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
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.
