|Rajasekaran, Kanniah - Rajah|
Submitted to: Peanut Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/28/2008
Publication Date: 10/22/2009
Citation: Niu, C., Akasaka-Kennedy, P., Faustinelli, P., Joshi, M., Rajasekaran, K., Yang, H., Chu, Y., Cary, J.W., Ozias-Akins, P. 2009. Antifungal activity in transgenic peanut (Arachis hypogaea L.) conferred by a nonheme chloroperoxidase gene. Peanut Science. 36(2):126-132. Interpretive Summary: Preharvest contamination of peanuts with aflatoxin due to infection of pods by Aspergillus flavus is a major problem in the peanut industry. Peanut is one of the most important, protein-rich edible crops and the product should be free from the highly toxigenic and carcinogenic aflatoxins. Our objective is to reduce aflatoxin contamination through genetic engineering of peanut through the introduction of anti-fungal genes. Progress has been slow, however, because of the relative inefficiency of peanut transformation and untested response of Aspergillus to the expression of anti-fungal genes in peanut tissues. Recently, we successfully transformed peanut with a chloroperoxidase gene from the bacterium Pseudomonas pyrrocinia that has been demonstrated to have antifungal properties by Rajasekaran et al. (2000; Plant Cell Rep 19:333) in transgenic tobacco. We have focused our analysis on progeny from one line that showed about 50% reduction in A. flavus growth using an in vitro assay with extracts from peanut tissues and edible cotyledons. We also demonstrated that progeny containing the transgene retain the antifungal properties of the parental line. Our research highlights the need and importance for successful gene insertion technologies for enhancing peanut resistance to aflatoxin- producing fungi.
Technical Abstract: A nonheme chloroperoxidase gene (cpo-p) from Pseudomonas pyrrocinia, a growth inhibitor of mycotoxin-producing fungi, was introduced into peanut via particle bombardment. The expression of the cpo-p gene is predicted to increase pathogen defense in peanut. Embryogenic peanut tissues were bombarded with gold particles coated with plasmid pRT66 cpo-p DNA, which includes the cpo-p and hygromycin phosphotransferase (hph) genes, under the control of a double CaMV 35S and a single CaMV 35S promoter, respectively. Selection for hygromycin-resistant somatic embryos was performed on a liquid medium containing 10-20 mg/l hygromycin 3-4 days after bombardment. The integration and expression of the cpo-p gene was confirmed by Southern, northern and western blot analyses. In vitro bioassay using crude protein extracts from transgenic R0, R1, and R4 plants showed inhibition of Aspergillus flavus hyphal growth, which could translate to a reduction in aflatoxin contamination of peanut seed.