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Title:Analysis of ABA-regulated expression of the maize Glb1 gene in tobacco seeds and maize cells
Author(s):Liu, Siqing
Doctoral Committee Chair(s):Briskin, Donald P.
Department / Program:Crop Sciences
Discipline:Crop Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Agriculture, Agronomy
Biology, Genetics
Abstract:The expression and regulation of the Glb1 gene was examined in both transgenic tobacco plants as well as maize P3377 cells. For transgenic studies, a ppGlb1GUS construct, consisting of 1402 bp 5$\sp\prime$ flanking sequence of Glb1, 1919 bp GUS (coding sequence, and 283 bp 3$\sp\prime$ Nos terminator, was cloned into Bin19 vector and introduced into tobacco plants by Agrobacterium-mediated transformation. Histochemical GUS assays of R$\sb{\rm o}$ tobacco mature seeds indicate that the Glb1 promoter drives GUS expression in ABA treated seeds, but not in water treated ones. Further GUS assays of the R$\sb1$ seeds at different developmental stages revealed that without ABA treatment, the Glb1 promoter drives GUS expression in immature seeds. The results from both R$\sb{\rm o}$ and R$\sb1$ tobacco plants indicated that GUS expression in tobacco is embryo specific. To study the accumulation of GLB1 proteins in tobacco plants, the plasmid ppConGlb1 was constructed by inserting the 2010 bp Glb1cDNA fragment into an expression cassette from the $\alpha\sp\prime$-subunit of the soybean $\beta$-conglycinin gene consisting of 866 bp 5$\sp\prime$ flanking sequence and 361 bp 3$\sp\prime$ terminator of the gene. The ppConGlb1 cassette was then cloned into the Bin19 vector and transformed into tobacco plants. The results of immunoassays of the tobacco R$\sb1$ seeds using whole maize globulin antisera indicate that the GLB1 protein is expressed in immature tobacco seeds and GLB1 is correctly processed to its mature form.
For transient studies, serial promoter deletions were generated and linked with the GUS reporter gene. The promoter deletion-GUS fusions were introduced into the maize P3377 cell line by the particle bombardment technique. GUS assays indicate that treatment of maize p3377 suspension cells with ABA is required for Glb1-driven GUS expression, and that the $-$295 bp sequence of the Glb1 promoter is sufficient for ABA-regulated expression of GUS. Compared with $-$295GUS, which gave the highest GUS activity among all the deletions, $-$1391GUS shows relatively low expression. This could be indicative of an upstream silencer element in the Glb1 promoter between $-$1391 and $-$805. Further studies indicate that the Glb1-driven GUS activity of bombarded maize P3377 cells depends on the levels of exogenous ABA. When the cells were exposed to 0 to 50 $\mu$M ABA, the Glb1 gene expression increases with the increased ABA level, but the ABA requirement can be saturated when 100 to 300 $\mu$M ABA is applied. To examine the functionality of a putative ABA response element (Em1a), site-directed mutagenesis of the Em1a sequence was performed by a "two step" PCR. The mutated plasmids ABm1 and ABm2 were transformed into P3377 cells, and GUS activity was undetectable after bombardment with the mutated plasmids. This result indicated that the mutations of Em1a element abolished the ABA responsiveness of the Glb1promoter to exogenous ABA, and that the Em1a sequence in Glb1 5$\sp\prime$ regulatory region is responsible for the positive ABA regulation of the gene expression.
Issue Date:1994
Rights Information:Copyright 1994 Liu, Siqing
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9503258
OCLC Identifier:(UMI)AAI9503258

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