Browsing by Subject "Luciferase"
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Item Identification of regulators of heat shock-inducible gene in Arabidopsis(2012-12) Ruan, Qing Xia; Shi, Huazhong; Xie, Zhixin; Paul, PareBy using forward genetic screening, we identified an Arabidopsis mutant LL729 showing reduced expression of the luciferase reporter gene driven by the heat stress inducible promoter of AtHsp18.2 gene. Extensive phenotyping of the LL729 mutant revealed that LL729 is resistant to the toxic non-selected herbicide methyl viologen. Through positional cloning, we found that the mutation responsible for the LL729’s luciferase imaging and methyl viologen resistant phenotypes is localized in At5g05630 encoding a polyamine uptake transporter named PUT3. We deduce that the mutation resulting in a change of ser252 to asn252 in the PUT3 reduced the transport of methyl viologen as well as polyamines into Arabidopsis.Item The role of HDA6 and FRY2 in gene regulation and abiotic stress response in arabidopsis(2011-05) Wang, Hui; Shi, Huazhong; Pare, Paul; Xie, ZhixinGene regulation is a central process in plant response to environmental stresses. Many genes are either up- or down-regulated under stress conditions, which are mediated through signaling pathways starting from perceiving the stress signals. To identify components important for abiotic stress signaling response, a forward genetic approach was employed by using the luciferase reporter gene fused with the SOT12 promoter, an abiotic stress responsive promoter from Arabidopsis. The seeds of the homozygous transgenic line were subjected to EMS mutagenesis and mutants with altered luciferases expression were identified. In this study, two mutants, named 1502 and 1005, were selected for further characterization. Map-based cloning revealed that 1502 mutant harbors a mutation in the gene encoding the Histone Deacetylase 6 (HDA6) and 1005 mutant possesses a mutation in the gene encoding the previously identified protein FRY2. Genetic complementation verified that these mutations are indeed responsible for the mutant phenotypes. Thus, 1502 mutant was renamed to hda6 and 1005 mutant was renamed to fry2. hda6 mutant showed no obvious phenotypes under NaCl or several hormonal treatments. Interestingly, growth of hda6 mutant plants under normal conditions displayed segregated growth phenotypes with no stable segregation ratio, which suggests an epigenetic effect of the hda6 mutation on normal plant growth and development. hda6 mutant exhibited substantially higher luciferase expression than wild type plants with or without NaCl treatment. The increased luciferase expression was attributed to the increased transcript level in hda6 mutant plants. Analysis of 5’capping and polyadenylation site selection of the luciferase mRNA suggested that HDA6 is involved in the regulation of these co-transcription processes, which may partly contribute to the increased transcript level of luciferase gene in hda6 mutant. Yeast two-hybrid screening for HDA6 and FRY2 interacting proteins was attempted and a number of putative interacting proteins were identified. However, protein-protein interaction needs to be further verified in both yeast and plant cells.