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

This dissertation had three components: (a) identification of genes involved in heat stress-related processes derived from a tropical plant known as the diesel tree (Copaifera officinalis) by means of an expressed sequence tag (EST) library, (b) stable transformation of Arabidopsis thaliana with C. officinalis heat stress-related genes, and (c) assessing the degree to which C. officinalis heat-stress associated genes provided thermotolerance in transgenic A. thaliana. This dissertation had important plant biotechnological components as well as agriculturally important themes. Global climate change is predicted to result in elevated temperatures over the next century; thus, 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 was that the sequences from C. officinalis cDNA were 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 - 80°C so individual wells containing the E. coli harboring the binary vector could be picked and grown overnight for subsequent plasmid miniprep. Plasmids were transferred into Agrobacterium tumefaciens for generating transgenic A. thaliana lines. This created a streamlined 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 genes from the tropical plant C. officinalis, which is novel. The results suggested that overexpression of C. officinalis heat stress-related genes in A. thaliana helped confer thermotolerance. Furthermore, the sequenced E. coli clones harboring C. officinalis genes can be readily obtained and investigated in future studies, which offers a rich resource for transgenic studies.