT in transgenic Chlorella conferred resistance to the antibiotic chloramphenicol and enabled growth in selective media. Overall efficiency for the transformation was estimated to be approximately 0.03%, which is relatively high compared with other available Chlorella transformation systems. Expression of CAT was induced in the presence of nitrate and inhibited in the presence of ammonium as a sole nitrogen source. This study presented an inducible recombinant gene expression system, also providing more gene regulation elements with potential for biotechnological applications.

Apoptosis is one of the main drivers of the decline in developmental potential of porcine oocytes after vitrification. However, which apoptotic pathways are engaged after vitrification remains poorly understood. To distinguish among the possible apoptotic pathways induced by vitrification of MII stage porcine oocytes, this study detected activity and expression levels of several key proteins and genes in both the death receptor and mitochondrial pathways using in situ fluorescence staining and real-time PCR (RT-PCR) following the addition of specific inhibitors of either the death receptor or the mitochondrial apoptotic pathway (Z-IETD-FMK or Z-LEHD-FMK, respectively) into the incubation solution. Survival and parthenogenetic developmental ability were also examined. The results showed the following: (i) compared with the vitrified group, the activities of pan-caspase, caspase 3, caspase 8 and caspase 9 as well as the early apoptotic rate were significantly lower in the Z-IETD-FMK and Z-LEHD-FMK groups (p < .05); (ii) After vitrification, mitochondrial ΔΨm decreased from 1.33 for fresh oocytes to 0.90 for vitrified oocytes, while the addition of Z-IETD-FMK or Z-LEHD-FMK following vitrification increased mitochondrial ΔΨm to 1.09 and 1.05, respectively (p < .05); (iii) Relative expression levels of apoptotic-related genes from both the death receptor pathway (caspase 8 and TNF-α) and the mitochondrial pathway (caspase 9, Bcl-2 and CuZnSOD) changed substantially after the addition of Z-IETD-FMK or Z-LEHD-FMK into the incubation solution; (iv) Survival, cleavage and blastocyst rates were higher in the Z-IETH-FMK or Z-LEHD-FMK groups than in the vitrified group (p < .05). In summary, both the death receptor and the mitochondrial apoptotic pathways participate in the apoptosis of MII stage porcine oocytes after vitrification.

Intramuscular fat (IMF) and subcutaneous fat (SCF) are important traits affecting the economics of the pork industry, in which less SCF and more IMF content is desirable. However, the mechanisms that regulate IMF and SCF content are not clear yet. In this study, we demonstrate that KLF3 (Krüppel-like factor 3) was negatively correlated with IMF content in the longissimus dorsi muscle of Erhualian pigs. In addition, the expression level of KLF3 was significantly higher in IMF than SCF. Overexpression and knockdown experiments revealed that KLF3 could suppress adipocyte differentiation in vitro by downregulating adipogenic markers, including PPARG, C/EBPA, and FABP4. Luciferase activity analysis proved that miR-32-5p was able to suppress KLF3. Notably, miR-32-5p level was negatively correlated to KLF3 mRNA level in both IMF and SCF tissues. The same relationship was proved in samples with different IMF content. Further studies showed that miR-32-5p could promote adipocyte differentiation via inhibiting KLF3. Our results suggest that the miR-32-5p-KLF3 pathway is involved in the regulation of differential fat deposition of IMF and SCF tissues.

Intramuscular fat (IMF) is considered as the fat deposited between muscle fibers. The extracellular matrix microenvironment of adipose tissue is of critical importance for the differentiation, remodeling and function of adipocytes. Therefore, in this study we extracted the muscle tissue centrifugal fluid (MTF) of the longissimus dorsi of Erhualian pigs to mimic the microenvironment of intramuscular pre-adipocytes. MTF of pigs with low intramuscular fat level can inhibit pig intramuscular pre-adipocytes differentiation. Then, proteomics technology (iTRAQ) was used to analyze the MTF with different IMF content, and it was found that individuals with high IMF had low ACAT2 (Acyl-CoA: cholesterol acyltransferases 2) levels, while individuals with low IMF had high ACAT2 levels. Significant changes took place in the pathways involved in coenzyme A, which are closely related to fat and cholesterol metabolism. Therefore, we speculate that ACAT2, as an important element involved in cholesterol metabolism, may become a potential molecular marker for the mechanism of pig intramuscular preadipocytes differentiation. Overexpression of ACAT2 in pig intramuscular pre-adipocytes can inhibit their differentiation, while adding ACAT2 inhibitor avasimibe can rescue the process. Knockdown of srebp2 or ldlr, which are two key genes closely related to ACAT2 and cholesterol metabolism, can inhibit pig intramuscular pre-adipocytes differentiation. Overall, our results suggest that ACAT2 is a novel negative regulator of intramuscular adipocyte differentiation through regulation of pparγ, cebpα signaling and srebp2/ldlr signaling involved in cholesterol metabolism.

