A Functional Analysis of microRNAs in Nicotiana tabacum

MicroRNAs (miRNAs) are a newly discovered class of endogenous post-transcriptional gene regulators that are typically 20-22 nucleotides in length that do not code for proteins. MiRNAs regulate gene expression by either inhibiting protein translation, or by targeting messenger RNAs (mRNAs) for cleavage. MiRNAs are highly evolutionarily conserved and have been found in many plants such as corn, soybeans, rice, and Arabidopsis. Nicotiana tabacum, or cultivated tobacco, is an important economic, agricultural, and research crop that provided approximately $4.4 billion dollars to the United States economy in 2007. Although much genetic research has already been dedicated to tobacco, hardly any research regarding the role of miRNAs has been performed. In this project, the primary objective was to provide a functional analysis of miRNAs in Nicotiana tabacum. First, the expression profiles of miRNAs and their targets were generated for different organs through the use of quantitative real time PCR (qRT-PCR). It was found that all the miRNAs and targets that were tested were differentially expressed throughout different tissue types in tobacco. In particular, miR159 was found to be expressed the highest in all tissue types tested. Secondly, very few reference gene analyses have been performed in tobacco, therefore a reference gene analysis in tobacco was conducted. In this analysis, 12 housekeeping genes were tested for their effectiveness in serving as reference genes for gene expression analyses in tobacco. QRT-PCR was used to quantify the amount of expression of each candidate reference gene, and a new comprehensive reference gene analysis tool, RefFinder, was used to rank the candidate reference genes based on the stability of their expression. The housekeeping genes that show the least amount of variability in expression were deemed appropriate reference genes for use. Through the use of RefFinder, GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and PP2A (protein phosphatase 2A) were identified as being the most relaible reference genes for use in tobacco gene expression studies. Finally, microRNAs have been identified that mediate stress responses in plants to abiotic factors, but there have been few studies conducted on the effects nanoparticles may have on tobacco growth, development, and microRNA expression. To study this, tobacco seedlings were exposed to varying concentrations of aluminum oxide nanoparticles and tested to see which miRNAs were significantly changed in expression. It was found that the growth and development of tobacco seedlings was significantly adversely affected by increasing concentrations of aluminum oxide nanoparticles. In addition, several miRNAs were identified that may play a significant role in mediating plant responses to nanoparticles stress due to the magnitude of up regulation in expression. Overall, by providing a functional analysis of miRNAs in tobacco, these results will further help scientists to understand how plants react to their environment and will allow the further use of miRNA-mediated biotechnology to further improve crop yield and quality.M.S