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1.
Methods Mol Biol ; 2050: 175-179, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31468492

RESUMO

Electroporation refers to the application of high strength electric pulse to create transient pores in the membrane, thereby enabling the passage of hydrophilic molecules into the cells. Based on the properties of cell and cell wall, the electroporation parameters vary among the algal species. Here, we demonstrated the optimized protocol for successful introduction of recombinant DNA (~5000 bp) into Nannochloropsis oceanica. The linearized recombinant plasmid that harbors eGFP and Bh-sle as the reporter and marker gene, respectively, was electroporated into the electrocompetent N. oceanica cells at voltage of 2200 V, 50 µF, resistance at 600 Ω using electroporator, and the transformed cells were then screened by molecular analysis. The report exemplifies a straightforward and reliable electroporation strategy for generating transgenic N. oceanica cells.

2.
Bioresour Technol ; : 122351, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31708386

RESUMO

This study investigates the prospective of utilizing kitchen wastewater and food wastes, biofuels industry byproducts as alternative water and carbon sources. Kitchen wastewater did not impede cellular growth rate of the evolved Phaeodactylum strain E70, which indicates its potential as an alternative to freshwater resources. Among the organic wastes assessed, food waste hydrolysate significantly increased cell growth. Supplement of crude glycerol in cultivation medium enhances the total fatty acid content. Mixed food waste hydrolysate and crude glycerol remarkably increased both the cell density and total fatty acid content. Also, the supplement of butylated hydroxytoluene alleviated the oxidative stress induced by impurities in organic wastes and concomitantly increased microalgal total fatty acids and polyunsaturated fatty acids content. The experimental results reported in this study show that a waste-based biorefinery could lead to utilization of organic waste resources for the efficient production of value-added products.

3.
Biotechnol J ; : e1900135, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31464064

RESUMO

Microalgae have long been considered as potential biological feedstock for the production of wide array of bioproducts, such as biofuel feedstock because of their lipid accumulating capability. However, lipid productivity of microalgae is still far below commercial viability. Here, a glucose-6-phosphate dehydrogenase from the oleaginous microalga Nannochloropsis oceanica is identified and heterologously expressed in the green microalga Chlorella pyrenoidosa to characterize its function in the pentose phosphate pathway. It is found that the G6PD enzyme activity toward NADPH production is increased by 2.19-fold in engineered microalgal strains. Lipidomic analysis reveals up to 3.09-fold increase of neutral lipid content in the engineered strains, and lipid yield is gradually increased throughout the cultivation phase and saturated at the stationary phase. Moreover, cellular physiological characteristics including photosynthesis and growth rate are not impaired. Collectively, these results reveal the pivotal role of glucose-6-phosphate dehydrogenase from N. oceanica in NADPH supply, demonstrating that provision of reducing power is crucial for microalgal lipogenesis and can be a potential target for metabolic engineering.

4.
Bioresour Technol ; 289: 121720, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31271916

RESUMO

Haematococcus pluvialis is a main biological resource for the antioxidant astaxanthin production, however, potential modulators and molecular mechanisms underpinning astaxanthin accumulation remain largely obscured. We discovered that provision of ethanol (0.4%) significantly triggered the cellular astaxanthin content up to 3.85% on the 4th day of treatment. Amongst, 95% of the accumulated astaxanthin was esterified, particularly enriched with monoesters. Ultrastructural analysis revealed that ethanol altered cell wall structure and physiological properties. Antioxidant analyses revealed that astaxanthin accumulation offset the ethanol induced oxidative stress. Ethanol treatment reduced carbohydrates while increased lipids and jasmonic acid production. Transcriptomic analysis uncovered that ethanol orchestrated the expression of crucial genes involved in carotenogenesis, e.g. PSY, BKT and CRTR-b were significantly upregulated. Moreover, methyl jasmonic acid synthesis was induced and played a major role in regulating the carotenogenic genes. The findings uncovered the novel viewpoint in the intricate transcriptional regulatory mechanisms of astaxanthin biosynthesis.


