Searching for potential binding partners of arabidopsis β-amylase9 using yeast 2-hybridization

Author

Sheikh Hossain, James Madison UniversityFollow

Creative Commons License

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Date of Graduation

Spring 2018

Document Type

Thesis

Degree Name

Bachelor of Science (BS)

Department

Department of Biology

Advisor(s)

Jonathan D. Monroe

Kimberly H. Slekar

Linette Watkins

Abstract

In plants, starch is a major carbon and energy storage compound. Starch is made as a product of photosynthesis while plants are in light and is degraded at night. Our lab is interested in the cellular mechanism of starch degradation in plants and for our studies we use Arabidopsis thaliana as a model. β-amylases are primarily responsible for the hydrolysis of starch in plants and a total of nine β-amylases genes are encoded in Arabidopsis thaliana. These nine genes are identified as BAM1-9. BAM9 is located in the chloroplast where starch is located, and is present is all flowering plants but it is not catalytically active. It was published that BAM9 was very strongly expressed in the transition between nighttime and daytime. BAM9 may have an altered activity as a regulatory protein. Since regulatory proteins function by interacting with other proteins, we used the yeast two-hybrid system to attempt to identify interacting protein(s). As a first step, yeast cells were transformed with a BAM9 bait plasmid. Next, a cDNA library screen was performed where yeast cells with the bait were transformed with a library that encodes for potentially interacting proteins. The expression of reporter genes was observed. Next, sequencing and bioinformatics analysis were used to identify the potentially interacting proteins(s) Twenty one unique hits were detected from both screens. Lastly false positives were then eliminated by a series of control experiments. Partial but overlapping sequences of AMY3 were identified four times in the bioinformatics analysis and a conserved coil region of AMY3 was identified in each of the prey plasmids. Obtaining more comprehensive information about protein-to-protein interactions will aid our understanding of BAM9’s function.

Recommended Citation

Hossain, Sheikh, "Searching for potential binding partners of arabidopsis β-amylase9 using yeast 2-hybridization" (2018). Senior Honors Projects, 2010-2019. 568.
https://commons.lib.jmu.edu/honors201019/568