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Zhu X.,Shanghai JiaoTong University | Chen L.,Shanghai Normal University | Shen P.,Development Center for Science and Technology | Jia J.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | And 2 more authors.
Journal of Agricultural and Food Chemistry | Year: 2011

Protein-based detection methods, enzyme-linked immunosorbent assay (ELISA) and lateral flow strip, have been widely used for rapid, spot, and sensitive detection of genetically modified organisms (GMOs). Herein, one novel quantum dot-based fluorescence-linked immunosorbent assay (QD-FLISA) was developed employing quantum dots (QDs) as the fluorescent marker for the detection of the Cry1Ab protein in MON810 maize. The end-point fluorescent detection system was carried out using QDs conjugated with goat anti-rabbit secondary antibody. The newly developed Cry1Ab QD-FLISA assay was highly specific to the Cry1Ab protein and had no cross-reactivity with other target proteins, such as Cry2Ab, Cry1F, and Cry3Bb. The quantified linearity was achieved in the value range of 0.05-5% (w/w). The limits of detection (LOD) and quantification (LOQ) of the QD-FLISA were 2.956 and 9.854 pg/mL, respectively, which were more sensitive than the conventional sandwich ELISA method. All of the results indicated that QD-FLISA was a highly specific and sensitive method for the monitoring of Cry1Ab in GMOs. © 2011 American Chemical Society.

Gao M.-J.,Jiangnan University | Zheng Z.-Y.,Jiangnan University | Wu J.-R.,Jiangnan University | Dong S.-J.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | And 4 more authors.
Applied Microbiology and Biotechnology | Year: 2012

Effective expression of porcine interferon-α (pIFN-α) with recombinant Pichia pastoris was conducted in a bench-scale fermentor. The influence of the glycerol feeding strategy on the specific growth rate and protein production was investigated. The traditional DO-stat feeding strategy led to very low cell growth rate resulting in low dry cell weight (DCW) of about 90 g/L during the subsequent induction phase. The previously reported Artificial Neural Network Pattern Recognition (ANNPR) model-based glycerol feeding strategy improved the cell density to 120 g DCW/L, while the specific growth rate decreased from 0.15 to 0.18 to 0.03-0.08 h -1 during the last 10 h of the glycerol feeding stage leading to a variation of the porcine interferon-α production, as the glycerol feeding scheme had a significant effect on the induction phase. This problem was resolved by an improved ANNPR model-based feeding strategy to maintain the specific growth rate above 0.11 h -1. With this feeding strategy, the pIFN-α concentration reached a level of 1.43 g/L, more than 1.5-fold higher than that obtained with the previously adopted feeding strategy. Our results showed that increasing the specific growth rate favored the target protein production and the glycerol feeding methods directly influenced the induction stage. Consequently, higher cell density and specific growth rate as well as effective porcine interferon-α production have been achieved by our novel glycerol feeding strategy. © 2011 Springer-Verlag.

Jin H.,Jiangnan University | Liu G.,Jiangnan University | Ye X.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Duan Z.,Jiangnan University | And 2 more authors.
Biochemical Engineering Journal | Year: 2010

Porcine interferon-α (pIFN-α) production by recombinant Pichia pastoris with standard induction strategy at 30°C often suffers problems such as low antiviral activity, long cells adaptation period, etc. To solve these problems, a combinational induction strategy by simultaneously controlling induction temperature at 20°C and dissolved oxygen concentration (DO) over 50% was proposed and the relevant fermentation runs were conducted in a 5l bioreactor. With this control strategy, pIFN-α antiviral activity could be continuously enhanced and eventually reached a level of 3.62×107IU/ml, which was about 16-fold of the maximum obtained when induction was done at 20°C but without DO control, and more than 100-fold of the maximum obtained with the standard induction strategy at 30°C. The novel control strategy could enhance alcohol oxidase (AOX) activity and relieve oxygen supply limitation in oxidative phosphorylation reaction to accelerate ATP regeneration simultaneously. As a result, the metabolic flux towards pIFN-α synthesis was enhanced and the adaptation period was shortened, enabling the entire system to be operated in a most efficient way. © 2010 Elsevier B.V.

