[ Objective] The FaOLtr2 （ Frago^ia ananassa osmotin-like protein） is a functional homolog of PR5-1ike protein. This study was undertaken to produce recombinant FaOLP2 and to identify its antifungal activity. [ Meth...[ Objective] The FaOLtr2 （ Frago^ia ananassa osmotin-like protein） is a functional homolog of PR5-1ike protein. This study was undertaken to produce recombinant FaOLP2 and to identify its antifungal activity. [ Method] The ORF of FaOLP2 （ accession number DQ325524） was cloned into pET22b vector to con- stroct the pET22b-FaOLP2 plasmid. The recombinant mature FaOLP2 was expressed in E. coli Rosetta-gami B （DE3） by inducing with I nunol/L IPTG and found exclusively in insoluble inclusion bodies. As FaOLP2 requires the correct formation of eight disulfide bonds, but there were no obvious effect to correctly form these by expression at different temperatures and high osmotic pressure （ supplemented Betaineand and D-Sorbitol）, we used an in vitro method to refold E. coli expressed FaOLP2 by gradually elution using reduced：oxidized gluthatione redox buffer, followed by 8 mol/L urea solubilized His6-tagged mature FaOLP2 protein, which was affinity-purified by an immobilized-metal （Ni2＋ ） affinity chromatography （IMAC） column. [ Result] This method generated biologically active conformations of the recombinant mature FaOLP2 that displayed antifungal activity against Ustilaginoides virens, a plant pathogenic fungus, which causes rice false smut. [ Conclusion] This study laid the foundation for further biotechnological application of the novel protein.展开更多
We developed a molecular marker for MAS of mungbean resistant varieties against CLS from the consensus sequence(MB-CLsRG)of identified RGAs(MB-ClsRCaG1 and MB-ClsRCaG2).The MB-CLsRG sequence-specific primer pair was u...We developed a molecular marker for MAS of mungbean resistant varieties against CLS from the consensus sequence(MB-CLsRG)of identified RGAs(MB-ClsRCaG1 and MB-ClsRCaG2).The MB-CLsRG sequence-specific primer pair was used to screen Cercospora leaf spot(CLS)resistant varieties of mungbean in genomic analysis that showed congruency with phenotypic screening.Validation of molecular marker linkage with CLS resistance was performed using rtPCR in transcriptomic analysis.The sequenced PCR products showed 100%homology with MB-CLsRG sequence and putative disease resistance proteins that confirmed the linkage of molecular marker with CLS resistance in mungbean.The antifungal potential of MB-CLsRG gene encoding protein was assessed.The MB-CLsRG gene sequence was cloned in the E.coli expression vector for recombinant protein production.The recombinant protein was then investigated for its in vitro antifungal potential against Cercospora canescens.The in vitro investigation showed strong antifungal activity of recombinant protein as it restricted the growth of fungal mycelial mass.The results validated the linkage of developed marker with CLS-resistant mungbean varieties;therefore,it can be used to screen resistant varieties from a large population in MAS.Moreover,the recombinant protein of the MB-CLsRG gene sequence revealed antifungal potential,which proved the gene sequence could be suitable to use in transgenic plants technology to develop fungal-resistant transgenic crops.展开更多
An antifungal protein named PaAFP was isolated and purified from the seeds of Pachyrrhizus erosus by extraction with PB buffer, S Sepharose Fast Flow, CM 52 cellulose and Sephadex G 75 column chromatography. On potato...An antifungal protein named PaAFP was isolated and purified from the seeds of Pachyrrhizus erosus by extraction with PB buffer, S Sepharose Fast Flow, CM 52 cellulose and Sephadex G 75 column chromatography. On potato sucrose agar medium, the purified protein obviously inhibited the growth of the important edible fungus pathogens Trichoderma viride and Chrysosporium luteum at 6 2 μg and 12 5 μg per disc respectively. The molecular weight was about 14 kD by SDS PAGE and HPLC. The isoelectric point was pH7 5. Analysis of the composition of amino acids showed that this protein was rich in Asx, but lacking in Met.展开更多
