1 CLONING AND CHARACTERIZATION OF aflR GENE INVOLVED IN AFLATOXIN BIOSYNTHESIS FROM LOCAL STRAINS OF Aspergillus sojae Maetrese Arianne

J. Beley Mindanao State University Iligan Institute of Technology Tibanga, Iligan City 9200

Background of the Study Aspergillus flavus is an imperfect filamentous fungus that is an opportunistic pathogen causing invasive and non-invasive aspergillosis in humans, animals, and insects. It is one of the most abundant and widely distributed soil-borne molds that can be found anywhere on earth. It is described as a saprophytic fungus that is capable of surviving on many organic nutrient sources like plant debris, tree leaves, decaying wood, animal fodder, cotton, compost piles, dead insect and animal carcasses, outdoor and indoor air environment (air ventilation system), stored grains, and even human and animal patients (Klich M. 1998). Its optimal range for growth is at 28 - 37 C and can grow in a wide range of temperatures from 12 to 48 C and its heat tolerance nature contributes to its pathogenicity on humans and other warm blooded animals (Bennett JW et al., 1986). The fungus mostly exists in the form of mycelium or asexual conidia spores. Under adverse conditions such as dry and poor nutrition, the mycelium congregates to form resistant structures called sclerotia. The fungus over-winters either as spores or as sclerotia. The sclerotia germinate t o form new colonies when growth conditions are favorable (Cotty PJ. 1988). A. flavus produces many secondary metabolites including aflatoxins, the most toxic and most potent carcinogenic natural compounds that cause aflatoxicosis and induce cancers in mammals. In addition, it is a weak and opportunistic pathogen of many crops (corn, cotton,

2 peanuts, and treenuts) and contaminates them with aflatoxins. This ubiquitous mold not only reduces yield of agricultural crops but decreases the quality of the harvested grains. Due to A. flavus infection to the crops and aflatoxin contamination in grains, hundreds of millions of dollars are lost to the world economy annually (Jiujiang et. at., 2005). Aflatoxins are a group of structurally related toxic secondary metabolites produced mainly by certain strains of A. flavus and A. parasiticus. The aflatoxins, B1, B2, G 1 and G2 (AFB1, AFB2, AFG1 and AFG2) are the major four toxins among at least 16 structurally related toxins (Goldblatt LA. 1969). A. flavus produces aflatoxins B1 and B2. Other toxic compounds produced by A. flavus are cyclopiazonic acid, kojic acid, -nitropropionic acid, aspertoxin, aflatrem and aspergillic acid. A. parasiticus produces aflatoxin G1 and G2, in addition to B1 and B2, but not cyclopiazonic acid (Jiujiang 2005). Aflatoxin B1 is predominant, the most toxic and most potent hepatocarcinogenic natural compound ever characterized (Squire RA. 1989). Aflatoxin M1 is a major metabolic product of aflatoxin B1 in animals and is usually excreted in the milk and urine of dairy cattle and other mammalian species that have consumed aflatoxincontaminated food or feed. There is a positive regulatory gene, aflR (Chang et. al., 1993; Payne et al., 1993), which is required for transcriptional activation of most, if not all, of the structural genes (Chang et al., 1995; Ehrlich et al., 1998) by binding to the palindromic sequence 5-TCGN5CGA-3 in the promoter region of the structural genes in A parasiticus, A. flavus and in A. nidulans (Ehrlich and Yu et al., 1996). Adjacent to the aflR gene, a gene, aflS (aflJ), is also involved in the regulation of transcription (Chang 2004 & Meyers et al 1993). Finally, the laeA gene, for loss of aflR expression, was shown to be involved in the global regulation of secondary metabolites,

3 aflatoxins, sterigmatocystin (ST), penicillin and gliotoxin, in several fungal species (Bok et al., 2004).

Objectives of the Study The main objective of this study is to isolate and characterize the alfR gene of the fungi Aspergillus flavus that involves in the biosynthesis of aflatoxin. Specifically, it aims to: 1. grow Aspergillus sojae isolate to different solid-state culture conditions 2. determine the presence and expression of aflR gene involved in aflatoxin biosynthesis in A. sojae. 3. determine the sequence of alfR gene in local strains of A. flavus 4. determine the copy number of the alfR homolog in the local strains of A. flavus genome; and 5. characterize the gene sequence through bioinformatics.

Significance of the Study The researcher will add a significant knowledge on the genetics of aflatoxin biosynthesis involving alfR gene of the local strain A. sojae since it has limited genetic information. The species could be used to facilitate transformation systems in modern biotechnology and microbiology since it is nontoxigenic and easy to cultivate. Furthermore, because of the important genes it contains that regulate production of toxins, it could be possible to use these genes to regulate or even inhibit the production of toxins, not only to fungi but to other toxinproducing microorganisms, hence, it will provide vital clues for engineering commercial crops

4 resistant to fungal infection by incorporating antifungal genes that may prevent aflatoxin contamination. Characterization of the gene sequence through bioinformatics enables early detection of mis-breeding, genetic drift and enables correlation of genetic information with phenotype. This set of information could serve as a guide in genetic engineering.

Scope and Limitation of the Study The study will focus on the isolation, cloning and characterization of the aflR gene which is involved in the biosynthesis of aflatoxin among local isolates of Aspergillus sojae from the National Culture Collection, UPLB. Genomic DNA isolation will be done to A. sojae culture and DNA by PCR shall be amplified by following the manufacturer's (QIAGEN PCR Kit) instructions. The DNA fragments generated by PCR will be separated by means of agarose gel electrophoresis. DNA samples will be sent to MACROGEN, Korea for sequencing and sequence analysis will be done using bioinformatics open software (NCBI and EMBL).