Inheritance of GFP-Bt transgenes from Brassica napus in backcrosses with three wild B. rapa accessions.

Transgenes from transgenic oilseed rape, Brassica napus (AACC genome), can introgress into populations of wild B. rapa (AA genome), but little is known about the long-term persistence of transgenes from different transformation events. For example, transgenes that are located on the crop's C chromosomes may be lost during the process of introgression. We investigated the genetic behavior of transgenes in backcross generations of wild B. rapa after nine GFP (green fluorescent protein)-Bt (Bacillus thuringiensis) B. napus lines, named GT lines, were hybridized with three wild B. rapa accessions, respectively. Each backcross generation involved crosses between hemizygous GT plants and non-GT B. rapa pollen recipients. In some cases, sample sizes were too small to allow the detection of major deviations from Mendelian segregation ratios, but the segregation of GT:non-GT was consistent with an expected ratio of 1:1 in all crosses in the BC1 generation. Starting with the BC2 generation, significantly different genetic behavior of the transgenes was observed among the nine GT B. napus lines. In some lines, the segregation of GT:non-GT showed a ratio of 1:1 in the BC2, BC3, and BC4 generations. However, in other GT B. napus lines the segregation ratio of GT:non-GT significantly deviated from 1:1 in the BC2 and BC3 generations, which had fewer transgenic progeny than expected, but not in the BC4 generation. Most importantly, in two GT B. napus lines the segregation of GT:non-GT did not fit into a ratio of 1:1 in the BC2, BC3 or BC4 generations due to a deficiency of transgenic progeny. For these lines, a strong reduction of transgene introgression was observed in all three B. rapa accessions. These findings imply that the genomic location of transgenes in B. napus may affect the long-term persistence of transgenes in B. rapa after hybridization has occurred.

Modeling the structure of the type I peritrophic matrix: characterization of a Mamestra configurata intestinal mucin and a novel peritrophin containing 19 chitin binding domains.

Twelve to fourteen integral proteins were found to reside in the Type I peritrophic matrix (PM) of Mamestra configurata (bertha armyworm) larvae. Several methods were employed, including de novo peptide sequencing, the generation of a midgut-specific EST database and immunological screening, which led to the isolation of cDNAs encoding two integral PM proteins. McPM1, the largest PM protein described to date at 202 kDa, was comprised of a concatamer of 19 chitin binding domains (CBD), 12 of which resided within a central repetitive region consisting of six iterations of a two CBD module. The protein was found to reside within the PM primarily as several lower molecular weight, presumably proteolytically processed, forms. McMUC1 was similar in structure to other insect intestinal mucins (IIM) and was highly glycosylated. The expression of both proteins was restricted to the larval midgut. Lower molecular weight proteins that may represent non- and partially glycosylated forms of McMUC1 were also recognized by an anti-McMUC1 antiserum. These were preferentially degraded upon ingestion of M. configurata multi-capsid nucleopolyhedrovirus by larvae, possibly by a viral-encoded metalloprotease. A molecular model of PM structure is presented featuring the interaction of McPM1 with chitin inter-fibril junctions and McMUC1 with the extended chains in the internodal regions. The potential for interaction between PM proteins via intermolecular disulfide bond formation and through association of CBD with N-linked glycans is discussed.

Midgut proteases from Mamestra configurata (Lepidoptera: Noctuidae) larvae: characterization, cDNA cloning, and expressed sequence tag analysis.

The activities of digestive protease within the midgut of Mamestra configurata (bertha armyworm) larvae were examined using specific substrates and protease inhibitors. The bulk of the activity was associated with serine proteases comprising trypsin-, chymotrypsin-, and elastase-like enzymes. At least 10-15 serine protease isozymes were detected using one-dimension gelatin gel electrophoresis. Cysteine or aspartic protease activities were not present; however, amino- and carboxypeptidase activities were associated with the midgut extract. Midgut proteases were active in the pH range of 5.0-12.0 with peaks at pH 7.5 and 11.0. In general, the middle region of the midgut exhibited a higher pH (approximately 8.0) than either the posterior or anterior regions (approximately 7.3-7.7). Moulting larvae possessed a neutral gut pH that was 0.5-1.5 units below that of feeding larvae. Degenerate PCR and expressed sequence tag (EST)-based approaches were used to isolate 30 distinct serine protease encoding cDNAs from a midgut-specific cDNA library including 8 putative trypsins, 9 chymotrypsins, 1 elastase, and 12 whose potential activities could not be determined. cDNAs encoding three amino- and two carboxypeptidases were also identified. Larvae feeding upon artificial diet containing 0.2% soybean trypsin inhibitor experienced a significant delay in development.

Transgenic Bt-producing Brassica napus: Plutella xylostella selection pressure and fitness of weedy relatives.

Release of transgenic insect-resistant crops creates the potential not only for the insect pest to evolve resistance but for the escape of transgenes that may confer novel or enhanced fitness-related traits through hybridization with their wild relatives. The differential response of diamondback moth (Plutella xylostella) populations in eastern and western Canada to Bt-producing (GT) Brassica napus and the potential for enhanced fitness of GT B. napus and weedy GT Brassica rapa x B. napus hybrid populations (F1, BC1, BC2) were studied. Comparative bioassays using neonates and 4th instars showed that GT B. napus and GT B. rapa x B. napus hybrids are lethal to larvae from both populations. No measurable plant fitness advantage (reproductive dry weight) was observed for GT B. napus (crop) and GT B. rapa x B. napus hybrid populations at low insect pressure (1 larva per leaf). At high insect densities (>10 larvae per leaf), vegetative plant weight was not significantly different for GT B. napus and non-GT B. napus, whereas reproductive plant weight and proportion of reproductive material were significantly higher in GT B. napus. Establishment of the Bt trait in wild B. rapa populations may also increase its competitive advantage under high insect pressure.