Assuntos
Clorofíceas/metabolismo , Ciclopentanos/metabolismo , Etanol/farmacologia , Oxilipinas/metabolismo , Clorofíceas/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Xantofilas/biossíntese
5.
Sci Total Environ ; 690: 911-922, 2019 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-31302555

RESUMO

In the saline-affected ecosystem, fungi have huge potential to promote growth, induce disease resistance and enhance tolerance against salt-stress of host plants. Since areas of plowland are gradually decreasing, the reclamation of coastal saline lands could play a crucial role in maintaining agricultural productivity and crop security globally. Therefore, it is of great significance to explore the fungal diversity in the coastal saline ecosystem. Here, we collected saline soil samples from unflooded areas and tidal flat areas, the two typical distinct landforms in coastal saline ecosystems, and used ITS metabarcoding to depict the diversity of fungal communities. We found that fungal species evenness had a remarkably higher variation from the tidal flat compared to unflooded soil samples. Furthermore, we also confirmed that the fungal niches differentiation reports in the coastal saline ecosystem. Our ITS based DNA sequencing revealed that both unflooded and tidal flat soil were mainly composed of amplicon sequence variants (ASVs) belonging to Ascomycota (93.43% and 86.91% respectively). Based on our findings, understanding the associations and distinctions of fungal microbiome between unflooded soil and tidal flat could provide the basis for the development of reclamation in coastal saline lands.


Assuntos
Código de Barras de DNA Taxonômico , Ecossistema , Fungos/genética , Microbiologia do Solo , Agricultura , Ascomicetos , Biodiversidade , DNA Fúngico , Fungos/classificação , Micobioma , Nitrogênio , Plantas , Solo
6.
Biotechnol Bioeng ; 116(11): 3006-3015, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31282986

RESUMO

There has been growing interest in using microalgae as production hosts for a wide range of value-added compounds. However, microalgal genetic improvement is impeded by lack of genetic tools to concurrently control multiple genes. Here, we identified two novel strong promoters, designated Pt202 and Pt667, and delineated their potential role on simultaneously driving the expression of key lipogenic genes in Phaeodactylum tricornutum. In silico analyses of the identified promoter sequences predicted the presence of essential core cis elements such as TATA and CAAT boxes. Regulatory role of the promoters was preliminarily assessed by using GUS reporter which demonstrated strong GUS expression. Thereafter, two key lipogenic genes including malic enzyme (PtME) and 5-desaturase (PtD5b), were overexpressed by the two promoters Pt202 and Pt667, respectively, in P. tricornutum. Combinatorial gene overexpression did not impair general physiological performance, meanwhile neutral lipid content was remarkably increased by 2.4-fold. GC-MS analysis of fatty acid methyl esters revealed that eicosapentaenoic acid (EPA; C20:5) was increased significantly. The findings augment a crucial kit to microalgal genetic tools that could facilitate the multiple-gene expression driven by various promoters, and promote microalgae for industrial bioproduction.

7.
Fish Shellfish Immunol ; 90: 173-179, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31059815

RESUMO

It is well documented that diarrhetic shellfish poisoning (DSP) toxins have strong genetic toxicity, cytotoxicity and oxidative damage to bivalve species. However, these toxic effects seem to decrease with the extension of exposure time and the increment of the toxin concentration, the mechanism involved remained unclear, though. In this paper, we found that expression of the genes related to cytoskeleton and Nrf2 signaling pathway displayed different changes over time in the gill of Perna viridis after exposure to DSP toxins-producing microalga Prorocentrum lima. During the short-term exposure (3 h and 6 h), KEAP1 gene expression was significantly up-regulated, coupled with up-regulation of MRP, ABCB1 and CAT transcriptions and down-regulation of GPx1 and NQO1 mRNA. After longer exposure to high density of P. lima, Nrf2 was significantly up-regulated, accompanied with up-regulation of Nrf2 pathway related genes such as NQO1, SOD, GST-ω and ABCB1, whereas KEAP1 was down-regulated. TUBA1C and TUBB1 transcripts were significantly down-regulated after short-term exposure of P. lima, but both of them were up-regulated at 96 h after exposure to high density of P. lima. Paraffin section demonstrated that P. lima had a strong damage on the gill of mussels during the short-term exposure. However, the negative effect to the gill decreased, and the gill restored after longer exposure (96 h). Taking together, we proposed that P. lima had a negative impact on cytoskeleton of mussel gill tissue, could cause oxidative damage to the gills. However, longer exposure of P. lima in high density could activate Nrf2 signaling pathway, thereby reducing the influence of toxin on mussel. Our study might provide a novel clue for the resistance mechanism of shellfish to DSP toxins.


