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
As the main structural protein of oil body,OLEOSIN is highly expressed only during seed development. OLEOSIN promoter is a very useful tool for seed-specific gene engineering and seed bioreactor designing. The B3 doma...As the main structural protein of oil body,OLEOSIN is highly expressed only during seed development. OLEOSIN promoter is a very useful tool for seed-specific gene engineering and seed bioreactor designing. The B3 domain transcription factor leafy cotyledon2 (LEC2) plays an important role in regulating seed development and seed-specific gene expression. Here,we first report how seed-specific B3 domain transcription factor leafy cotyledon2 (LEC2) efficiently activates OLEOSIN expression. The central promoter region of OLEOSIN,responsible for seed specificity and LEC2 activation,was determined by 5'-deletion analysis. Binding experiments in yeast cells and electrophoretic mobility shift assays showed that LEC2 specifically bound to two conserved RY elements in this region. In transient expression assays,mutation in either RY element dramatically reduced LEC2 activation of OLEOSIN promoter activity,while double mutation abolished it. Analysis of the distribution of RY elements in seed-specific genes activated by LEC2 also supported the idea that genes containing neighboring RY elements responded strongly to LEC2 activation. Therefore,we conclude that two neighboring RY elements are essential for efficient LEC2 activation of OLEOSIN expression. These findings will help us better utilize seed-specific promoter activity.展开更多
Oleosin, caleosin and steroleosin are normally expressed in developing seed cells and are targeted to oil bodies. In the present work, the cDNA of each gene tagged with fluorescent proteins was transiently expressed i...Oleosin, caleosin and steroleosin are normally expressed in developing seed cells and are targeted to oil bodies. In the present work, the cDNA of each gene tagged with fluorescent proteins was transiently expressed into tobacco protoplasts and the fluorescent patterns observed by confocal laser scanning microscopy. Our results indicated clear differences in the endocellular localization of the three proteins. Oleosin and caleosin both share a common structure consisting of a central hydrophobic domain flanked by two hydrophilic domains and were correctly targeted to lipid droplets （LD）, whereas steroleosin, characterized by an N-terminal oil body anchoring domain, was mainly retained in the endoplasmic reticulum （ER）. Protoplast fractionation on sucrose gradients indicated that both oleosin and caleosin- green fluorescent protein （GFP） peaked at different fractions than where steroleosin-GFP or the ER marker binding immunoglobulin protein （BiP）, were recovered. Chemical analysis confirmed the presence of triacylglycerols in one of the fractions where oleosin-GFP was recovered. Finally, only oleosin- and caleosin-GFP were able to reconstitute artificial oil bodies in the presence of triacylglycerols and phospholipids. Taken together, our results pointed out for the first time that leaf LDs can be separated by the ER and both oleosin or caleosin are selectively targeted due to the existence of selective mechanisms controlling protein association with these organelles.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 Grants from Toyota Motor Corporation of Japan and the National Special Project of Transgenic Organisms (Grant No. 2008ZX08010-001) of the Chinese Government
文摘As the main structural protein of oil body,OLEOSIN is highly expressed only during seed development. OLEOSIN promoter is a very useful tool for seed-specific gene engineering and seed bioreactor designing. The B3 domain transcription factor leafy cotyledon2 (LEC2) plays an important role in regulating seed development and seed-specific gene expression. Here,we first report how seed-specific B3 domain transcription factor leafy cotyledon2 (LEC2) efficiently activates OLEOSIN expression. The central promoter region of OLEOSIN,responsible for seed specificity and LEC2 activation,was determined by 5'-deletion analysis. Binding experiments in yeast cells and electrophoretic mobility shift assays showed that LEC2 specifically bound to two conserved RY elements in this region. In transient expression assays,mutation in either RY element dramatically reduced LEC2 activation of OLEOSIN promoter activity,while double mutation abolished it. Analysis of the distribution of RY elements in seed-specific genes activated by LEC2 also supported the idea that genes containing neighboring RY elements responded strongly to LEC2 activation. Therefore,we conclude that two neighboring RY elements are essential for efficient LEC2 activation of OLEOSIN expression. These findings will help us better utilize seed-specific promoter activity.
文摘Oleosin, caleosin and steroleosin are normally expressed in developing seed cells and are targeted to oil bodies. In the present work, the cDNA of each gene tagged with fluorescent proteins was transiently expressed into tobacco protoplasts and the fluorescent patterns observed by confocal laser scanning microscopy. Our results indicated clear differences in the endocellular localization of the three proteins. Oleosin and caleosin both share a common structure consisting of a central hydrophobic domain flanked by two hydrophilic domains and were correctly targeted to lipid droplets （LD）, whereas steroleosin, characterized by an N-terminal oil body anchoring domain, was mainly retained in the endoplasmic reticulum （ER）. Protoplast fractionation on sucrose gradients indicated that both oleosin and caleosin- green fluorescent protein （GFP） peaked at different fractions than where steroleosin-GFP or the ER marker binding immunoglobulin protein （BiP）, were recovered. Chemical analysis confirmed the presence of triacylglycerols in one of the fractions where oleosin-GFP was recovered. Finally, only oleosin- and caleosin-GFP were able to reconstitute artificial oil bodies in the presence of triacylglycerols and phospholipids. Taken together, our results pointed out for the first time that leaf LDs can be separated by the ER and both oleosin or caleosin are selectively targeted due to the existence of selective mechanisms controlling protein association with these organelles.
