GC-rich coding sequences reduce transposon-like, small RNA-mediated transgene silencing

Area of Science:

• Plant molecular biology
• Genetic engineering
• Biotechnology

Background:

• Transgene silencing is a significant hurdle in genetic engineering, limiting the stable expression of introduced genes in plants.
• The molecular mechanisms underlying transgene silencing, particularly heritable silencing, are not fully understood.
• Current strategies for transgene design often fail to account for factors that promote silencing.

Purpose of the Study:

• To investigate the impact of transgene design parameters on heritable gene silencing in plants.
• To identify sequence features that can enhance transgene expression and stability across generations.
• To develop strategies for reducing the risk of transgene loss in genetically modified crops.

Main Methods:

• Analysis of Arabidopsis plants harboring transgenes for microalgal polyunsaturated fatty acid (PUFA) synthase.
• Evaluation of small RNA (sRNA)-mediated silencing and transposon-like silencing mechanisms.
• Testing the effect of regulatory sequence diversification and coding sequence (CDS) GC content.
• Assessing transgene expression, sRNA accumulation, and DNA methylation in Arabidopsis.
• Experimentation in maize using transgenes expressing Bacillus thuringiensis (Bt) crystal (Cry) proteins with CDS recoding.

Main Results:

• Repetitive regulatory elements and canola-biased PUFA synthase transgenes induced aggressive silencing via sRNAs and transposon-like activity.
• Diversified regulatory sequences and native microalgal CDSs with higher GC content improved transgene expression and stability.
• Higher GC content in CDSs led to reduced sRNA accumulation and DNA methylation, enhancing trans-generational stability.
• In maize, CDS recoding to higher GC content increased Cry protein and transcript accumulation.
• Transgene sequence composition directly influences silencing, affecting expression levels and heritable stability.

Conclusions:

• Transgene sequence composition, particularly GC content and regulatory element diversity, is critical for mitigating gene silencing.
• Optimizing transgene design by increasing GC content and diversifying regulatory elements can significantly improve expression levels and long-term stability.
• These findings provide practical design strategies to enhance the efficacy and reliability of transgenic crops.