ARS | Publication request: Steps toward discovering the function and expression of multiple drug resistance genes in "Arabidopsis thaliana"

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: October 15, 2004
Publication Date: November 10, 2004
Citation: Mooers, M., Cheng, N-H., Hirschi, K. 2004. Steps toward discovering the function and expression of multiple drug resistance genes in "Arabidopsis thaliana" [abstract]. Proceedings in the Annual Biomedical Research Conference for Minority Students, November 10-13, 2004, Dallas, Texas. G107, p. 435.

Technical Abstract: The ATP-binding cassette (ABC) superfamily is the largest protein family identified in all organisms. It is a highly conserved domain responsible for the ATP-dependent transport of substances including ions, carbohydrates, xenobiotics, drugs, and peptides. Also, the subfamily, multidrug resistance (MDR) genes, specifically "mdr1" in human cancer cells, are able to significantly reduce the effectiveness of chemotherapy drugs. Studying these genes and their physiological functions in other organisms, such as the model plant "Arabidopsis thaliana", can expand the knowledge for possible enhancement in the nutritional and medical value of plants. As a start toward the discovery of the specific functions of multidrug-resistant genes, three genes from the 20 MDR-like genes in Arabidopsis were chosen. Each of these genes, "AtMDR4, AtMDR10, AtMDR20", are located on different branches of the phylogenic tree. Therefore, we hypothesize that each of the chosen genes will have a different expression and function. As an initial approach to finding each gene's function, RT-PCR was used to analyze the gene expression in different tissues and after exposure to various metal treatments. The results of the RT-PCR for the "AtMDR4" gene showed high-expression in the root and stem and very low expression in the flower. For "AtMDR20", the leaf, stem, and flower tissues displayed a very high expression. The "AtMDR10" gene, however, was only expressed in the stem. In the metal-treated plants (water, calcium, sodium, nickel, manganese, and MS), "AtMDR4" showed high expression in sodium, nickel, and manganese, and a decreased expression in MS salts. The "AtMDR20" gene had a slightly increased expression in calcium, manganese, and nickel. Since only young plants with little stem were used for this RT-PCR, it is logical that the AtMDR10 did not show expression in any treatment. Another method used to find function and expression of a gene is to observe the phenotype of T-DNA insertion mutants. For that reason, the second half of this project was to isolate homozygote T-DNA insertion mutants for the Arabidopsis ABC transporters "AtMDR4, AtMDR10, AtMDR20". Using a PCR-based approach to screen a T-DNA insertion population of seedlings, heterozygote and some homozygote plants were identified. For the "AtMDR4" gene, only three heterozygote lines were identified and isolated. There was one heterozygote and four homozygote lines identified and isolated for the "AtMDR10" gene. Two heterozygote and nine homozygote lines were isolated with the "AtMDR20" T-DNA insertion. Currently, the PCR-based screening process is continuing in search of more homozygote T-DNA insertion mutants. These homozygote mutants will be examined for phenotypic abnormalities as well as being analyzed on a biological, biochemical, and genetic level. Once there is conclusive data on the expression of these genes, advances toward the physiological gene function can take place.