2009 Annual Report
1a.Objectives (from AD-416)
Identify cotton plastid promoters that demonstrate high expression levels in both green and non-green plant tissues for use in development of cotton plastid transformation vectors. Determine levels of expression of reporter genes in both green (leaf and outer boll) and non-green (cottonseed and root) cotton tissues under control of select, engineered plastid promoters. Generate cotton plastid transformation vectors that place antifungal genes and selectable marker genes under control of selected cotton plastid promoters and transform cotton. Test transformed tissues for expression of antifungal genes and selectable marker genes under both light and dark growth conditions. Perform in planta bioassays for antifungal activity in transplastomic cotton plants.
1b.Approach (from AD-416)
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.
Efforts are underway to add specific antifungal genes into the deoxyribonucleic acid (DNA) of certain organelles (plastids) of cotton as a means to reduce A. flavus invasion and subsequent aflatoxin contamination of cottonseed. Successful production of the antifungal protein/peptide will depend in large part on the ability of the machinery controlling expression of the antifungal gene to generate high levels of gene expression in the plastid genomes present in cells of both green and non-green tissues. A total of 18 genes from the cotton plastid genome were assayed for their levels of expression in a number of green (e.g. leaf and stem) and non-green (e.g. seed) tissues at different growth stages using standard techniques. Two genes in particular, matK and rrn16, demonstrated high level expression at almost all growth stages and tissues. Real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) technology was used to confirm the data, and though both matK and rrn16 were expressed in all tissues and times tested, the rrn16 gene expression was found to be much higher than matK. Therefore, when specific antifungal piece of DNA or specific gene needs to be added to cotton plastids, the transfer mechinery (vectors) will utilize the relevant-part (promoter) of the rrn16 gene to drive high level expression of the antifungal genes in both green and non-green cotton tissues. Development of transgenic cottons expressing antifungal genes will be used to control aflatoxin contamination in cottonseed. Research progress is monitored by phone, email, and site visits.