Analysis of multiple silencing processes of the transgene-induced gene silencing in higher plants

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A tobacco endoplasmic retliculum ω-3 fatty acid desaturase (NtFAD3) catalyzes the formation of α-linolenate. In a transgenic tobacco plant (S44), the NtFAD3 mRNA transcribed from the transgene was accumulated. We fractionated the polysomes into 12 fractions by sucrose-density-gradient centrifugation. The transgene-derived NtFAD3 mRNAs were found in the fractions with low sucrose densities, indicating that the synthesis of NtFAD3 protein was partially inhibited. On the other hand, the level of endogenous NtFAD3 mRNA was a trace in the S44 plants, and aberrantly spliced products from the endogenous NtFAD3 gene were detected. These aberrantly spliced products would be derived from the inhibition of splicing of the 5th intron of endogenous NtFAD3 gene. The splicing of internal intron (5th intron) was difficult when this intron was introduced into tobacco cells by particle bombardment, suggesting that splicing of this intron would require another factor, such as exon-intron or intron-intron interaction. Our data suggest that in the, S44 plants, the regulation for splicing of the 5th intron would be perturbed by the transgene-induced gene silencing.