Establishment of a transformation procedure to study the role of trypsin inhibitors in soybean

Abstract

The major serine proteinase inhibitors Kunitz and Bowman-Birk-type trypsin are key anti-nutrients responsible for the low nutritional value of raw soy cake, the by product of oil expression from soybean. Traditionally, proteinase inhibitors are eliminated from soy cake through intensive heating, which is highly costly. The long term goal is to generate soybean seeds devoid of trypsin inhibitors through tissue culture and genetic modification of soybean. The RNAi technology has been selected in this study as a technique for down-regulation or silencing these two major serine trypsin inhibitors. Conserved regions, which have been identified by searching NCBI and EMBL database, were targeted for down regulation. Seed specific promoters were also isolated to drive the expression of hairpin constructs designed to down-regulate selected conserved regions of the inhibitors in soybean seeds. RNAi silencing constructs were designed for use in soybean transformation. Ultimately, a tissue culture and transformation protocol for a local soybean variety PAN 512 was established for transformation with two designed RNAi constructs. Suitability of selected promoters was tested by attaching promoters to the gus gene and evaluating specificity of seed expression after soybean transformation using the Agrobacterium tumefaciens strain EHA101. Future work will focus on further optimisation of the transformation protocol and generation of transformed plants carrying the designed silencing vectors. Copyright