A study on optimization of pat gene expression cassette for maize transformation

Area of Science:

• Plant molecular biology
• Genetic engineering
• Crop science

Background:

• The phosphinothricin acetyltransferase (pat) gene is crucial for herbicide resistance and as a selectable marker in plant transformation.
• Maize transformation frequency using pat is notably lower than other markers like epsps, pmi, or ppo, hindering efficient event production.

Purpose of the Study:

• To identify and characterize a new variant of the pat gene for improved transformation efficiency in maize.
• To address the bottleneck of low transformation frequency associated with the pat gene in maize genetic engineering.

Main Methods:

• Codon optimization of the pat gene for expression in monocot plants (maize).
• Construction and testing of 16 different pat gene variants.
• Evaluation of transformation frequency and PAT protein levels.
• Assessment of herbicide tolerance in transgenic maize plants.

Main Results:

• A new codon-optimized pat gene variant demonstrated over a 2x higher transformation frequency compared to an older version optimized for dicots.
• PAT protein levels were significantly elevated, up to 40-fold higher than controls.
• Generated T0 transgenic maize plants exhibited excellent tolerance to ammonium glufosinate herbicide at high application rates (4x and 8x).

Conclusions:

• Codon optimization of the pat gene is an effective strategy to enhance maize transformation efficiency.
• The developed pat variant provides a more robust selectable marker and herbicide trait gene for maize genetic improvement.
• This advancement facilitates the efficient generation of transgenic events, particularly for complex genetic constructs.