Testing a CRISPR/Cas9 Construct Against Candidate B3 Gene Involved in NFS in Medicago Truncatula

Presenter: Emily Naughton

Group Members: Maitri Gandhi

Faculty Sponsor: Dong Wang

School: UMass Amherst

Research Area: Genetics

Session: Poster Session 1, 10:30 AM - 11:15 AM, 163, C31

ABSTRACT

In an effort to utilize the abundant nitrogen in Earth’s atmosphere, legume plants will often form symbiotic relationships with bacteria in a process known as nitrogen-fixing endosymbiosis (NFS). Through the formation of nodules on the plant roots, NFS allows bacteria to convert atmospheric nitrogen into ammonia for plant use and provides carbon and other forms of energy for bacterial growth. Despite over a century’s worth of ongoing research on NFS, the molecular mechanisms underlying this complex process are still not fully understood, especially the specific gene expressions that allow bacteria to enter the root nodules. Using previous research identifying B3 as an evolutionarily conserved gene, we hypothesize that B3 in the model plant species Medicago truncatula (Mt) is essential for bacterial internalization into root nodules for NFS. To test this, we have successfully designed and cloned a double gRNA CRISPR-Cas9 construct targeting B3 in Mt. We are currently working towards analyzing the knockout potential of this construct and ultimately using a LacZ reporter assay to visualize any disruptions to bacterial entry into root nodules due to the B3 knockout. As there are increased agricultural demands for human consumption, the need for synthetic fertilizers derived from fossil fuels has also increased. In order to reduce the negative environmental impact of increased fossil fuel usage, it is important to better understand the genes essential for NFS. This will allow scientists to genetically engineer the NFS system into non-legume plants, leading to more sustainable agricultural practices.

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