The genomic DNA sequence encoding soybean 24 kDa oleosin and its promoter were cloned andanalyzed for investigation of the potentials of the oleosin acted as a carrier forproduction of recombinant proteins in plant. T...The genomic DNA sequence encoding soybean 24 kDa oleosin and its promoter were cloned andanalyzed for investigation of the potentials of the oleosin acted as a carrier forproduction of recombinant proteins in plant. The -300 box, GA-rich, G-box, SEF-3, SEF-4, RY box, ABA box, CAn and TATA box were found in the upstream region of the soybeanoleosin gene, which shows the functional oleosin promoter available. Homology comparisonreveals that the soybean 24 kDa oleosin shares the highest identity with the soybeanoleosin isoform A (U09118, GenBank), reaching to 98.4% in nucleotide. A soybean oleosin-hirudin fusion gene driven by the oleosin promoter was constructed and inserted intoplant binary expression vector. The intact tobacco plantlets were transformed by meansof vacuum infiltration approach, with the Agrobacterium tumefaciens harboring the abovevector. The transient correct expression of oleosin-hirudin fusion gene was identifiedby SDS/PAGE, western blotting and enterokinase treatment.展开更多
In the late phase of Bombyx mori nucleopolyhedrovirus(BmNPV)infection,a large amount of polyhedra appear in the infected cell nucleolus,these polyhedra being dense protein crystals protecting the incorporated virions ...In the late phase of Bombyx mori nucleopolyhedrovirus(BmNPV)infection,a large amount of polyhedra appear in the infected cell nucleolus,these polyhedra being dense protein crystals protecting the incorporated virions from the harsh environment.To investigate whether the foreign protein could be immobilized into the polyhedra of BmNPV,two recombinant baculoviruses were generated by a novel BmNPV polyhedrin-plus(polh +)Bac-to-Bac system,designated as vBmBac(polh +)-enhanced green fluorescent protein(EGFP)and vBmBac(polh +)-LacZ,which can express the polyhedrin and foreign protein simultaneously.Light microscopy analysis showed that all viruses produced polyhedra of normal appearance.Green fluorescence can be apparently detected on the surface of the vBmBac(polh +)-EGFP polyhedra,but not the BmNPV polyhedra.Fluorescence analysis and anti-desiccation testing confirmed that EGFP was embedded in the polyhedra.As expected,the vBmBac(polh +)-LacZ polyhedra contained an amount of LacZ and had a higherβ-galactosidase activity.Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting were also performed to verify if the foreign proteins were immobilized into polyhedra.This study provides a new inspiration for efficient preservation of useful proteins and development of new pesticides with toxic proteins.展开更多
基金supported by a grant from the National High Tech R&D Program(863 Program)of China(2001AA2121).
文摘The genomic DNA sequence encoding soybean 24 kDa oleosin and its promoter were cloned andanalyzed for investigation of the potentials of the oleosin acted as a carrier forproduction of recombinant proteins in plant. The -300 box, GA-rich, G-box, SEF-3, SEF-4, RY box, ABA box, CAn and TATA box were found in the upstream region of the soybeanoleosin gene, which shows the functional oleosin promoter available. Homology comparisonreveals that the soybean 24 kDa oleosin shares the highest identity with the soybeanoleosin isoform A (U09118, GenBank), reaching to 98.4% in nucleotide. A soybean oleosin-hirudin fusion gene driven by the oleosin promoter was constructed and inserted intoplant binary expression vector. The intact tobacco plantlets were transformed by meansof vacuum infiltration approach, with the Agrobacterium tumefaciens harboring the abovevector. The transient correct expression of oleosin-hirudin fusion gene was identifiedby SDS/PAGE, western blotting and enterokinase treatment.
基金supported by the National Basic Research Program(973)of China(No.2012CB114600)the Zhejiang Provincial Natural Science Foundation(No.Y3110058)the Public Agricultural Program of Zhejiang Province(No.2012C32G2010076),China
文摘In the late phase of Bombyx mori nucleopolyhedrovirus(BmNPV)infection,a large amount of polyhedra appear in the infected cell nucleolus,these polyhedra being dense protein crystals protecting the incorporated virions from the harsh environment.To investigate whether the foreign protein could be immobilized into the polyhedra of BmNPV,two recombinant baculoviruses were generated by a novel BmNPV polyhedrin-plus(polh +)Bac-to-Bac system,designated as vBmBac(polh +)-enhanced green fluorescent protein(EGFP)and vBmBac(polh +)-LacZ,which can express the polyhedrin and foreign protein simultaneously.Light microscopy analysis showed that all viruses produced polyhedra of normal appearance.Green fluorescence can be apparently detected on the surface of the vBmBac(polh +)-EGFP polyhedra,but not the BmNPV polyhedra.Fluorescence analysis and anti-desiccation testing confirmed that EGFP was embedded in the polyhedra.As expected,the vBmBac(polh +)-LacZ polyhedra contained an amount of LacZ and had a higherβ-galactosidase activity.Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting were also performed to verify if the foreign proteins were immobilized into polyhedra.This study provides a new inspiration for efficient preservation of useful proteins and development of new pesticides with toxic proteins.
