Chimeric gene expression using maize intron in cultured cells of breadwheat

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genetics

Background:

• Efficient gene expression in cultured plant cells is crucial for genetic engineering.
• Electroporation is a common method for introducing DNA into plant protoplasts.
• The role of introns in enhancing heterologous gene expression in wheat remains an area of investigation.

Purpose of the Study:

• To evaluate the efficiency of different DNA constructs for reporter gene expression in breadwheat protoplasts.
• To determine the impact of promoter choice and intron presence on CAT gene activity.
• To identify optimal DNA construct features for enhanced heterologous gene expression in Triticum aestivum.

Main Methods:

• Breadwheat (Triticum aestivum) protoplasts were generated and cultured.
• High voltage electrical pulses (electroporation) were used to introduce four different DNA constructs containing the CAT reporter gene.
• Constructs varied in promoter elements (35S or mannipine synthase) and the inclusion of a maize intron sequence.

Main Results:

• CAT gene activity was measured 40-45 hours post-electroporation.
• A construct featuring a maize intron between the 35S promoter and the CAT gene exhibited significantly higher CAT activity (30-185 fold increase) compared to constructs without an intron.
• The mannipine synthase promoter showed lower activity than the 35S promoter across tested constructs.

Conclusions:

• The inclusion of a maize intron sequence is critical for achieving high levels of heterologous gene expression in breadwheat protoplasts.
• Intron-mediated enhancement is a key factor for efficient gene delivery and expression in cultured wheat cells.
• These findings provide valuable insights for designing effective gene constructs for wheat genetic transformation.