This Small Business Technology Transfer (STTR) Phase II project seeks to identify new and improved promoters to create enhanced genetically modified crops. Plant biotechnology relies on the insertion of promoter-gene constructs into plants. The promoter is the portion of DNA that controls when and where a gene is expressed. The relatively few plant promoters in use today have significant limitations including inconsistent effects across different growing conditions and a lack of predictability. This project involves developing and implementing a novel pipeline for promoter discovery that starts with a sophisticated bioinformatics analysis to identify high confidence promoter candidates. Using fluorescent reporters and confocal imaging, these candidates are assessed in transgenic plants for cell-type-specific expression, developmental-stage-specific expression, and responsiveness to environmental stimuli. This pipeline was validated in the Phase I component of the project where four novel and patentable constitutive promoters were identified.
The broader impacts of this research are the development of superior genetically modified crops. Genetically modified plants already play an important role in world agricultural production and will play a central role in averting widespread food shortages in the future. In addition, substantial research is being conducted to improve bioenergy crops though genetic engineering. Genetically enhanced bioenergy crops are predicted to play a key role in reducing our dependence on fossil fuels and in cutting greenhouse gas emissions. A critical innovation that will facilitate advances in all of these areas will be the introduction of new and enhanced plant promoters.
Biotech crops play an important role in US agricultural production today, and will play a critical role in averting widespread food shortages in the future. Biotech crops are developed through the introduction of transgenes which confer beneficial traits such as insect resistance. To create the beneficial trait, the transgene must produce a functional protein through a process known as gene expression. For maximal benefit, it is often important for large amounts of the protein to be produced throughout the plant under a wide variety of field conditions. The goal of our NSF funded research project was to identify and develop new gene regulatory elements that promote a high level of transgene expression in crops under many different conditions. Several such elements were identified and characterized in depth during the course of this project. Our initial work used the dicot plant Arabidopsis because it is widely studied and easy to work with. Regulatory elements derived from Arabidopsis are expected to work in agriculturally important dicot crops like soybean and cotton. However, dicot regulatory elements generally to not work well in monocots like corn which is the most important crop in the US. Therefore, the latter stages of our research focused on developing similar tools to direct high levels of gene expression in monocots. For this purpose we chose to work with green millet which the scientific community considers a model monocot because it is easier to study than corn. To establish the groundwork necessary to achieve our research goals, methods for transferring genes into millet were optimized, and the natural genes that are normally active in various tissues throughout the growth cycle of this plant were cataloged. We then developed and applied computational methods to identify potential gene regulatory elements and demonstrated that several of these function to increase gene expression in millet. In summary, our NSF funded research has achieved the goal of generating a number of regulatory sequences that can be used to consistently provide a high level of transgene expression in both dicot and monocot crops. Additionally, a broader impact of this work is that the knowledge gained will be useful reference material for the growing community of researchers that work with green millet.
