Maize transformation efficiency increase via RNAi-mediated suppression of chromatin-associated genes.

摘要

Chromatin remodeling plays a crucial role in gene regulation and expression. A more complete understanding of the role of chromatin effects on transgene integration and expression is needed to facilitate plant transformation experiments and to understand mechanisms of lateral DNA transfer. Suppression of chromatin-associated genes (chromatin genes) was therefore hypothesized to increase maize transformation efficiencies via increased transgene integration or reduced transgene silencing. RNA interference (RNAi)-inducing inverted-repeat (IR) vectors targeting maize chromatin genes SGB101, HON102, MBD109, SDG119, CHR110, GTC101, and SRT101 were transformed into maize via biolistics in replicated experiments to test for effects of chromatin gene suppression on primary transformation efficiency. The maize chromatin gene, SGB101, is the probable maize ortholog of yeast ARD1, an N-terminal acetyltransferase required for telomere and mating-loci silencing. Over the course of twelve independent, replicated experiments, the average stable transformation rate for the IR vector targeting SGB101 was 8.5%, and was statistically significantly higher than the IR backbone base vector, pMCG161, efficiency of 4.3%, representing a two-fold increase in transformation efficiency. The maize chromatin gene, HON102, encodes a linker histone protein within the Histone H1 homology group. The average stable transformation rate for the IR vector targeting HON102 was 7.5%, and was also statistically significantly higher than the average transformation rate of 4.2% for pMCG161 over eleven independent, replicated experiments. T1 RNAi lines were then re-transformed with a control vector and stable transformation efficiencies compared between sibling 'null' and RNA-silenced embryos; however, the low overall transformation efficiencies generated with the nptII selectable marker led to insufficient data for significant conclusions to be drawn. The role of HON102 and the histone H1 family in maize transformation was further elucidated by the suppression of either HON102 or HMGA102 individually, or both genes simultaneously. Suppression of HON102 resulted in a statistically significant increase in the average stable transformation efficiency (9.0%) when compared with the average pMCG161 baseline efficiency of 4.1%. These experiments demonstrate that suppression of maize chromatin genes that are hypothesized to affect chromatin conformation and/or gene silencing can lead to increased stable maize transformation efficiencies of at least two-to-three fold over that of the control.