Modulation of plant gene expression using antisense technology and gene amplification: Alteration of shikimate pathway gene expression
Abstract
The effect of genetic alterations on carbon flow through the shikimate pathway, using gene amplification or antisense RNA expression, was examined. Transgenic potato plants containing a chimeric 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) antisense gene were produced to examine the role(s) of DAHPS in plant development. Wound-induced DAHPS gene expression (enzyme activity, protein, and mRNA) was reduced in wounded tuber tissue of three independent transgenic plants expressing DAHPS antisense RNA. DAHPS mRNA levels were reduced in shoot tips and/or stem tissue of five antisense plants. These plants had reduced stem diameter and/or stem lignification compared to untransformed plants. These alterations may in part be due to reduced carbon flow through the shikimate pathway. Gene amplification allows an organism to increase the expression of a gene(s), particularly in response to environmental stress. The stability and expression of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) genes was examined in glyphosate resistant tobacco cells and regenerated plantlets. Resistant cells grown without glyphosate for over 3 years stably maintained high levels of EPSPS gene amplification; however, resistance to glyphosate declined 7-fold compared to cells continously grown on glyphosate. Similarly, plantlets regenerated from resistant cells retained levels of EPSPS gene amplification comparable to that in resistant cells, but resistance to glyphosate was greatly reduced. EPSPS mRNA levels in plantlets were reduced relative to those in resistant cells. Regenerated plantlets were altered morphologically; this was correlated with an increased genome size. The potential of utilizing gene amplification to increase the expression of nonselectable genes was examined. A transgenic petunia cell culture containing neomycin phosphotransferase II (npt II) and $\beta$-glucuronidase (GUS) genes was used in selection experiments to obtain kanamycin resistant cell lines. Resistance was correlated with npt II gene amplification and increased npt II mRNA levels. There was also a concurrent co-amplification of the linked GUS gene, resulting in increased GUS mRNA levels and enzyme activity. Amplified genes were stably maintained and GUS enzyme activity stably overexpressed in the absence of selective pressure. Amplification was found to occur within eleven growth cycles. In one cell line, gene amplification was accompanied by DNA rearrangement.
Degree
Ph.D.
Advisors
Weller, Purdue University.
Subject Area
Botany|Molecular biology|Biology
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