Lorraine Braun

Stable Bacillus thuringiensis (Bt) toxin content in interspecific F1 and backcross populations of wild Brassica rapa after Bt gene transfer

Stable expression of a transgene may lead to increased fitness for wild plants after acquiring the transgene via crop–weed hybridization. Here, we investigate the stability of Bt toxin content in wild Brassica rapa acquiring the Bt gene from Bt Brassica napus. The Bt toxin content in nine Bt‐expressing B. napus lines was 0.80–1.70 µg/g leaf tissue throughout the growing season. These nine lines were crossed with three accessions of wild B. rapa and the Bt gene was successfully transferred to interspecific hybrids (F1) and successive backcross generations (BC1 to BC4). The Bt toxin level in F1 and BC progenies containing the Bt gene remained at 0.90–3.10 µg/g leaf tissue. This study indicates that the Bt gene can persist and be stably expressed in wild B. rapa.

Mamestra configurata serpin-1 homologues: cloning, localization and developmental regulation

A screen of a Mamestra configurata (bertha armyworm) midgut cDNA library identified three types of cDNA clones that resemble the Manduca sexta serpin-1 gene family. Two serpins, 1b and 1c, possess a common conserved serpin amino terminal scaffold domain but bear no similarity to any members of the M. sexta gene family within the reactive centre loop. These serpins differ from one another by only two amino acids in the reactive centre loop (S(363)-->P) and serpin signature (M(369)-->T) regions. The other member, denoted serpin-1a, is closely related to the M. sexta serpin-1Z. M. configurata serpins as a group were expressed in all insect developmental stages including eggs, larvae and adult moths. Within larvae, serpin gene expression was restricted to the early to middle instar developmental phase and mainly in the fat body and hemocytes. Stress imposed by starvation strongly induced expression in fat body and to a lesser degree in alimentary organs, nervous system and Malphigian tubules. Conversely, starvation decreased expression in hemocytes. Wounding or inoculation with bacteria did not induce serpin gene transcription but did lead to the formation of higher and lower molecular weight forms, presumably serpin-protease complexes and resultant truncated serpin, respectively. Two dimensional PAGE and western blotting analysis revealed at least 12 distinct serpins consisting primarily of neutral, but also highly acidic and basic isoforms, as well as additional high and low molecular weight immuno-reactive species. Serpins-1b/1c are the more prominent serpin isoforms and are expressed predominantly in the fat body and subsequently exported to the hemolymph as revealed by western blotting and immunolocalization. The serpin-1b/1c isoform was found only as the fully glycosylated species within the hemolymph. Hemolymph protease activity was comprised mostly of serine proteases whose overall activity increased dramatically at the onset of the molt concomitant with a sharp decline in serpin gene expression.

Changes in cysteine protease activity and localization during midgut metamorphosis in the crucifer root maggot (Delia radicum)

We show that differential localization and/or activation of two cysteine protease activities occur at the onset of dipteran midgut metamorphosis. A 26 kDa cysteine protease activity was associated specifically with midgut tissues of late third instar larvae. Starvation of mid third instar larvae simulated the onset of prepupation and resulted in loss of the 26 kDa protease activity. A cDNA clone encoding a cysteine protease, termed DrCP1, was isolated and shown to be highly similar to those from Sarcophaga peregrina and Drosophila melanogaster (DmCP1). DrCP1 mRNA was present in all developmental stages including eggs, larvae, pupae and adults, but was highly induced at the onset of the larval-pupal transition and thereafter. The DrCP1 protein is localized to the exterior of the midgut tissues during the onset of the prepupal transition period, possibly in response to ecdysone. Analysis of transcription factor binding sites associated with the DmCP1 promoter indicated that elements exist that allow for both ecdysone-mediated as well as tissue-specific regulation. Based upon these and other studies we propose: (1) that the expression, activity and localization of the DrCP1-like cysteine proteases are highly regulated throughout development; and, (2) that cysteine protease activities are involved in aspects of tissue reconstruction at the onset of and during metamorphosis.