Study of auxin responsive gene promoters in transient expression assays

Abstract

Transfection assays are an important tool for biochemists; the comparatively quick technique allows experimenters to see relative levels of expression of a particular gene in the presence or absence of a plant hormone and/or effector. Transfection assays can be used to study the promoter region of a gene to determine the importance of specific cis-regulatory elements in the control of gene expression. This is accomplished by fusing the promoter region with a reporter gene. A reporter gene allows the expression level of the promoter to be monitored by fluorescence or by other observable properties. Transfection can also be used to study growth substances and various cellular signaling pathways. This technique involves isolating protoplasts from cells such as carrot suspension cells. In order to use these suspension cells, the cell walls must be digested; thus the cells become protoplasts. The experimental DNA (effector and reporter genes) is then introduced into protoplasts by chemical methods. The protoplasts are incubated in the dark and the relative reporter gene activity is then read on a fluorometer. The goal of this project is to determine if two Aux/IAA genes are auxin inducible and what influence specific effectors have on their expression using carrot protoplasts to do transient transfection assays. We study auxin responsive gene promoters because many of them contain auxin responsive cis-regulatory elements designated as Auxin Response Elements (AuxREs), which contain the sequence TGTCTC. These promoters are regulated by at least two groups of transcription factors known as Auxin Response Factors (ARFs) and Aux/IAA proteins. One group of auxin inducible genes is Aux/IAA genes. We studied the Aux/IAA17 and Aux/IAA19 promoter regions by amplifying them through PCR and cloning them upstream of the ß-glucuronidase (GUS) reporter gene open reading frame (ORF). This reporter gene was used because the GUS activity can be detected through fluorescence. The GUS gene was terminated by a nopaline synthase (NOS) terminator cloned downstream of the GUS gene. The effectors used in these studies consisted of ARF proteins translated from ARF effector genes during the transfection process. Results showed that the Aux/IAA19 gene (which has 3 cis-regulatory AuxREs) was inducible by auxin. The ARF effectors influenced the expression of both Aux/IAA genes by positively or negatively regulating their expression. However, auxin was found to have no affect on Aux/IAA17 gene expression. This correlates with the fact that Aux/IAA17 has no AuxREs in its promoter region.