The use of antisense RNA to inhibit gene expression in plant crown gall tumors
Abstract
Traditional approaches for studying gene function are not always applicable to multicellular eukaryotes. This is especially true for plants where there is currently no reliable method for gene replacement by homologous recombination. Inhibition of gene expression by artificial antisense RNA genes is one possible alternative for the study of gene function. RNA-RNA interactions are prevalent in eukaryotic cellular functions and there are many examples of natural antisense RNA regulatory mechanisms in prokaryotes. Antisense RNA has been used to explore gene functions in a variety of animal and fungal systems. This research was aimed at extending the use of antisense RNA to plant systems and to explore its usefulness in studying genes involved in complex developmental mechanisms. The tms1 and tms2 genes from the T-DNA of Agrobacterium tumefaciens were chosen as the target genes for these experiments. The expression of the tms1 and tms2 genes in transformed plant cells, and the elevated synthesis of IAA that is catalyzed by their gene products, contributes to the abnormal proliferation of plant cells in crown gall tumors. Antisense RNA genes were constructed containing various fragments of the tms1 or tms2 genes, and were introduced into plant cells along with the tumor-inducing T-DNA. Experiments using Kalanchoe diagremontiana as the host plant for A. tumefaciens infections revealed that antisense RNAs complementary to the tms2 transcript were effective in reducing the expression of the tms2 gene to the extent that a change in tumor phenotype resulted. Antisense RNAs complementary to the tms1 transcript had no effect on tms1 gene expression as measured by tumor phenotype. In contrast, experiments using Nicotiana tabacum as a host for infection showed that none of the antisense RNA genes were effective in reducing the expression of the target tms genes to the extent that it induced a change in tumor phenotype.
Degree
Ph.D.
Advisors
Gelvin, Purdue University.
Subject Area
Molecular biology
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