Genetic Engineering Cotton for Enhanced Resistance to Aspergillus Flavus

Approach:
Total RNA isolated from developing cotton plants and cottonseed will be hybridized with PCR-generated probes for selected cotton plastid genes using standard Northern hybridization technology. The promoters from those genes that demonstrate high levels of expression in green and/or non-green cotton tissues based on Northern hybridization results will be cloned and characterized using standard molecular biological methods. Promoter fragments of select plastid genes will be fused to reporter genes (GUS, GFP, etc.) and transformed into tobacco and cotton plastids in order to identify the minimal functional promoter sequences. While tobacco plastid transformation protocols have been developed, the same cannot be said of cotton, and protocols will have to be optimized for this plant. Once an efficient cotton plastid transformation system has been developed, cotton plastid will be transformed with transformation vectors in which reporter, antifungal, and selectable marker genes are placed under control of selected cotton plastid gene promoters. Transplastomic cotton plants will be analyzed for expression and production of reporter, antifungal, and selectable marker genes by standard molecular biological techniques (PCR, Northern and Western blotting). In planta antifungal bioassays will be performed to determine levels of resistance to A. flavus, as well as other cotton fungal pathogens.