Event Title

Ancestral Sequence Reconstruction of Rubisco Activase from Zea Maize to Improve Thermal Stability

Session Number

Project ID: CHEM 01

Advisor(s)

Dr. Angela Ahrendt, Illinois Mathematics and Science

Dr. Sarah Stainbrook, Washington University in St Louis

Discipline

Arts & Humanities

Start Date

20-4-2022 8:50 AM

End Date

20-4-2022 9:05 AM

Abstract

Rising global temperatures due to climate change threaten the productivity of key agricultural staple crops such as maize corn, thus having negative implications for food security and production. Increased heat stress impairs plant function by decreasing the efficiency of rubisco activase (RCA), a critical enzyme responsible for net photosynthesis. To address this issue, we performed Ancestral Sequence Reconstruction (ASR) on the RCA gene sequence from Zea maize to form a mutated sequence with improved thermostability. ASR is a technique which uses software to generate a most likely ancestor to a protein sequence, which is often more stable than the wild type. Through ASR, it is possible to enhance the thermostability, promiscuity, and activity of proteins. Several rounds of ASR were performed with different combinations of Archaeplastida species, including those genetically furthest and closest from maize on the phylogenetic tree. From this, two generated sequences were synthetically constructed by a commercial lab. The sequences were cloned into an expression vector, and the protein variants will be expressed, purified, and analyzed for improved heat tolerance compared to the maize wild-type RCA.

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Apr 20th, 8:50 AM Apr 20th, 9:05 AM

Ancestral Sequence Reconstruction of Rubisco Activase from Zea Maize to Improve Thermal Stability

Rising global temperatures due to climate change threaten the productivity of key agricultural staple crops such as maize corn, thus having negative implications for food security and production. Increased heat stress impairs plant function by decreasing the efficiency of rubisco activase (RCA), a critical enzyme responsible for net photosynthesis. To address this issue, we performed Ancestral Sequence Reconstruction (ASR) on the RCA gene sequence from Zea maize to form a mutated sequence with improved thermostability. ASR is a technique which uses software to generate a most likely ancestor to a protein sequence, which is often more stable than the wild type. Through ASR, it is possible to enhance the thermostability, promiscuity, and activity of proteins. Several rounds of ASR were performed with different combinations of Archaeplastida species, including those genetically furthest and closest from maize on the phylogenetic tree. From this, two generated sequences were synthetically constructed by a commercial lab. The sequences were cloned into an expression vector, and the protein variants will be expressed, purified, and analyzed for improved heat tolerance compared to the maize wild-type RCA.