Molecular characterisation of the HMG CoA reductase gene from Neotyphodium Lolii : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Genetics at Massey University, Palmerston North, New Zealand
3-Hydroxy-3-methylglutaryl Coenzyme A reductase (Hmg) catalyses the conversion of HMG CoA to mevalonic acid: the first step of the isoprenoid biosynthetic pathway. This pathway produces a wide variety of primary metabolites which are involved in many different cellular processes. Neotyphodium endophytes in association with the grass host are known to produce a range of secondary metabolites including the indole diterpenoids (eg paxilline and lolitrem) and the ergopeptine alkaloids (eg ergovaline). Given that these pathways are upregulated in planta the availability of mevalonic acid, be it from fungal or plant source, will be important in controlling the levels of the different toxins synthesised. The aim of this work was to clone the fungal endophyte hmg and characterise the promoter to enable study of its regulation in planta via reporter gene studies. Using degenerate primers designed against conserved regions of other hmg genes a 359 bp fragment was amplified from the Neotyphodium lolii isolate Lpl9, which grows in perennial ryegrass (Lolium perenne). DNA sequencing confirmed that the sequence amplified was part of a unique hmg gene. Southern hybridisations suggest that there is a single copy of hmg in strain Lpl9 (a haploid) but two copies in strain Lpl (an interspecific hybrid; Schardl et al. 1994). The fragment of N. lolii hmg was used to screen a λGEM-12 genomic library of Lpl9 and four positive overlapping clones were isolated. Fragments of one clone. λJD12, were subcloned, sequenced and a physical map of this region of the genome was constructed. The entire sequence of hmg was determined using primer walking and was found to encode a 1188 amino acid polypeptide. From comparison to other Hmg proteins the catalytic domain has been shown to be highly conserved while the amino-terminal domain, containing transmembrane regions is divergent with very little sequence similarity near the translation start site and promoter region. Using RT-PCR analysis the hmg gene was shown to consist of two open reading frames separated by a 73 bp intron. RT-PCR was also used to determine the location of the transcriptional start site and this is supported by the presence of putative CAAT and TATA consensus sequences. With the promoter region identified and characterised further analysis of the regulation of hmg in planta can be undertaken.