Structure of the amplified 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate-resistant carrot cells and attempts to isolate the anthranilate synthase gene from plants
Suh, Hyang
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Permalink
https://hdl.handle.net/2142/22417
Description
Title
Structure of the amplified 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate-resistant carrot cells and attempts to isolate the anthranilate synthase gene from plants
Author(s)
Suh, Hyang
Issue Date
1992
Doctoral Committee Chair(s)
Widholm, Jack M.
Department of Study
Biology
Discipline
Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Chemistry, Biochemistry
Biology, Plant Physiology
Language
eng
Abstract
Analysis of the structure of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) DNA of carrot suspension cultured cell lines selected for glyphosate resistance was carried out to determine the mechanism of gene amplification in this plant system. Cloning of the EPSPS gene and 5$\sp\prime$ flanking sequences was carried out and two different DNA structures were revealed. The 13 kbp clone contained only one copy of the EPSPS gene while the 16 kbp clone contained an inverted duplication of the gene. Southern blot analysis with cloned carrot DNA as a probe showed that only the non-inverted repeated DNA structure was present in all of the cell lines during the selection process and the inverted repeat (IR) was present only in highly amplified DNA. The two structures were present in about equal numbers in the highly amplified line, TC 35G. The presence of the IR was further verified by resistance to Sl nuclease hydrolysis after denaturation and rapid renaturation, showing foldback DNA. This is the first report of an IR in amplified DNA of a target enzyme gene in selected plant cells.
In attempts to isolate the anthranilate synthase (AS) gene from plants, antibodies were prepared from the purified carrot protein and purified bacterial AS to be used to screen a gene expression library. Carrot AS activity in native PAGE was localized and the region of enzyme activity was used as an antigen to raise antibodies in a rabbit. That antibody, denoted anti-Gel II, and an antibody against AS from Serratia marcescens did not inhibit AS activity of carrot. Anti-Gel II reacted to show two major bands of 65 kd and 67.5 kd in western blots of carrot, tobacco and potato, which could be good candidates to be the large subunit of AS as known by subunit size studies with microorganisms.
Bacterial and fungal AS genes were used to probe Southern blots of genomic DNA of many plant species to see if the heterologous probe can be used to screen the genomic DNA library. The approach using heterologous probes was not adequate for the isolation of the AS gene of plants due to the low homology of amino acids and nucleic acids sequences of the AS gene.
Attempts were made to isolate the Blue fluorescent gene (Bf1) which could be the AS gene by transposon tagging.The cosegregation of the restriction fragment with the mutant phenotype was followed with the Orf1 fragment of the En element as probe. We could not observe any bands cosegregating with the mutable allele with one parental line and two mutable lines using many methylation sensitive enzymes. Thus we could not clone the gene so this project has been discontinued.
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