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

ORCID

https://orcid.org/0000-0002-0261-3762

Date of Graduation

7-13-2022

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

Advisor(s)

Dr. Jonathan Monroe

Abstract

A noncanonical β-amylase7 (BAM7) gene found in some basal angiosperms and some monocots, including corn, appears to encode two functionally distinct proteins. Phylogenetic evidence suggests that BAM7 originated from a fusion event that combined a BZR1-like DNA-binding domain to the 5’ end of BAM2 about 400 million years ago (mya). In Arabidopsis thaliana (At), AtBAM7 is a catalytically-inactive transcription factor found in the nucleus and AtBAM2 is a catalytically-active, tetrameric starch hydrolase found in the chloroplast. These proteins are encoded on different chromosomes. The corn (Zea mays, Zm) genome does not contain a BAM2 gene, however its ZmBAM7 gene shares all the conserved residues necessary for catalysis in BAMs, some of which are lacking in AtBAM7. Furthermore, we found an in-frame methionine in the first intron of ZmBAM7 that begins a putative chloroplast transit peptide. We hypothesized that the ZmBAM7 gene contains two transcriptional starts sites about 1000 bp apart, the first for a longer BAM7 protein (ZmBAM7-L) and the second for a shorter BAM2-like protein (ZmBAM7-S). Using 5’ RACE, we found that corn tissues contain two populations of transcripts consistent with the predicted ZmBAM7-L and ZmBAM7-S. Intron-spanning RNA seq data from NCBI further supported the hypothesis. We fused the coding sequences for the two proteins to GFP and transformed Arabidopsis plants to determine their subcellular locations. GFP fused to ZmBAM7-S is targeted to chloroplasts in transgenic Arabidopsis plants, whereas GFP fused to the DNA-binding domain of ZmBAM-L is targeted to nuclei, confirming the function of the predicted localization signals. Moreover, previous work in our lab demonstrated that ZmBAM7-S encodes a tetrameric protein with catalytic properties like AtBAM2. Since its emergence 400 mya the “dual-function” BAM7 has been retained in at least 15 species including basal angiosperms, some monocots, and basal eudicots. In four other lineages including ferns, gymnosperms, some monocots, and most eudicots the dual-function gene appear to have become duplicated and subfunctionalized leading to two distinct genes, each retaining only one of the original functions, such as in Arabidopsis. It is possible that other genomes contain genes like ZmBAM7 that contain cryptic transcriptional start sites encoding currently unannotated proteins.

Available for download on Thursday, July 11, 2024

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