Assuntos
Antioxidantes/metabolismo , Dinoflagelados/fisiologia , Toxinas Marinhas/efeitos adversos , Fator 2 Relacionado a NF-E2/genética , Perna (Organismo)/genética , Animais , Elementos de Resposta Antioxidante/imunologia , Fator 2 Relacionado a NF-E2/metabolismo , Perna (Organismo)/efeitos dos fármacos , Perna (Organismo)/enzimologia , Perna (Organismo)/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologia , Regulação para Cima
8.
Ecotoxicol Environ Saf ; 180: 192-201, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31085430

RESUMO

As a main marine phycotoxin, okadaic acid (OA) is mainly responsible for diarrheic shellfish poisoning (DSP), through specifically inhibiting phosphatase (PP1 and PP2A). It has been shown that isotope labelled-OA could cross the placental barrier in mice. However, it remains obscure how OA exposure could affect the formation of neural crest cells (NCCs), especially cranial NCCs in early embryo development. Here, we explored the effects of OA exposure on the generation of neural crest cells during embryonic development using the classic chick embryo model. We found that OA exposure at 100 nM (80.5 µg/L) could cause craniofacial bone defects in the developing chick embryo and delay the development of early chick embryos. Immunofluorescent staining of HNK-1, Pax7, and Ap-2α demonstrated that cranial NCC generation was inhibited by OA exposure. Double immunofluorescent staining with Ap-2α/PHIS3 or Pax7/c-Caspase3 manifested that both NCC proliferation and apoptosis were restrained by OA exposure. Furthermore, the expression of Msx1 and BMP4 were down-regulated in the developing chick embryonic neural tubes, which could contribute the inhibitive production of NCCs. We also discovered that expression of EMT-related adhesion molecules, such as Cadherin 6B (Cad6B) and E-cadherin, was altered following OA exposure. In sum, OA exposure negatively affected the development of embryonic neural crest cells, which in turn might result in cranial bone malformation.


Assuntos
Inibidores Enzimáticos/toxicidade , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Crista Neural/efeitos dos fármacos , Ácido Okadáico/toxicidade , Animais , Apoptose/efeitos dos fármacos , Caderinas/metabolismo , Embrião de Galinha , Regulação para Baixo , Desenvolvimento Embrionário/efeitos dos fármacos , Crista Neural/citologia , Crista Neural/embriologia , Tubo Neural/efeitos dos fármacos , Tubo Neural/metabolismo , Crânio/anormalidades
9.
Mar Drugs ; 17(5)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052268

RESUMO

Prorocentrum lima is a typical benthic toxic dinoflagellate, which can produce phycotoxins such as okadaic acid (OA). In this study, we identified three ABC transporter genes (ABCB1, ABCC1 and ABCG2) and characterized their expression patterns, as well as OA production under different environmental conditions in P. lima. We found that the three ABC transporters all showed high identity with related ABC proteins from other species, and contained classical features of ABC transport proteins. Among them, ABCG2 was a half size transporter. The three ABC transporter genes displayed various expression profiles under different conditions. The high concentration of Cu2+ could up-regulate ABCB1, ABCC1 and ABCG2 transcripts in P. lima, suggesting the potential defensive role of ABC transporters against metal ions in surrounding waters. Cu2+, in some concentration, could induce OA production; meanwhile, tributyltin inhibited OA accumulation. The grazer Artemia salina could induce OA production, and P. lima displayed some toxicity to the grazer, indicating the possibility of OA as an anti-grazing chemical. Collectively, our results revealed intriguing data about OA production and the expression patterns of three ABC transporter genes. However, we could not find any significant correlation between OA production and expression pattern of the three ABC transporters in P. lima. Our results might provide new molecular insights on the defensive responses of P. lima to the surrounding environment.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Dinoflagelados/metabolismo , Dinoflagelados/crescimento & desenvolvimento , Meio Ambiente , Toxinas Marinhas/química , Ácido Okadáico/metabolismo , RNA Mensageiro
10.
Ecotoxicol Environ Saf ; 176: 178-185, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-30927639

