The next challenge in plant biology is to discern the function of the many genes revealed through the various genome sequencing projects. One important approach for understanding gene function is to study the consequence of removing or knocking out the function of a specific gene. In the model plant Arabidopsis, efficient methods are available to knock out many genes of interest. Functional analysis, however, is often frustrated by genetic redundancy or the presence of multiple copies of the same or closely related genes. Particularly prevalent in plants are tandem gene duplications, and as many as 15% of Arabidopsis genes are organized in tandem arrays. A targeted mutagenesis protocol will be developed to augment existing approaches for understanding Arabidopsis gene function, particularly for genes for which functional analysis has been confounded by genetic redundancy. The approach uses zinc finger nucleases (ZFNs) - chimeric proteins made up of a zinc finger array fused to the DNA cleavage domain of Fokl endonuclease. Zinc finger arrays of high affinity and specificity can be engineered to recognize virtually any target gene. Upon cleavage of the target DNA by the ZFN, the broken ends are repaired inefficiently, resulting in locus-specific mutations. In initial experiments, a model zinc finger nuclease and a reporter gene containing the cognate target site will be used to optimize methods for efficient recovery of heritable ZFN-induced mutations. Next, ZFNs will be engineered to recognized native Arabidopsis loci, including those that give observable phenotypes when mutated. ZFNs will also be engineered for a locus where mutatations do not result in an observable phenotype. To recover mutations at such a locus, robust DNA amplification and sequence-based detection methods will be employed. Lastly, a tandem array of duplicate genes will be targeted. Deletion of the array will be accomplished by engineering ZFNs to cleave within the outermost genes. The frequency of recovering deletions and individual mutations will be determined to assess the relative efficiency of this approach. In addition to Arabidopsis, this mutagenesis approach will also be valuable for plant species such as rice and maize, where genome sequencing projects are ongoing and tandem genes are abundant. Moreover, the ability to generate and regulate specific chromosomal double-strand breaks will facilitate studies of chromosome structure and DNA repair mechanisms in plants. The URL for the web site where protocols as well as results of gene knock-out studies can be accessed is www.beckmancenter.umn.edu/html/2010.html.
Understanding the function of plant genes is critical if plants are to be fully harnessed to meet the world's burgeoning need for fuel, feed and industrial raw materials. Although significant progress has been made in discerning plant gene function, existing approaches have inherent limitations, particularly with respect to ascribing function to redundant or duplicated genes. The goal of this project is to implement an efficient targeted mutagenesis approach to overcome these limitations. This project will train undergraduate interns and graduate students for work in plant molecular biology. Students from underrepresented groups will be recruited to the project through programs designed to enhance minority participation in science.
In the coming decades, considerable demand will be placed on plant agriculture to provide the food, fiber and fuel needed to sustain an expanding world population. Coupled to this demand for increased agricultural productivity is the challenge of growing crops in an increasingly unpredictable climate and on marginal lands that previously have not been cultivated. In the past decade, scientists have determined the genetic blueprint of a variety of plants, including several important crop species. One goal of plant molecular genetics is to determine how this genetic blueprint dictates plant growth and development. Once these processes are better understood, this information can be used to create new crop varieties that will meet the burgeoning demands placed on modern agriculture. The specific goal of this research project sponsored by the National Science Foundation was to develop methods to make specific alterations in the plant genetic code. Such targeted alterations provide a powerful approach for determining how a plant’s genetic blueprint (or genome) gets deciphered. The focus of study was the model plant Arabidopsis thaliana – the first plant species for which the DNA sequence of the entire genome was determined. To create precise alterations in the genetic code of Arabidopsis, proteins called zinc finger nucleases were engineered that recognize specific DNA sequences in the Arabidopsis genome. The zinc finger nucleases make a break in the targeted Arabidopsis DNA, and this break gets repaired imprecisely, resulting in mutations that are incorporated at the break site. The mutations alter the way the plant grows and develop, and therefore they inform us of the role played by that particular segment of the genetic code in the life of the plant. Mutagenesis with zinc finger nucleases proved very effective, making it possible to make targeted modifications in many Arabidopsis genes. Since reporting these findings, others have used the method to make specific alterations in the genomes of a variety of species, including crops such as soybean and rice, suggesting the method has broad utility.
