Synonymous mutation gene design to overexpress native enzymes in plants while bypassing posttranscriptional-gene-silencing mechanisms
Heckart, Douglas Lee
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Plants have gene-silencing mechanisms to prevent overexpression (OE) of native genes. OE of transgenes with a high level of identity to a native gene can trigger post-transcriptional-gene-silencing (PTGS) where mRNA is degraded into small interfering RNA (siRNA) approximately 21-nt long. In the present study, we tested transgene design using synonymous mutations to bypass PTGS. The acetyl coenzyme A carboxylase (ACCase) coding sequence was cloned from creeping bentgrass (Agrostis stolonifera L.) and used as a template for gene design. An additional non-synonymous mutation was included in the first position of the 1781 codon causing an isoleucine to leucine substitution known to provide resistance to ACCase-inhibition herbicides. Bentgrass calli were inoculated with the bentgrass or modified ACCase with or without the 1781 mutation. Six HR events were obtained from calli transformed with ACCase with synonymous mutations and the 1781 SNP. Whole plant dose response showed transgenic plants were resistant to the ACCase-inhibiting herbicide, sethoxydim. Transcription of the modified gene was confirmed, showing PTGS was avoided. Additionally, sethoxydim-resistant bentgrass was selected in vitro to compare to transgenic plants. Four sethoxydim-resistant events were selected and the whole plant dose response confirmed high levels of sethoxydim resistance. Sequencing confirmed the 1781 leucine mutation. Grass-derived promoters and terminators were tested for ability to control GUSPlus reporter gene expression. Maize (Zea mays L.) centromere-specific sequences were evaluated for promoter activity. The long terminal repeats (LTR) from retrotransposons CRM1 and CRM2, and the centromere repeat element CentC, were cloned from maize and evaluated for promoter activity. The CentC and the CRM LTRs were fused to GUSPlus gene. Constructs contained the luciferase reporter gene as an internal control to normalize GUS expression. Possible transcription terminator sequences were cloned from several grass species and tested in place of T-NOS, controlling GUSPlus transcription. All constructs were evaluated using a transient expression assay by microprojectile bombardment of rice callus tissue. Promoter activity of CentC was not significantly different from the CaMV 35S promoter. Among the terminator sequences evaluated, GUS expression was highest with the switchgrass pvubi2 3’UTR and was not significantly different from T-NOS.