RESUMO

Diarrhetic shellfish poisoning (DSP) toxins are key shellfish toxins that cause diarrhea, vomiting and even tumor. Interestingly, bivalves such as Perna viridis have been reported to exhibit some resistances to alleviate toxic effects of DSP toxins in a species-specific manner. Nevertheless, the molecular mechanisms underlying the resistance phenomenon to DSP toxins, particularly the mechanistic role of CYP450 is scant despite its crucial role in detoxification. Here, we exposed P. viridis to Prorocentrum lima and examined the expression pattern of the CYP450 and our comprehensive analyses revealed that P. lima exposure resulted in unique expression pattern of key CYP450 genes in bivalves. Exposure to P. lima (2 × 105 cells/L) dramatically orchestrated the relative expression of CYP450 genes. CYP2D14-like mRNA was significantly down-regulated at 6 h in gill, but up-regulated at 2 h in digestive gland compared with control counterparts (p < 0.05), while CYP3A4 mRNA was increased at 12 h in gill. After exposure to P. lima at 2 × 106 cells/L, the expression of CYP3A4 mRNA was significantly increased in digestive gland at 2 h and 12 h, while CYP2D14-like was up-regulated at 6 h. Besides, CYP3L3 and CYP2C8 also exhibited differential expression. These data suggested that CYP3A4, CYP2D14-like, and even CYP3L3 and CYP2C8 might be involved in DSP toxins metabolism. Besides, provision of ketoconazole resulted in significant decrement of CYP3A4 in digestive gland at 2 h and 12 h, while the OA content significantly decreased at 2 h and 6 h compared to control group without ketoconazole. These findings indicated that ketoconazole could depress CYP3A4 activity in bivalves thereby altering the metabolic activities of DSP toxins in bivalves, and also provided novel insights into the mechanistic role of CYP3A4 on DSP toxins metabolism in bivalves.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Dinoflagelados/metabolismo , Toxinas Marinhas/toxicidade , Perna (Organismo)/enzimologia , Intoxicação por Frutos do Mar , Poluentes da Água/toxicidade , Animais , Sistema Enzimático do Citocromo P-450/genética , Brânquias/efeitos dos fármacos , Brânquias/enzimologia , Perna (Organismo)/efeitos dos fármacos , Alimentos Marinhos/análise
11.
Sci Adv ; 5(1): eaau3795, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30729156

RESUMO

Commercialization of algal lipids and biofuels is still impractical owing to the unavailability of lipogenic strains and lack of economically viable oil extraction strategies. Because lipogenesis is governed by multiple factors, success in generating industrial-suitable algal strains using conventional strategies has been limited. We report the discovery of a novel bZIP1 transcription factor, NobZIP1, whose overexpression results in a remarkable elevation of lipid accumulation and lipid secretion in a model microalga Nannochloropsis oceanica, without impairing other physiological properties. Chromatin immunoprecipitation-quantitative PCR analysis revealed that the key genes up- and down-regulated by NobZIP1 are involved in lipogenesis and cell wall polymer synthesis, respectively, which, in turn, induce lipid overproduction and secretion. Among these regulated genes, UDP-glucose dehydrogenase was shown to alter cell wall composition, thus also boosting lipid secretion. In summary, these results offer a comprehensive strategy for concurrent lipid overproduction and secretion, strongly increasing the commercial potential of microalgae.

