A study on optimization of pat gene expression cassette for maize transformation
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Phosphinothricin acetyltransferase gene (pat) is an important selectable marker and also a key herbicide trait gene in several commercial products. In maize, the transformation frequency (TF) using pat as a selectable marker is the lowest among the commonly used marker options including epsps, pmi or ppo. Low pat transformation efficiency can become a major bottleneck in our ability to efficiently produce large numbers of events, especially for large molecular stack vectors with multiple trait gene cassettes. The root cause of the lower efficiency of pat in maize is not well understood and it is possible that the causes are multifaceted, including maize genotype, pat marker cassette, trait gene combinations and selection system. In this work we have identified a new variant of pat gene through codon optimization that consistently produced a higher transformation frequency (> 2x) than an old version of the pat gene that has codons optimized for expression in dicot plants. The level of PAT protein in all 16 constructs was also found multifold higher (up to 40 fold) over that of the controls. All of the T0 low copy transgenic plants generated from the 16 different constructs showed excellent tolerance to ammonium glufosinate herbicide spray tests at 4x and 8x recommended field application rates (1x = 595 g active ingredient (ai)/hectare of ammonium glufosinate) in the greenhouse.
KeywordsMaize transformation Agrobacterium Codon optimization Pat selectable marker
Help rendered by Rachel Whinna, Pei Su, Ping Wu, Sabrina Patton, Melissa Murray, Jamie McCuiston, Yaping Jiang, Lucy Qin and Yoshimi Barron towards this work are gratefully acknowledged. We are thankful to Liang Shi, Heng Zhong and Kasi Azhakanandam for project support and technical discussions, respectively.
The study was funded by Syngenta’s internal resources.
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