Shi L.,Nanjing Agricultural University | Fang X.,Nanjing Agricultural University | Li M.,Nanjing Agricultural University | Mu D.,Nanjing Agricultural University | And 3 more authors.
World Journal of Microbiology and Biotechnology | Year: 2012

In this study, we report the development of a simple and efficient system for genetic transformation of the medicinal fungus Ganoderma lucidum. Various parameters were optimized to obtain successful Agrobacterium tumefaciens-mediated transformation. Co-cultivation of bacteria and protoplast at a ratio of 1,000:1 at 25°C in medium containing 0. 2 mM acetosyringone was found to be the optimum condition for high efficiency transformation. Four plasmids, each carrying a different promoter driving the expression of an antibiotic resistance marker, were tested. The construct carrying the Ganoderma lucidum glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter showed good transformation efficiency, whereas constructs with the GPD promoter from ascomycetes were ineffective. Our analysis showed that over 70% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. We were able to detect the expression of EGFP and GUS reporter genes in the Ganoderma lucidum transformants by fluorescence imaging and histochemical staining assays respectively. Our results demonstrate a new transgenic approach that will facilitate Ganoderma lucidum research. © 2011 Springer Science+Business Media B.V.

Tian Y.-S.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Xu J.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Xiong A.-S.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Zhao W.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | And 4 more authors.
Applied Microbiology and Biotechnology | Year: 2012

Although a large number of AroA enzymes (5-enopyruvylshikimate-3-phosphate synthase [EPSPS]) have been identified, cloned and tested for glyphosate resistance, only AroA variants derived from Agrobacterium tumefaciens strain CP4 have been successfully used commercially. We have now used a polymerase chain reaction (PCR)-based two-step DNA synthesis (PTDS) method to synthesize an aroA gene (aroA H. orenii ) from Halothermothrix orenii H168 encoding a new EPSPS similar to AroA A. tumefaciens CP4. AroA H. orenii was then expressed in Escherichia coli and key kinetic values of the purified enzyme were determined. Kinetic analysis of AroA H. orenii indicated that the full-length enzyme exhibited increased tolerance to glyphosate compared with E. coli AroA E. coli while retaining a high affinity for the substrate phosphoenolpyruvate. Transgenic Arabidopsis plants containing aroA H. orenii were resistant to 15 mM glyphosate. Site-directed mutagenesis showed that residues Thr355Ser affected the affinity of AroA H. orenii for glyphosate, providing further evidence that specific amino acid residues are responsible for differences in enzymatic behavior among different AroA enzymes. © 2011 Springer-Verlag.

Fu X.-Y.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Zhao W.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Xiong A.-S.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Tian Y.-S.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | And 3 more authors.
Applied Microbiology and Biotechnology | Year: 2013

Triphenylmethane dyes are extensively utilized in textile industries, medicinal products, biological stains, and food processing industries, etc. They are generally considered as xenobiotic compounds, which are very recalcitrant to biodegradation. The widespread persistence of such compounds has generated concerns with regard to remediation of them because of their potential carcinogenicity, teratogenicity, and mutagenicity. In this study, we present a system of phytoremediation by Arabidopsis plants developed on the basis of overexpression of triphenylmethane reductase (TMR) from the Citrobacter sp. The morphology and growth of TMR transgenic Arabidopsis plants showed significantly enhanced tolerances to crystal violet (CV) and malachite green (MG). Further, HPLC and HPLC-MS analyses of samples before and after dye decolorization in culture media revealed that TMR transgenic plants exhibited strikingly higher capabilities of removing CV from their media and high efficiencies of converting CV to non-toxic leucocrystal violet (LCV). This work indicates that microbial degradative gene may be transgenically exploited in plants for bioremediation of triphenylmethane dyes in the environment. © 2012 Springer-Verlag.

Tian Y.-S.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Xu J.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Xiong A.-S.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Zhao W.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | And 3 more authors.
Applied and Environmental Microbiology | Year: 2011

A mutant of 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified after four rounds of DNA shuffling and screening. Its ability to restore the growth of the mutant ER2799 cell on an M9 minimal medium containing 300 mM glyphosate led to its identification. The mutant had mutations in seven amino acids: E145G, N163H, N267S, P318R, M377V, M425T, and P438L. Among these mutations, N267S, P318R, and M425T have never been previously reported as important residues for glyphosate resistance. However, in the present study they were found by site-directed mutagenesis to collectively contribute to the improvement of glyphosate tolerance. Kinetic analyses of these three mutants demonstrated that the effectiveness of these three individual amino acid alterations on glyphosate tolerance was in the order P318R > M425T > N267S. The results of the kinetic analyses combined with a three-dimensional structure modeling of the location of P318R and M425T demonstrate that the lower hemisphere's upper surface is possibly another important region for glyphosate resistance. Furthermore, the transgenic Arabidopsis was obtained to confirm the potential of the mutant in developing glyphosate-resistant crops. © 2011, American Society for Microbiology.