12.
Bioresour Technol ; 277: 204-210, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30630660

RESUMO

Microalgae have emerged as the potential source for value-added products such as polyunsaturated fatty acids (PUFAs). Metabolic engineering of multiple metabolic pathways has promoted eicosapentaenoic acid (EPA) production in microalgae, however, further improvement is warranted owing to the burgeoning demand. Here we improved the microalgal strains by adaptive evolution under hyposalinity treatment, which showed that 70% salinity potentiated the algae to enhance PUFAs. To exploit the maximal PUFA production potential of evolved strains, we subjected evolved algae to light, temperature and fulvic acid treatment. Amongst, fulvic acid (15 mg/L) enhanced growth and achieved the highest EPA content (13.9%) in the evolved diatom. Fulvic acid enhanced antioxidant potential and unprecedently governed the expression of PUFA and lipid biosynthetic genes. Collectively, this investigation demonstrates the efficacy of adaptive evolution empowered by fulvic acid and exemplifies a feasible strain improving strategy to harness the biotechnological potential of microalgae.


Assuntos
Benzopiranos/metabolismo , Ácido Eicosapentaenoico/análogos & derivados , Microalgas/metabolismo , Diatomáceas/metabolismo , Ácido Eicosapentaenoico/biossíntese , Temperatura Ambiente
13.
Biotechnol Biofuels ; 11: 318, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30479663

RESUMO

Background: Metabolic engineering has emerged as a potential strategy for improving microalgal lipid content through targeted changes to lipid metabolic networks. However, the intricate nature of lipogenesis has impeded metabolic engineering. Therefore, it is very important to identify the crucial metabolic nodes and develop strategies to exploit multiple genes for transgenesis. In an attempt to unravel the microalgal triacylglycerol (TAG) pathway, we overexpressed two key lipogenic genes, glycerol-3-phosphate acyltransferase (GPAT1) and lysophosphatidic acid acyltransferase (LPAT1), in oleaginous Phaeodactylum tricornutum and determined their roles in microalgal lipogenesis. Results: Engineered P. tricornutum strains showed enhanced growth and photosynthetic efficiency compared with that of the wild-type during the growth phase of the cultivation period. However, both the cell types reached stationary phase on day 7. Overexpression of GPAT1 and LPAT1 increased the TAG content by 2.3-fold under nitrogen-replete conditions without compromising cell growth, and they also orchestrated the expression of other key genes involved in TAG synthesis. The transgenic expression of GPAT1 and LPAT1 influenced the expression of malic enzyme and glucose 6-phosphate dehydrogenase, which enhanced the levels of lipogenic NADPH in the transgenic lines. In addition, GPAT1 and LPAT1 preferred C16 over C18 at the sn-2 position of the glycerol backbone. Conclusion: Overexpression of GPAT1 together with LPAT1 significantly enhanced lipid content without affecting growth and photosynthetic efficiency, and they orchestrated the expression of other key photosynthetic and lipogenic genes. The lipid profile for elevated fatty acid content (C16-CoA) demonstrated the involvement of the prokaryotic TAG pathway in marine diatoms. The results suggested that engineering dual metabolic nodes should be possible in microalgal lipid metabolism. This study also provides the first demonstration of the role of the prokaryotic TAG biosynthetic pathway in lipid overproduction and indicates that the fatty acid profile can be tailored to improve lipid production.

14.
Appl Microbiol Biotechnol ; 102(24): 10803-10815, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30349933

RESUMO

Commercial production of biofuel from oleaginous microalgae is often impeded by their slow growth rate than other fast-growing algal species. A promising strategy is to genetically engineer the fast-growing algae to accumulate lipids by expressing key lipogenic genes from oleaginous microalgae. However, lacking of strong expression cassette to transform most of the algal species and potential metabolic target to engineer lipid metabolism has hindered its biotechnological applications. In this study, we engineered the oxidative pentose phosphate pathway (PPP) of green microalga Chlorella pyrenoidosa for lipid enhancement by expressing a glucose-6-phosphate dehydrogenase (G6PD) from oleaginous diatom Phaeodactylum tricornutum. Molecular characterization of transformed lines revealed that heterologous PtG6PD was transcribed and expressed successfully. Interestingly, subcellular localization analyses revealed that PtG6PD was targeted to chloroplasts of C. pyrenoidosa. PtG6PD expression remarkably elevated NADPH content and consequently enhanced the lipid content without affecting growth rate. Collectively, this report represents a promising candidate to engineer lipid biosynthesis in heterologous hosts with notable commercial significance, and it highlights the potential role of plastidial PPP in supplying lipogenic NADPH in microalgae.

