Expression and properties of diacylglycerol acyltransferase from cell-suspension cultures of oilseed rape.

The expression of diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) with predicted molecular mass of 56.9. kDa (BnDGAT1) was examined using microspore-derived cell suspension cultures of oilseed rape (Brassica napus L. cv Jet Neuf). As well, a recombinant histidine-tagged N-terminal fragment of BnDGAT1 [BnDGAT1((1-116))His(6)], which was relatively hydrophilic, was partially characterized. A temporal increase in DGAT activity occurred within a 24 h period following transfer of cells from 6% (w/v) sucrose to 14% (w/v) sucrose. Western blotting indicated that the abundance of BnDGAT1 protein was closely correlated with DGAT activity. BnDGAT1 mRNA also exhibited a temporal increase within the 24 h period following transfer of cells into higher sucrose concentrations, but the transcript level was not closely associated with DGAT activity as BnDGAT1 protein. The fragment BnDGAT1(1-116)His(6) interacted with [1-(14)C]oleoyl-CoA, suggesting that the N-terminal region of BnDGAT1 may have a role in binding cellular acyl-CoA.

Detection of additional restriction fragment length polymorphisms among the weakly virulent (nonaggressive) and highly virulent (aggressive) isolates of Leptosphaeria maculans.

Isolates of Leptosphaeria maculans were analyzed for their genetic relatedness based on DNA restriction fragment length polymorphisms (RFLPs), employing as Southern hybridization probes a combination of heat shock responsive genes (hsp70 and hsp80 from Neurospora crassa), the cutinase gene of Magnaporthe grisea, and cloned genomic DNA sequences from a virulent strain. Southern hybridization analysis revealed a high frequency of DNA polymorphism. Restriction fragments generated by each enzyme-probe combination resulted in distinct banding patterns, clearly separating the isolates into two groups. The cutinase gene probe did not reveal any polymorphisms. Although the majority of the probes used displayed RFLP profiles unique to each group, a nonaggressive isolate, LmA, showed additional genetic characteristics in common with the virulent pathotype.

An electroporation-based system for high-efficiency transformation of germinated conidia of filamentous fungi.

A rapid and efficient electroporation procedure has been developed for transformation of germinating conidia of filamentous fungi. Pretreatment of conidial preparations with a cell wall weakening agent, such as beta-glucuronidase, was found to be essential for successful transformation. Using the qa-2+ gene of Neurospora crassa, encoding the catabolic dehydroquinase, as a selectable marker with a double-mutant host strain, auxotrophic for aromatic amino acids, integration of the plasmid was observed to be predominantly at ectopic chromosomal sites. Cotransformation with the qa-2+ gene and a plasmid containing a heat shock gene sequence (hsp70 of N. crassa) suggested integration site preference. High efficiencies of transformation to hygromycin resistance were achieved employing the bacterial hygromycin B phosphotransferase gene with N. crassa, the patulin-producer Penicillium urticae, and the causal agent of blackleg disease of crucifers, Leptosphaeria maculans. The economically important species Aspergillus oryzae was similarly transformed to benomyl resistance with the benomyl-resistant beta-tubulin gene of N. crassa as a dominant selectable marker.