基金Supported by Zhejiang Province Natural Science Foundation(Y307591,Y3110288)Key Scientific and Technological Innovation Team Program of Zhejiang Province(2010R50028)
文摘[ Objective] The FaOLtr2 （ Frago^ia ananassa osmotin-like protein） is a functional homolog of PR5-1ike protein. This study was undertaken to produce recombinant FaOLP2 and to identify its antifungal activity. [ Method] The ORF of FaOLP2 （ accession number DQ325524） was cloned into pET22b vector to con- stroct the pET22b-FaOLP2 plasmid. The recombinant mature FaOLP2 was expressed in E. coli Rosetta-gami B （DE3） by inducing with I nunol/L IPTG and found exclusively in insoluble inclusion bodies. As FaOLP2 requires the correct formation of eight disulfide bonds, but there were no obvious effect to correctly form these by expression at different temperatures and high osmotic pressure （ supplemented Betaineand and D-Sorbitol）, we used an in vitro method to refold E. coli expressed FaOLP2 by gradually elution using reduced：oxidized gluthatione redox buffer, followed by 8 mol/L urea solubilized His6-tagged mature FaOLP2 protein, which was affinity-purified by an immobilized-metal （Ni2＋ ） affinity chromatography （IMAC） column. [ Result] This method generated biologically active conformations of the recombinant mature FaOLP2 that displayed antifungal activity against Ustilaginoides virens, a plant pathogenic fungus, which causes rice false smut. [ Conclusion] This study laid the foundation for further biotechnological application of the novel protein.
基金The Higher Education Commission(HEC)funded this work under“Transcriptomics Based Understanding of Cercospora Leaf Spot Resistance in Mungbean and Disease Management through Nanotechnology,”Project No.7425.
文摘We developed a molecular marker for MAS of mungbean resistant varieties against CLS from the consensus sequence(MB-CLsRG)of identified RGAs(MB-ClsRCaG1 and MB-ClsRCaG2).The MB-CLsRG sequence-specific primer pair was used to screen Cercospora leaf spot(CLS)resistant varieties of mungbean in genomic analysis that showed congruency with phenotypic screening.Validation of molecular marker linkage with CLS resistance was performed using rtPCR in transcriptomic analysis.The sequenced PCR products showed 100%homology with MB-CLsRG sequence and putative disease resistance proteins that confirmed the linkage of molecular marker with CLS resistance in mungbean.The antifungal potential of MB-CLsRG gene encoding protein was assessed.The MB-CLsRG gene sequence was cloned in the E.coli expression vector for recombinant protein production.The recombinant protein was then investigated for its in vitro antifungal potential against Cercospora canescens.The in vitro investigation showed strong antifungal activity of recombinant protein as it restricted the growth of fungal mycelial mass.The results validated the linkage of developed marker with CLS-resistant mungbean varieties;therefore,it can be used to screen resistant varieties from a large population in MAS.Moreover,the recombinant protein of the MB-CLsRG gene sequence revealed antifungal potential,which proved the gene sequence could be suitable to use in transgenic plants technology to develop fungal-resistant transgenic crops.
文摘An antifungal protein named PaAFP was isolated and purified from the seeds of Pachyrrhizus erosus by extraction with PB buffer, S Sepharose Fast Flow, CM 52 cellulose and Sephadex G 75 column chromatography. On potato sucrose agar medium, the purified protein obviously inhibited the growth of the important edible fungus pathogens Trichoderma viride and Chrysosporium luteum at 6 2 μg and 12 5 μg per disc respectively. The molecular weight was about 14 kD by SDS PAGE and HPLC. The isoelectric point was pH7 5. Analysis of the composition of amino acids showed that this protein was rich in Asx, but lacking in Met.