15.
Biochem Biophys Res Commun ; 505(4): 1003-1009, 2018 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-30309658

RESUMO

Giant-cell tumor (GCT) of the bone is an invasiveness and high recurrent bone tumor that is considered borderline or potentially malignant. To explore the molecular mechanism leading to bone destruction and identify novel targets for treatment, we conducted silencing of miR-223 and miR-19a in stromal giant cells and identified TWIST and Runx2 as their target genes. We investigated the impact of these microRNAs and their target genes on stromal giant cells that promote the differentiation of monocyte/macrophages into osteoclast cells and recruitment to the bone microenvironment, which in turn enhances the bone destruction capacity of GCT. MiR-223 and miR-19a were found to regulate the expression of TWIST and Runx2, influence the RANKL-RANK pathway and the expression of MCP-1, and finally regulate the pathophysiological process of osteolytic bone destruction. Our results indicate that re-expression of miR-223 and miR-19a induces an inhibitory effect on the bone destruction capacity of GCT, suggesting that re-expression of miR-223 and miR-19a can be a novel strategy for the treatment of GCT.

16.
Biotechnol J ; : e1800220, 2018 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-30076758

RESUMO

Chrysolaminarin, the primary polysaccharide reservoir in some marine algae, has attracted much attention due to its broad health properties. However, its biosynthetic pathway and regulation mechanisms have rarely been reported which hinders the improvement of production efficiency. Therefore, this study aims to identify key metabolic nodes in the chrysolaminarin biosynthetic pathway. A phosphoglucomutase (PGM) in the model microalga Phaeodactylum tricornutum, revealing its critical role in chrysolaminarin biosynthesis is identified. PGM overexpression significantly elevates chrysolaminarin content by 2.54-fold and reaches 25.6% of cell dry weight; while algal growth and photosynthesis are not impaired. Besides, PGM overexpression up- and down-regulates the expression of chrysolaminarin and lipid biosynthetic genes, respectively. Microscopic analysis of aniline blue stained cells reveals that overproduced chrysolaminarin localized predominantly in vacuoles. Lipidomic analyses reveal that PGM overexpression significantly reduces the lipid content. The findings reveal the critical role of PGM in regulating the carbon flux between carbohydrate and lipid biosynthesis in microalgae, and provide a promising candidate for high efficiency production of chrysolaminarin.

17.
Ecotoxicol Environ Saf ; 162: 365-375, 2018 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-30007186

RESUMO

Blooms of Aureococcus anophagefferens, referred to as brown tides are responsible for massive mortalities and recruitment failure of some bivalves. However, the molecular mechanisms underlying the toxicity remain elusive despite its biological significance, and the information currently available on the molecular effects is still insufficient. In this study, to evaluate the toxicity and associated mechanism of A. anophagefferens on bivalves, we analyzed the protein expression profiles in digestive glands of the A. anophagefferens-exposed Perna viridis by using iTRAQ. A total of 3138 proteins were identified in the digestive glands of A. anophagefferens-exposed P. viridis based on iTRAQ. Amongst, a repertoire of 236 proteins involved in cell, cell part, catalytic activity, metabolic process, biological regulation, immune system process, and response to stimulus were found to be differentially expressed. Functional analysis of the differentially expressed proteins demonstrated that innate immune system of P. viridis was activated, and some proteins associated with stress response and lipid metabolism were induced after exposure to A. anophagefferens. Additionally, MDA content, SOD activity and GSH-Px activity was increased significantly in the digestive gland of A. anophagefferens-exposed P. viridis. Taken together, our results indicated that the A. anophagefferens could induce oxidative stress, activate complement system and alter fat acid metabolism of P. viridis.