Abstract The marine diatom Phaeodactylum tricornutum , a widely used forage species, has a storage lipid content of up to 30% dry cell weight. To explore the mechanism behind the high storage lipid accumulation in this diatom, acetyl‐CoA carboxylase (ACCase), which catalyzes the first committed step of the fatty acid biosynthetic pathway, was characterized in this study. A homogeneous type of ACCase ( PtACC ) was identified from P. tricornutum by homology searches. The first exon of the ACCase gene ( PtACC‐1 ) was cloned. PtACC‐1 was fused with a Myc epitope tag and cloned into plasmid pMD18 driven by the LacZ promoter and expressed in Escherichia coli . The expression of the PtACC‐1‐Myc protein was verified by Western blot. The neutral lipid content in transformed E. coli increased substantially by twofold as determined by Nile red fluorescent dye staining. Concomitantly, ACCase activity increased by 1.72‐fold. The fatty acid composition, analyzed by GC‐MS, demonstrated a significant difference in the ratio of saturated fatty acids and monounsaturated fatty acids (MUFAs). MUFAs of PtACC‐1 expressing cells increased by 13%. This study represents the first characterization of the key domains of ACCase from a diatom and demonstrates high neutral lipid accumulation in E. coli expressing PtACC‐1, providing an additional genetic resource with the potential for biodiesel development.

The developmental potential of vitrified porcine oocytes is very lower, and apoptosis is considered as one of the key factors involved.To investigate the effects of apoptotic inhibitor Z-VAD-FMK addition into the incubation medium after warming on apoptosis and developmental ability of vitrified porcine MII-stage oocytes.The activities of several caspases, mitochondrial membrane potential (ΔΨm) and early apoptotic levels were measured. Parthenogenetic developmental ability and relative expression levels of apoptosis related genes were also detected.Caspase activity and early apoptotic level of the Z-VAD-FMK group were significantly lower than those of the group without Z-VAD-FMK addition, but were much higher than those of fresh group (P < 0.05). The ΔΨm of Z-VAD-FMK group was 1.19, higher than the vitrified group (0.91) and lower than the fresh group (1.33). The cleavage rate and blastocyst rate after parthenogenetic activation in the Z-VAD-FMK group were much higher than those in the vitrified group, and much lower than those in the fresh group (P < 0.05). Vitrified porcine oocytes exhibited increased expression of pro-apoptotic genes (caspase 3, 8, 9, TNF-α) and decreased genes expression levels of anti-apoptotic genes (Bcl-2, CuZnSOD), and the Z-VAD-FMK addition in incubaiton medium significantly decreased the transcripts levels of caspase 3,8,9, Bax, TNF-α and increased Bcl-2 and CuZnSOD genes expression.The addition of apoptotic inhibitor Z-VAD-FMK into the incubation medium after warming improved the in vitro developmental ability of vitrified porcine oocytes by increasing mitochondrial function, reducing apoptotic level and changing apoptosis-elated gene expression.

The survival of porcine oocytes is still very low after cryopreservation.To investigate whether and when the mitochondrial function of vitrified porcine oocytes could be recovered post-thaw.Mitochondrial potential, ROS level, ATP content, apoptotic rate, caspase activity, and parthenogenetics developmental ability of thawed porcine oocytes were measured after culture in vitro for 0, 1, 2 or 4 h.Mitochondrial potential after 2 h and 4 h post-thaw culture were 1.19 and 1.26, significantly lower than that of fresh oocytes but much higher than the groups cultured for 0 h and 1 h (P<0.05). Cryopreservation increased the ROS level in oocytes considerably, which decreased only after 2 to 4 h incubation following thaw. ATP content increased gradually over time and recovered to the level comparable to that of fresh oocytes after 4 h. Pan caspase levels increased after cryopreservation and reached the highest level at 1 h incubation. Thereafter it decreased to a low value, but still higher than fresh oocytes. Oocytes showing an early apoptotic event decreased upon 2 to 4 h incubation. The parthenogenetic cleavage and blastocyst rates were the highest (19.8% and 5.6%) after 2 h incubation.The recovery of mitochondrial function could complete after 2 to 4 h post-thaw incubation. Post-thaw incubation for 2 to 4 h reduced apoptotic events and improved parthenogenetic developmental ability of vitrified porcine MII stage oocytes.

