INDUCTION OF NITRATE REDUCTASE HOST GENE EXPRESSION HAS A NEGATIVE EFFECT ON THE EXPRESSION OF TRANSGENES DRIVEN BY THE NITRATE REDUCTASE PROMOTER

摘要

In two previous studies the expression of the tobacco nitrate reductase genes (Nia1 or Nia2) introduced into nitrate reductase deficient mutants or of glucuronidase reporter genes linked to the promoter of a tobacco Nia gene were characterized. In both cases no expression of the introduced genes was found in the leaves of approximately 85% of the transformants and only a low level of expression was detected in the remaining transformants ranging from 0.1 to 7% of the expression of host Nia genes. In this paper we show that reduced levels of transgene expression in leaves result from a negative effect of host Nia gene expression. Transformants have been generated carrying a neomycin phosphotransferase reporter gene (Npt) linked to a 1.4 kb promoter fragment (-1350 to +40) of the Nial gene by electroporation. The level of resistance to the antibiotic declined in transformed calli after 1 month of culture whereas a high level of resistance was recovered in cells freshly prepared from leaf protoplasts of the regenerated transgenic plants, conditions in which the expression of host Nia genes is severely down-regulated. The level of resistance in seedlings also declined after 1 month of growth when plants were grown on a medium containing nitrate whereas a high level of resistance was observed when plants were grown on a medium supplemented with ammonium as a sole nitrogen source, conditions that do not allow the induction of host Nia genes expression. The analysis of a series of progressive deletions covering the 5' end of the promoter down to position -92 showed that such a negative correlation between the expression of pNia1-Npt transgenes and host Nia genes is found in approximately 70% of the transformants, irrespective of the size of the deletion, A model is proposed to explain how the expression of host Nia genes can impede the expression of phria-driven transgenes. [References: 20]