The use of LAC fusions to analyse the regulation of NOD gene region of Rhizobium loti : a thesis presented in partial fulfilment of the requirements for the Degree of Master of Science in genetics at Massey University, Palmerston North, New Zealand
Two approaches where used in the analysis of common and host specific nod gene expression in Rhizobium loti strains NZP2213 and NZP2037.
The first approach using the Tn3-HoHol transposon to generate lacZ transcriptional/translational fusions, produced 290 insertions within the 8.3kb EcoRI nod fragment of R.loti strain NZP2213. The position and orientation of all but one of these insertions was determined using restriction enzyme mapping and hybridisation. The sites of the insertion and orientation were generally found to be random.
The lacZ fusions were transferred into R.loti strain NZP2213 where their B-galactosidase activity was measured in the presence and absence of Lotus tenuis seed exudate. All insertions had a low level of B-galactosidase activity that was the same as the controls. This activity was independent of position or orientation. This lack of expression could be a result of the fusions being in regions that are not transcribed ie not downstream of either a nod inducible or other promoter, or that the appropriate conditions for constitutive or inducible activity were not achieved.
The second approach to construct lacZ transcriptional fusions was less random and involved the cloning of three separate nod gene fragments:
i) a 4.1kb Sall fragment that overlaps the nod region of the 8.3kb EcoRI fragment of R.loti strain NZP2213,
ii) a 0.65kb EcoRl fragment isolated from the 4.1kb Sall fragment of R.loti strain NZP2213, and
iii) a 1.4kb Sall fragment isolated from the 7.1kb EcoRI nod region of R.loti strain NZP2037.
These three fragments (4.1kb, 0.65kb and 1.4kb) were isolated and cloned into pMP190, pMP220 and pMP190 respectively, in both orientations. Each lacZ fusion was transferred into the R.loti strains from which the fragment had originated, ie either NZP2213 or NZP2037. The B-galactosidase activity of these transconjugants was measured in the presence and absence of Lotus tenuis seed exudate.
The 4.1kb Sall construct from R.loti NZP2213 was found to have consititutive activity in both orientations indicating that at least two consititutive promoters are located on this fragment. The activity of one orientation, corresponding to pPN38, was twice that of the reverse orientation corresponding to pPN37.
The smaller 0.65kb EcoRI fragment, that lies within the larger 4.1kb Sall fragment, contains a "nod box" and part of a nodD-like gene (Scott et al., In prep.). No significant 13-galactosidase activity was observed in either orientation with or without seed extract. These experiments showed that the "nod box" alone was insufficient for plant inducible expression.
The 1.4kb Sall fragment from R.loti NZP2037, that was known to contain a nodA promoter (Emerson-Colins et al., pers. comm.) showed inducible expression for pPN39, corresponding to a fusion between nodA and lacZ. No significant activity was detected in the reverse orientation, pPN40, either with or without plant exudate.