Rajasekaran Kanniah, Sayler Ronald J, Majumdar Rajtilak, Sickler Christine M, Cary Jeffrey W
Southern Regional Research Center, USDA-ARS, Food and Feed Safety Research Unit;
Department of Plant Pathology, University of Arkansas.
J Vis Exp. 2019 Feb 15(144). doi: 10.3791/59169.
Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are potent carcinogens that substantially reduce crop value in maize and other oil rich crops like peanut besides posing serious threat to human and animal health. Different approaches, including traditional breeding, transgenic expression of resistance associated proteins, and RNA interference (RNAi)-based host-induced gene silencing of critical A. flavus gene targets, are being evaluated to increase aflatoxin resistance in susceptible crops. Past studies have shown an important role of α-amylase in A. flavus pathogenesis and aflatoxin production, suggesting this gene/enzyme is a potential target to reduce both A. flavus growth and aflatoxin production. In this regard, the current study was undertaken to evaluate heterologous expression (under control of the constitutive CaMV 35S promoter) of a Lablab purpureus L. α-amylase inhibitor-like protein (AILP) in maize against A. flavus. AILP is a 36-kDa protein, which is a competitive inhibitor of A. flavus α-amylase enzyme and belongs to the lectin-arcelin-α-amylase inhibitor protein family in common bean. In vitro studies prior to the current work had demonstrated the role of AILP in inhibition of A. flavus α-amylase activity and fungal growth. Fungal growth and aflatoxin production in mature kernels were monitored in real time using a GFP-expressing A. flavus strain. This kernel screening assay (KSA) is very simple to set up and provides reliable and reproducible data on infection and the extent of spread that could be quantified for evaluation of germplasm and transgenic lines. The fluorescence from the GFP strain is closely correlated to fungal growth and, by extension, it is well-correlated to aflatoxin values. The goal of the current work was to implement this previous knowledge in a commercially important crop like maize to increase aflatoxin resistance. Our results show a 35%-72% reduction in A. flavus growth in AILP-expressing transgenic maize kernels which, in turn, translated into a 62%-88% reduction in aflatoxin levels.
食品和饲料作物中的黄曲霉毒素污染是全球面临的一项重大挑战。由黄曲霉产生的黄曲霉毒素是强效致癌物，除了对人类和动物健康构成严重威胁外，还会大幅降低玉米及花生等其他富油作物的作物价值。目前正在评估包括传统育种、抗性相关蛋白的转基因表达以及基于RNA干扰（RNAi）的关键黄曲霉基因靶点宿主诱导基因沉默等不同方法，以提高易感作物对黄曲霉毒素的抗性。过去的研究表明，α-淀粉酶在黄曲霉致病机制和黄曲霉毒素产生中发挥着重要作用，这表明该基因/酶是减少黄曲霉生长和黄曲霉毒素产生的潜在靶点。在这方面，本研究旨在评估扁豆α-淀粉酶抑制剂样蛋白（AILP）在玉米中（在组成型CaMV 35S启动子控制下）的异源表达对黄曲霉的抗性。AILP是一种36 kDa的蛋白质，是黄曲霉α-淀粉酶的竞争性抑制剂，属于菜豆中的凝集素-阿塞林-α-淀粉酶抑制剂蛋白家族。在当前工作之前的体外研究已经证明了AILP在抑制黄曲霉α-淀粉酶活性和真菌生长中的作用。使用表达绿色荧光蛋白（GFP）的黄曲霉菌株实时监测成熟玉米粒中的真菌生长和黄曲霉毒素产生。这种玉米粒筛选试验（KSA）设置非常简单，可提供关于感染和传播程度的可靠且可重复的数据，这些数据可进行量化，以评估种质和转基因品系。GFP菌株发出的荧光与真菌生长密切相关，进而与黄曲霉毒素值也高度相关。当前工作的目标是将这些先前的知识应用于玉米这种具有商业重要性的作物，以提高其对黄曲霉毒素的抗性。我们的结果表明，表达AILP的转基因玉米粒中黄曲霉的生长减少了35% - 72%，这反过来又使黄曲霉毒素水平降低了62% - 88%。
