Transformation of Arabidopsis thaliana with heat stress-related genes from a Copaifera officinalis expressed sequence tag library

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Zwenger, Samuel R.
Scholarship & Creative Works @ Digital UNC, University of Northern Colorado
Biology, Agrobacterium, arabidopsis, binary library, copaifera officinalis, plant transformation, transgene, Biology; Agrobacterium; arabidopsis; binary library; copaifera officinalis; plant transformation; transgene
thesis / dissertation description
This dissertation has three components: (1) discovery of genes involved in heat stress-related processes derived from a tropical plant known as the diesel tree (C. officinalis) by means of an expressed sequence tag (EST) library, (2) stable transformation of Arabidopsis thaliana with C. officinalis heat stress-related genes and (3) assessing the degree to which C. officinalis heat-stress associated genes provide thermotolerance in transgenic A. thaliana. This dissertation project has important plant biotechnological components as well as agriculturally important themes. Global climate change is predicted to result in elevated temperatures over the next century and so studying the influence of heat stress upon plants is becoming increasingly important. Subsequently, in this work, it was hypothesized that heat stress-related transgenes derived from C. officinalis would provide some level of thermotolerance to A. thaliana. Various stress assays, which included both in vitro and in vivo assays, were performed to help determine the degree of thermotolerance in each transgenic line. A novel and key component in this project is that for the EST library C. officinalis cDNA was ligated into a plant binary vector, which eliminated the need to perform individual ligation reactions for each gene of interest (GOI). Sequencing plates were obtained and stored at -80C so that individual wells containing the E. coli harboring the binary vector could be picked and grown overnight for a subsequent plasmid miniprep. Plasmids were transfered into Agrobacterium tumefaciens for generation transgenic lines. This created a streamlined and highthroughput method of generating multiple transgenic plants, each with a unique GOI. Although others have overexpressed heat stress-related genes in A. thaliana, this project overexpressed C. officinialis plant genes, which is novel. The results suggest that overexpression of C. officinalis heat stress-related genes in A. thaliana help confer thermotolerance. Furthermore, the library clones obtained can be investigated in future studies, which offers a rich resource for transgenic studies.

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