Development of a high throughput, semi-quantitative dot blot to detect HME protein in ryegrass

Cookson R, Roberts N, Crowther T, Beechey-Gradwell Z, Cooney L, Bryan G and Winichayakul S

AgResearch Grasslands, Tennent Drive, Private Bag 11008, Palmerston North 4442, New Zealand.

High Metabolizable Energy (HME) technology utilizes two recombinant proteins to increase the energy content, change the fatty acid profile, and increase the biomass, of transformed plants. Over-expression of optimised Tropaeolum majus diacylglycerol acyltransferase (DGAT) and a modified oleosin from Sesamum indicum seeds allows excess lipid to be stored in cross-linked oil bodies in leaf tissue. Our HME ryegrass (Lolium perenne) breeding programme has resulted in large populations of T2 segregating lines, which require a fast, accurate assessment of HME protein presence in order to distinguish between null and HME seedlings. Here we describe the development of an immuno-dot blot system to detect sesame oleosin via a simple plant extraction, which can be configured for high throughput or formulated as a semi-quantitative assay for smaller numbers of samples. Due to the robustness of detection after various tissue treatments (including freeze drying), the assay offers an improved approach for detecting sesame oleosin in ryegrass blades, compared to SDS-PAGE immunoblotting. In addition, cost and speed of result per sample extracted are greatly improved. Validation of the protocol involved demonstration that the sesame oleosin protein level positively correlated with the HME traits and with Fatty Acid Methyl Esterification (FAMES) Gas Chromatography-Mass Spectrometry (GC-MS) data. Using generic laboratory equipment, this method provides a valuable tool for the rapid identification of plants expressing HME-based technologies.