ANALYSIS OF THE LUPIN NODULIN-45 PROMOTER - CONSERVED REGULATORY SEQUENCES ARE IMPORTANT FOR PROMOTER ACTIVITY

The promoter from the Lupinus angustifolius late nodulin gene, Nodulin -45, has been analysed to identify cis-elements and trans-acting facto rs. Various regions of the Nodulin-45 promoter, fused to the luciferas e reporter gene, were introduced into Lotus roots using an Agrobacteri um rhizogenes transformation procedure. The transgenic roots were then nodulated. The promoter region A (-172 to +13, relative to the transc ription start site) was capable of directing low-level expression of t he reporter gene and in a nodule-enhanced manner when compared to root s. The addition of region C (-676 to -345) resulted in a significant i ncrease in the expression within the nodule, whilst a low level of roo t expression was maintained. The C region, which confers this high-lev el nodule expression, contains the nodule consensus motifs AAAGAT and CTCTT. When region C was ligated to a minimal promoter element from th e unrelated asparaginase gene rather than the Nodulin-45 A region, nod ule-enhanced expression was still apparent, but at a much lower level. Mutation of the AAAGAT element in this construct resulted in a furthe r significant decrease of expression. Gel retardation assays revealed that a factor from lupin nodule nuclear extracts interacted with two s equences of the C region, The binding of the factor to both of these r egions could be removed by the addition of an oligonucleotide containi ng the AT-rich binding site for the soybean factor NAT2. This suggests that the lupin factor identified here is a NAT2, homologue. No factor binding was observed to the AAAGAT or CTCTT elements present in the C region.