FUNCTIONAL-ANALYSIS OF THE PROMOTER REGION OF A NODULE-ENHANCED GLUTAMINE-SYNTHETASE GENE FROM PHASEOLUS-VULGARIS L

The 5'-flanking region of gln-gamma, the nodule-enhanced glutamine synthetase gene from Phaseolus vulgaris L., has been analysed for cis-regulatory elements, using a series of 5' deletions and hybrid gln-gamma: : CaMV 35S promoters. The promoters were fused to the uidA reporter gene and their activities tested in two heterologous expression systems. In the first system, the chimaeric genes were transferred to Lotus corniculatus L. using Agrobacterium rhizogenes and their expression was studied in nodulated hairy roots. In the second system, the constructs were electroporated into tobacco mesophyll protoplasts. The results of the 5' deletion analysis showed that the sequence between -597 and -21 (relative to the ATG codon) was sufficient for nodule-specific expression of the chimaeric gene in nodulated hairy roots, and revealed the existence of at least two positive regulatory elements. Sequences located between -2000 and -597 were able to stimulate expression in nodules but not protoplasts, while the region from -597 to -354 enhanced expression in both nodules and protoplasts. Results obtained with the hybrid gln-gamma: :35S promoters showed that two overlapping restriction fragments (-516/-343 and -474/-293) were able to stimulate expression from a heterologous promoter in an orientation-dependent manner. Previous work has demonstrated the presence of conserved A/T-rich binding sites for nuclear proteins in the region between -516 and -446, and their possible role in regulating gln-gamma expression is discussed.