Assuntos
Proliferação Nociva de Algas , Perna (Organismo)/metabolismo , Estramenópilas/química , Animais , Exposição Ambiental , Imunidade Inata , Modelos Biológicos , Estresse Oxidativo , Perna (Organismo)/imunologia , Perna (Organismo)/fisiologia , Proteômica
18.
Mar Biotechnol (NY) ; 2018 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-29931608

RESUMO

Photosynthetic microalgae are of burgeoning interest in the generation of commercial bioproducts. Microalgae accumulate high lipid content under adverse conditions, which in turn compromise their growth and hinder their commercial potential. Hence, it is necessary to engineer microalgae to mitigate elevated lipid accumulation and biomass. In this study, we identified acetyl-CoA carboxylase (ACCase) in oleaginous microalga Phaeodactylum tricornutum (PtACC2) and expressed constitutively in the chloroplast to demonstrate the potential of chloroplast engineering. Molecular characterization of transplastomic microalgae revealed that PtACC2 was integrated, transcribed and expressed successfully, and localized in the chloroplast. Enzymatic activity of ACCase was elevated by 3.3-fold, and the relative neutral lipid content increased substantially by 1.77-fold, and lipid content reached up to 40.8% of dry weight. Accordingly, the number and size of oil bodies markedly increased. Fatty acid profiling showed that the content of monounsaturated fatty acids increased, while polyunsaturated fatty acids decreased. This method provides a valuable genetic engineering toolbox for microalgal bioreactors with industrial significance.

19.
Microb Cell Fact ; 17(1): 54, 2018 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-29618383

RESUMO

BACKGROUND: Microalgal metabolic engineering holds great promise for the overproduction of a wide range of commercial bioproducts. It demands simultaneous manipulation of multiple metabolic nodes. However, high-efficiency promoters have been lacking. RESULTS: Here we report a strong constitutive promoter Pt211 in expressing multiple target genes in oleaginous microalga Phaeodactylum tricornutum. Pt211 was revealed to contain significant cis-acting elements. GUS reporter and principal genes glycerol-3-phosphate acyltransferase (GPAT) and diacylglycerol acyltransferase 2 (DGAT2) involved in triacylglycerol biosynthesis were tested under driven of Pt211 in P. tricornutum. GUS staining and qPCR analysis showed strong GUS expression. DGAT2 and GPAT linked with a designed 2A sequence exhibited higher transcript abundances than WT, while algal growth and photosynthesis were not impaired. CONCLUSION: The total lipid content increased notably by 2.6-fold compared to WT and reached up to 57.5% (dry cell weight). Overall, our findings report a strong promoter and a strategy for coordinated manipulation of complex metabolic pathways.

20.
Neural Regen Res ; 13(2): 309-316, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29557382

RESUMO

Scar hyperplasia at the suture site is an important reason for hindering the repair effect of peripheral nerve injury anastomosis. To address this issue, two repair methods are often used. Biological agents are used to block nerve sutures and the surrounding tissue to achieve physical anti-adhesion effects. Another agent is glucocorticosteroid, which can prevent scar growth by inhibiting inflammation. However, the overall effect of promoting regeneration of the injured nerve is not satisfactory. In this regard, we envision that these two methods can be combined and lead to shared understanding for achieving improved nerve repair. In this study, the right tibial nerve was transected 1 cm above the knee to establish a rat tibial nerve injury model. The incision was directly sutured after nerve transection. The anastomotic stoma was coated with 0.5 × 0.5 cm2 chitosan sheets with betamethasone dipropionate. At 12 weeks after injury, compared with the control and poly (D, L-lactic acid) groups, chitosan-betamethasone dipropionate film slowly degraded with the shape of the membrane still intact. Further, scar hyperplasia and the degree of adhesion at anastomotic stoma were obviously reduced, while the regenerated nerve fiber structure was complete and arranged in a good order in model rats. Electrophysiological study showed enhanced compound muscle action potential. Our results confirm that chitosan-betamethasone dipropionate film can effectively prevent local scar hyperplasia after tibial nerve repair and promote nerve regeneration.

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