ISOLATION OF FATTY ACID BIOSYNTHETIC GENES FROM ARABIDOPSIS-A FIRST STEP IN PLANT OIL CONTENT MODIFICATION

THE FATTY ACID (FA) COMPOSITION OF OILSEED CROPS IS AN ECONOMICALLY IMPORTANT TRAIT AMENABLE TO MODIFICATION BY GENETIC ENGINEERING. FOR EXAMPLE, IT SHOULD BE POSSIBLE TO ENGINEER A CANOLA (RAPESEED) OIL WITH A LOWER CONTENT OF LINOLENIC ACID, AN UNDERSIRABLE COMPONENT THAT CAUSES RANCIDITY. TRANSPOSON TAGGING PROVIDES A POWERFUL GENETIC APPROACH TO ISOLATE GENES ENCODING ENZYMES THAT ARE DIFFICULT TO PURIFY, SUCH AS THOSE INVOLVED IN FA BIOSYNTHESIS. THE SMALL MODEL PLANT ARABIDOPSIS THALIANA IS A CLOSE RELATIVE OF RAPESEED. WE PROPOSE TO ISOLATE THE FAD3 GENE, RESPONSIBLE FOR THE FORMATION OF LINOLENIC ACID IN ARABIDOPSIS, AS A FIRST STEP TOWARDS THE MODIFICATION OF THE FA COMPOSITION IN CANOLA. WE WILL USE OUR ENGINEERED STREPTOMYCIN RESISTANCE ASSAY (SPT) TO VISUALLY DETECT TRANSPOSITION OF THE MAIZE TRANSPOSON ACTIVATOR (AC) IN ARABIDOPSIS. SOMATIC EXCISION OF AC FROM AN SPT::AC GENE PRODUCES GREEN SPOTS ON THE WHITE COTYLEDONS OF SEEDLINGS GERMINATED ON STREPTOMYCIN AND GERMINAL TRANSPOSTION RESULTS IN GREEN REVERTANT SEEDLINGS IN THE SAME ASSAY. THEREFORE, THE SPT::AC SYSTEM ALLOWS US TO SELECT PLANTS THAT HAVE UNDERGONE TRANSPOSITION. BY SELECTING GREEN REVERTANTS FROM PLANTS THAT CARRY AC CLOSELY LINKED TO FAD3,AND SELFING THEM, WE SHOULD BE ABLE TO IDENTIFY TAGGED FAD3 MUTATIONS AND CLONE THEM BY HOMOLOGY TO AC. THE ANALOGOUS GENE CAN BE CLONED FROM CANOLA BY HOMOLOGY TO THE ARABIDOPSIS GENE AND SUBSEQUENTLY MODIFIED AND REINTRODUCED TO THE OILSEED CROP.