PI: Burkhard Schulz (Purdue University) CoPI: Joseph Irudayaraj (Purdue University)
This project develops and tests a novel method for the detection and quantification of alternatively spliced genes in plants. Most genes in higher organisms are structured into exons and introns, the latter interrupting the protein coding sequence of the genes. Yet these introns are not always precisely spliced and removed from a precursor RNA during transcription. This results in different mRNA variants from the same gene. It greatly increases the variability and number of expressed proteins and explains why there are thousands more proteins than genes in a cell. It is becoming clear that this process of alternative splicing (AS) plays an important role in regulating developmental processes in plants. A precise quantification of different splice variants, also on the cellular level, is necessary in order to gain a deeper understanding of the role of this process. This project is an innovative approach to the detection and quantification of several AS generated mRNA variants in a single experiment. It is a highly sensitive method for detecting mRNA variants. This project combines nanotechnology based Surface Enhanced Raman Scattering (SERS) and the use of novel nanomaterials. SERS uses gold nanoparticles to enhance the Raman scattering signal of a laser beam by molecules (DNAs, proteins, etc) within nanometer distance of these signal-enhancing gold nanoparticles. The objectives of the project are: (1) to develop a nanotechnology toolbox for detection of mRNA splicing variants in plants; (2) to assess detection limits for quantification of AS; and, (3) to develop a SERS-based quantification strategy for the detection of splicing variants in small cell numbers on a sub-organ level.
This interdisciplinary project is a close collaboration between plant geneticists and biophysicists to quantitatively profile mRNA variants in plant cells. It will generate a foundation for the application of nanotechnology approaches in the broader field of plant sciences with impacts beyond basic research. The project will create new opportunities for students in engineering as well as plant sciences that will expand their educational portfolio in fields not part of their traditional curricula, opening new avenues for their career development. Outreach activities to the public and during research summer camps for middle school and high school students are projected. A reference website that publishes detailed protocols and additional background information will be established with access through www.hort.purdue.edu/hort/people/faculty/schulz.shtml. It will provide information on resources, newly developed nanomaterials and educational material to train students and postdoctoral researchers in nanotechnology. Protocols and procedures to analyze AS in plants using nanotechnology will also be made available to the scientific community through publications in appropriate journals and communications at scientific conferences.
