Thesis

Functional Characterization of Germacrene Synthase Gene in Arabidopsis thaliana and Helianthus annuus Under Heat-Stress

Elevated carbon dioxide levels caused by greenhouse gasses result in rising temperatures that have a detrimental impact on crop production, which cost farmers billions of dollars and loss in crop productivity. In response to abiotic stress, plants emit terpenes as a response to protect themselves from harsh environmental conditions. Germacrene synthase is an enzyme involved in the production of sesquiterpenes specifically, germacrene. The sesquiterpene gene, germacrene synthase provides thermotolerance in plants and are differentially expressed in plants during heat stress. Helianthus annuus plants were placed in a chamber at 42°C for 24 hours. qPCR was utilized to visualize the expression of the gene, germacrene synthase after heat stress. GC-MS was also utilized to analyze the expression of terpenes under heat stress conditions. Arabidopsis thaliana plants were genetically modified with a germacrene synthase gene by the Agrobacterium tumefaciens mediated floral dip method with the GV101 strain of Agrobacterium tumefaciens containing the recombinant pBINmgfp5-er vector. Approximately three weeks after the floral dip was conducted, seeds were collected and plated on MSO media with the antibiotic, kanamycin to select for transformants. All transgenic plants had NPTII gene, which indicated kanamycin resistance. After 14 days, surviving plants on kanamycin plates were selected and planted on soil. Approximately 4 weeks after maturation, DNA from the transformed plants were extracted, and PCR was performed using NPTII primers to confirm transgenic plants. For heat tolerance assay, the three transgenic Arabidopsis thaliana plants and an untreated wild-type plant were placed in a chamber at 42°C for 72 hours. After 72 hours, no significant signs of thermotolerance were observed in the transgenic plants. Further studies need to be done to prove that the gene, germacrene synthase provides thermotolerance in plants.

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