Triacylglycerol (TAG) production in the microspore derived (MD) cell suspension culture ofBrassica napus L. cv Jet Neuf was enhanced when the sucrose concentration in the growth medium was increased from 2 to 14 % (w/v). mRNA differential display by polymerase chain reaction was used to examine gene expression in cells grown at different sucrose concentrations in order to identify mRNAs which could be associated with oil formation. The anchored primer, T12AA, was used to screen one subset, representing approximately one twelfth of the transcript population, isolated from cultures grown in media supplemented to 2, 6 and 14 % (w/v) sucrose. Analysis of this mRNA subset revealed thirteen cDNAs which appeared to be upregulated as the sucrose concentration was increased. Cloning and sequencing revealed multiple cDNA fragments for each signal detected by differential display. RT-PCR analysis of sixteen different cDNAs revealed that eight encoded mRNAs which were upregulated in parallel to the increase in media sucrose. Comparison of the eight upregulated cDNAs to other sequences in GenBank revealed the following: (1) BSS8A had a 100% identity with the last 25 amino acids of an acyl carrier protein from Arabidopsis thaliana, (2) BSS1A displayed homology to a number of sequences of unknown function, (3) BSS1 IB displayed weak but significant homology to a number of sequences of unknown function, (4) BSS13A displayed homology to four members of the thioredoxin family from ,4. thaliana and (5) four Had no significant homology to previously reported sequences which makes them potential candidates to encode lipogenic enzymes. These results indicate that differential display of mRNA may be a simple and rapid method for the identification of sucrose-modulated gene expression changes in this system and for the characterization of novel sequences potentially encoding lipogenic proteins. / xxi, 256 leaves : ill. ; 28 cm.

Diacylglycerol acyltransferase (DGAT), an integral membrane protein of the endoplasmic reticulum, catalyses the final step in the sn-glycerol-3-phosphate pathway leading to triacylglycerol. Although DGAT has been cloned from a variety of species, including the major oilseed crop of Canada, canola (Brassica napus), little is known about the structure/function of the enzyme. BnDGAT1 is the major isoform of type I DGAT (DGAT-I) in microspore-derived cell suspension cultures of B. napus L. cv Jet Neuf, with the possible existence of a truncated form of BnDGAT1 known as BnDGAT2. In order to gain some insight into the topology of the enzyme, type I DGAT from B. napus was investigated using two approaches: (1) in vitro translation in the presence of microsomes and (2) immunochemical analyses of microsomes isolated from cell suspension cultures, both in combination with proteolytic mapping. Difficulties were encountered with the in vitro translation approach, possibly due to proper incorporation of the polypeptide into microsomal vesicles. Two cytocolic regions were identified in BnDGAT1, and one cytosolic region in putative BnDGAT2, using the immunochemical approach, thus providing some insight into the topology of B. napus DGAT-I. The results here support and nine and eight membrane-spanning topology for BnDGAT1 and BnGAT2, respectively. / xvii, 194 leaves ; 29 cm.