Peng Y.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Xiang H.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Chen C.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Zheng R.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | And 3 more authors.
International Journal of Biochemistry and Cell Biology | Year: 2013

MicroRNAs (miRNAs) are small ~22 nucleotide regulatory RNAs that regulate the stability and trans-lation of cognate messenger RNAs (mRNAs). MicroRNAs participate in the regulation of adipogenesis and identification of the full repertoire of MicroRNAs expressed in adipose tissue is likely to improveour understanding of adipose tissue growth and development significantly. In the present study, it isfound that miR-224-5p abundance decreases first and then increases during adipogenesis of 3T3-L1 cells.And early growth response 2 (EGR2) and Acyl-CoA synthetase long-chain family member 4 (ACSL4) aredirect targets of miR-224-5p. Further studies in mouse 3T3-L1 cell-line shows that miR-224-5p is a novelnegative regulator of adipocyte differentiation through post-transcriptional regulation of early growth response 2 during early adipogenesis. Furthermore, miR-224-5p could regulate fatty acid metabolism through Acyl-CoA synthetase long-chain family member 4 at terminal differentiation. It indicates thatmiR-224 plays different roles on different stages of adipogenesis. Crown Copyright © 2013 Published by Elsevier Ltd. All rights reserved.

Yu R.,Shanghai Key Laboratory of Agricultural Genetics and Breeding
Wei sheng wu xue bao = Acta microbiologica Sinica | Year: 2011

A new method used to heterologously express [FeFe]-hydrogenase in Escherichia coli was investigated in our present study. By homologous recombination, three assistant genes (hydE, hydF and hydG) for hydrogenase were integrated into the chromosome of E. coli BW 25113-10, in which all hydrogenase genes were inactivated. A hydrogenase structural gene hydA from Clostridium butyricum was used to test the hydrogenase maturation ability of the recombined E. coli. BW 25113-13. The corrected integration of the three assistant genes was confirmed by PCR, and RT-PCR results indicated that the three accessory genes were transcripted in the recombinant. The active expression of hydA indicated that the constitutively expressed accessory proteins could assist the maturation of the [FeFe]-hydrogenases. A simplified [FeFe]-hydrogenase expression recombinant E. coli BW25113-13 was constructed. It would lay foundations for the functional screening of [FeFe]-hydrogenases and the construction of novel hydrogen producing pathways in E. coli.

PubMed | Shanghai Key Laboratory of Agricultural Genetics and Breeding
Type: | Journal: Scientific reports | Year: 2017

Trichlorophenol (TCP) is a widely used and persistent environmentally toxic compound that poses a carcinogenic risk to humans. Phytoremediation is a proficient cleanup technology for organic pollutants. In this study, we found that the disulfide isomerase-like protein AtPDIL1-2 in plants is a good candidate for enhancing 2,4,6-TCP phytoremediation. The expression of AtPDIL1-2 in Arabidopsis was induced by 2,4,6-TCP. The heterologously expressed AtPDIL1-2 in Escherichia coli exhibited both oxidase and isomerase activities as protein disulfide isomerase and improved bacteria tolerance to 2,4,6-TCP. Further research revealed that transgenic tobacco overexpressing AtPDIL1-2 was more tolerant to high concentrations of 2,4,6-TCP and removed the toxic compound at far greater rates than the control plants. To elucidate the mechanism of action of AtPDIL1-2, we investigated the chemical interaction of AtPDIL1-2 with 2,4,6-TCP for the first time. HPLC analysis implied that AtPDIL1-2 exerts a TCP-binding activity. A suitable configuration of AtPDIL1-2-TCP binding was obtained by molecular docking studies using the AutoDock program. It predicted that the TCP binding site is located in the b-b domain of AtPDIL1-2 and that His254 of the protein is critical for the binding interaction. These findings imply that AtPDIL1-2 can be used for TCP detoxification by the way of overexpression in plants.

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