Both RNA level and translation efficiency are reduced by anti-sense RNA in transgenic tobacco

Abstract

The effect of anti-sense RNA on the expression of the bialaphos resistance (bar) gene which encodes phosphinothricin acetyl transferase (PAT), was analysed in tobacco. Transient expression studies revealed that an anti-bar RNA with sequence complementarity to the complete bar coding region, inhibits PAT synthesis. To quantify the phenomenon, SR1 tobacco cells were transformed twice to introduce first a hybrid bar gene with a reporter gene and in a second instance an anti-bar gene. A first cycle transformant and a double transformant derived herefrom in which PAT synthesis was reduced to only 8%, were studied in detail. The interference of the anti-sense gene with the expression of the bar gene is manifested at least two levels. First, the bar mRNA steady state level is significantly reduced relative to the parental whereas the transcript level of the reporter gene is unchanged. Comparison of bar mRNA levels in total and single stranded (ss) RNA preparations demonstrated that little if any stably base-pairing bar and anti-bar RNA accumulates. Secondly, a three fold reduction of PAT synthesis per bar mRNA is observed. This supposes that because of unstable interactions with the complementary anti-bar RNA either a substantial part of the bar mRNA detected does not enter the cytoplasm and/or that in the cytoplasm the bar mRNA is less efficiently translated. It is not clear if or how the reduced bar mRNA level is related to such unstable interactions.