Development of detection method for FAD3A gene edited high oleic acid soybean by duplex PCR technique

Abstract

Soybeans are one of the world's most economically important crops. It were classified in plants that are important food sources of protein and fat. However fat from soybeans is highly polyunsaturated fat which is the cause of shorter shelf life. In the industrial process, hydrogen is utilized to increase shelf life, but since hydrogenation produces trans fats (Tran-fatty acid), this type of fat has a huge impact on the consumer health. Because it may increase the risk of heart disease and high blood cholesterol. From the study of fatty acid biosynthesis pathway in soybeans, it was found that Fatty Acid Desaturase (FAD2) and (FAD3) genes were able to regulate the polyunsaturated fatty acid synthesis in soybeans. The purpose of this research is to develop a technique for detection the high-oleic-acid in the mutated soybeans which is undergone the duplex Polymerase chain reaction (PCR) technique based on the results of a primer pair test used in PCR reaction. The wild type soybean DNA was compared with the synthetic gene edited soybean DNA in the FAD3A gene in a single-gene reaction (Simplex PCR). Specific DNA band of FAD3A gene was not found in mutant. The LectinF and LectinR primer pairs were added to the PCR reaction in the FAD3_T1 primer pair in the duplex PCR reaction, and adjusted in the annealing step at 56 °C, The two DNA bands were most clearly visible in wild type soybean while in mutant showed single DNA band in gel electrophoresis.