Microalgae have been an emerging biofuel resource; however, the germplasm improvement has been slow due to the lack of molecular tools. Pyruvate dehydrogenase kinase (PDK) deactivates the pyruvate dehydrogenase complex (PDC) which catalyzes the oxidative decarboxylation of pyruvate. Acetyl-CoA production via PDC is important in plant tissues that are active in fatty acid synthesis. A 1261-bp cDNA of a putative PDK gene (PtPDK) was cloned from a diatom Phaeodactylum tricornutum, and PtPDK antisense knockdown transgenic diatoms were generated. Both PtPDK transcript abundance and enzyme activity were reduced significantly due to antisense knockdown of PtPDK. Neutral lipid content of transgenic diatom cells increased up to 82% as determined by Nile red staining, and fatty acid composition was not altered. Transgenic cells showed slightly lower growth rate but similar cell size with the wild type, hence retaining similar biomass productivity. This work first obtained a successful engineered diatom regulating a key gene involved in lipid metabolism. Our findings also provide powerful indications in enhancing microalgal lipid production by metabolic engineering for biofuel industry.

Seeds rich in protein in nature, are ideal bioreactors for economic production and storage of valueadded recombinant proteins and enzymes for industries. The upstream region of the seed storage protein gene is able to provide an attractive promoter for seed-specific expression of heterologous genes. Our previous research showed that 8S globulin occupied the majority of total soluble protein stored in seeds of mung bean ( Vigna radiata ), a rich source of protein, indicating that the promoter of this gene could be a seed-specific promoter with high activity. To improve the expression of heterologous proteins in plants to act as a bioreactor, the putative seed-specific promoter of 8S globulin gene was characterized in this study. Hence, this potential promoter of beta subunit gene of 8S globulin (8SGb) was isolated by genome walking. Analysis with various promoter prediction softwares showed that the promoter sequence possessed many common motifs related to gene transcription in the seed (such as W-box, ABRE element, E-box, etc.). The putative promoter was subsequently cloned into the binary vector pBI121-GFP by replacing the CaMV 35S promoter. The resultant construct designated as pBI-8SGb-GFP was transformed to mung bean cotyledon mesophyll protoplasts. Reporter gene GFP was expressed high in cotyledon protoplasts, which was detected by confocal microscopy, demonstrating the specific activity of 8SGb promoter in driving gene expression. This study also proved that the 8SGb promoter is an efficient regulatory element for plant seeds to act as a bioreactor. Key words : Seed-specific, promoter, genome walking, Vigna radiata.

To develop a method for determination of bisphenol A in human blood by fast phospholipid removal solid phase extraction method-ultra performance liquid chromatography tandem mass spectrometry( UPLC-MS/MS).Enzymatic hydrolysis( β-glucuronidase/arylsulfatase) and phospholipid removal solid phase extraction were used to treat the blood samples under the acidic condition, a Infinity Lab Poroshell 120 PFP column( 100 mm × 3. 0 mm, 2. 7 μm) was used for LC separation, ESI negative ion scan was used with multiple reaction monitoring( MRM) mode.The calibration curve was linear in the range of 0. 1-100 ng/mL for bisphenol A with correlation coefficients more than 0. 999. The limit of detection was 0. 05 ng/mL, the limit of quantity was 0. 15 ng/mL. The recoveries of the method for bisphenol A at three spiked levels of 0. 5, 5 and 50 ng/mL ranged from 87. 3%to 112. 1%. The relative standard deviation( RSD) of intra and inter day were range from3. 3%-8. 2%, 4. 9%-10. 7%( n = 6), respectively.The method is successfully applied in the analysis of bisphenol A in human blood with its simple operation, sensitivity and accuracy.