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Root Genes Related to KOIN In Arabidopsis Thaliana

Ella Fukazawa

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Background

  • Plant Roots
    • Absorption and Anchorage
    • Investigating genes’ functions and localization in the root
  • Arabidopsis Thaliana:
    • A small weed
    • Quick generation time
    • Efficient in a lab setting
  • Wallflower and Kinase on the Inside (KOIN)
    • Polar localized kinases
  • Double Mutants
    • Genes that are closely related
    • Have similar functions (redundant)

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(Kumar, 2014)

(Planck, 2014)

(Ryan et al., 2016), (Van Norman, 2021)

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Experimental Design

  • Steps
    • Gene Structure
    • Primers designed via BLAST
    • Homologs found (phylogenetic tree)
    • Guide RNAs designed (E-CRISP)
  • Target gene: KOIN/PLK4
    • AT5G58300
  • Guides designed in LRR and Kinase Domains
  • Target vs. Off-Target

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(Bioinformatics, 2021)

(Van Norman, 2021)

(TAIR, 2021 https://www.arabidopsis.org/)

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Gene Structure and Primers

  • KOIN is 3250 bp in length
    • Coding sequence is 1965 bp in length
  • Primer Pair 5
    • First Exon
  • Primer Pair 9
    • Second Exon

(TAIR, 2021 https://www.arabidopsis.org/)

(NCBI, 2021)

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Guide RNAs

LRR Domain

On Target:

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Off Target: N/A

Kinase Domain

On Target:

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Off Target:

Designed to knock out AT5G05160

Designed to knock out AT3G08680

(E-CRISP, 2021)

(E-CRISP, 2021)

(CRISPR-P, 2021)

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Primers are in green

Dark blue is LLR domain

Orange is kinase domain

Light blue is the on-target RNA sequences

Magenta is the off target RNA sequences

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Summary

  • Mutations could cause nonpolar localization
    • Meristems grow
      • More cell division
    • Change in function
      • Does not repress cell division

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(Van Norman, 2021)