Hypocotyl expression and light downregulation of the soybean tubulin gene, tubB1.
The tubB1 beta-tubulin gene of Glycine max (previously named s beta 1) is highly expressed only in rapidly elongating regions of etiolated seedling hypocotyls and this expression is strongly downregulated when the seedlings are exposed to light. Primer extension demonstrated that the gene was transcribed in these tissues and contained two sites of transcriptional initiation. To determine the mechanism regulating tubB1 expression, a chimeric reporter gene was constructed by fusing 5' upstream regions of tubB1 to a promoterless beta-glucuronidase (GUS) gene and these constructs were introduced into protoplasts by electroporation. Strong transient expression of the reporter gene was obtained after electroporation of chimeric constructs containing 1 kb of tubB1 5' upstream sequence into tobacco protoplasts. Deletion of the distal most 300 bp from the 5' sequence of tubB1 enhanced expression, suggesting the possibility of a negative transcriptional regulator in this region. Additional deletions of the 5' sequence reduced expression substantially. Constructs containing a tubB1 3' terminus were expressed at much lower levels than those containing a nopaline synthase (NOS) 3' terminus. The tubB1-GUS chimeric gene also was introduced into tobacco by Agrobacterium-mediated Ti plasmid transformation and the organ-specific expression pattern of the chimeric gene was determined in seedlings of the transgenic plants. Hypocotyls exhibited strong GUS activity when the seedlings were germinated in darkness, but lacked the GUS enzyme when the seedlings were germinated in the light.(ABSTRACT TRUNCATED AT 250 WORDS)
Tonoike
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Department of Developmental and Cell Biology, University of California, Irvine 92717.
Han
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Jongewaard
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Doyle
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Guiltinan
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Fosket
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D001483:Base Sequence; D015536:Down-Regulation; D018274:Electroporation; D005786:Gene Expression Regulation / Q000528:radiation effects*; D017343:Genes, Plant / Q000235:genetics*; D017930:Genes, Reporter; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D013025:Glycine max / Q000235:genetics* / Q000528:radiation effects; D014018:Tissue Distribution; D014026:Nicotiana / Q000235:genetics; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D014404:Tubulin / Q000096:biosynthesis / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D014404:Tubulin; D005966:Glucuronidase