Stable transgene expression and random gene silencing in wheat

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Epigenetics

Background:

• Maize ubiquitin promoter-intron driven pathogenesis-related (PR) protein genes (chitinase and beta-1,3-glucanase) were introduced into spring wheat 'Bobwhite'.
• Transgenic wheat lines were generated using a biolistic transformation approach.

Purpose of the Study:

• To investigate transgene expression stability and epigenetic regulation in wheat.
• To characterize the relationship between DNA methylation and gene silencing in transgenic plants.

Main Methods:

• Biolistic transformation of wheat with PR-protein genes.
• Analysis of transgene expression across generations (T0-T4) using molecular techniques.
• Methylation analysis using isoschizomeric enzymes (MspI and HpaII) and Southern blotting.
• Reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blotting, and Western blotting for gene expression analysis.

Main Results:

• Twenty out of 24 primary transgenic wheat lines exhibited gene silencing in subsequent generations.
• Two lines, genetically identical but differing in transgene expression, were selected for detailed study.
• Stable transgene expression was observed in one line with lower DNA methylation, while the other showed silencing correlated with higher CCGG sequence methylation.
• Treatment with 5-azacytidine did not reverse the silenced phenotype.

Conclusions:

• DNA methylation plays a crucial role in regulating transgene expression stability in wheat.
• Epigenetic modifications, specifically DNA methylation, can lead to heritable gene silencing in transgenic plants.
• Understanding methylation patterns is essential for developing stable transgenic crop varieties.