Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/1/2003
Publication Date: 10/1/2003
Citation: Brown, R.L., Brown-Jenco, C.S., Payne, G.A., Bhatnagar, D. 2003. Construction and preliminary evaluation of an Aspergillus flavus reporter gene construct as a potential tool for screening aflatoxin-resistance. Journal of Food Protection. 66(10):1927-1931. Interpretive Summary: Aflatoxins are poisons produced by the fungus, Aspergillus flavus, after it infects agricultural commodities, such as corn. An important strategy for controlling aflatoxin contamination of corn involves developing corn lines that have resistance to aflatoxin production. Breeders implementing this strategy are hindered from doing so by the high costs in time, money, and labor involved in screening corn lines for resistance. A genetic transformant of Aspergillus flavus was constructed that carries a reporter gene linked to a gene involved in the biosynthesis of aflatoxins. This linkage enables aflatoxin biosynthesis activity to be indirectly, yet easily monitored by the reporter gene. When this transformant was inoculated onto corn kernels, reporter gene activity ran parallel with aflatoxin production. This was also true for two other previously-constructed reporter gene transformants. These results indicate ethat it may be possible to monitor aflatoxin production in corn in an indirect, yet accurate, easy, and cost-saving way. This could enable more resistant corn lines to be discovered/developed, thus, saving growers millions of dollars through the control of aflatoxin contamination.
Technical Abstract: Aflatoxins are the most carcinogenic naturally occurring compounds known and are produced on such crops as maize, peanuts, cottonseed, and tree nuts. Effective strategies are not available to eliminate aflatoxin accumulation. To better understand the molecular biology of aflatoxin biosynthesis, genetic and molecular tools such as reporter gene systems have been developed. It was previously shown that a reporter construct containing the promoter of the A. flavus beta-tubulin gene fused to the E. coli beta-glucuronidase (GUS) is a reliable tool for indirectly measuring fungal growth in maize kernels. Since cost-saving alternatives to direct measurement of aflatoxin levels are needed to facilitate more widespread field-screening of maize lines, a new reporter gene construct utilizing the omtA gene promoter of the aflatoxin biosynthetic pathway was constructed and tested. Expression of GUS activity by this construct (omtA::GUS) paralleled aflatoxin accumulation in culture. In transformant GAP26-1, which harbors this construct, aflatoxin production and GUS expression on sucrose-containing medium also showed the same temporal pattern of induction. It was further shown that either GUS or GFP (the reporter gene that expresses green fluorescent protein) expression correlated with aflatoxin accumulation in corn kernels inoculated with GAP26-1 or with either of two previously-made reporter gene transformants carrying aflR::gfp or the cmv::gfp constructs. Further evaluation in laboratory and field trials may demonstrate that these constructs provide superior alternatives to direct aflatoxin quantification, when screening maize lines for resistance.