Antibody Production against the heterologous expressed PGIP1 to confirm the transgenic canola plants

Phytopathogenic fungi cause serious yield losses of oilseed crops including canola. Many phytopathogenic fungi produce polygalacturonase enzymes (PGs) which are thought to play an important role to degrade the pectin component of plant cell walls. On the other hand polygalacturonase-inhibiting proteins (PGIPs) which are cell wall bounded proteins are secreted by plants and they defend the cell walls against PGs. Due to the lack of commercial PGIP1 antibody, in this study PGIP1 was expressed in prokaryotic system to be used for production of antibody. The pgip gene was amplified using two primers RB and RB2H contaning NdeI and XhoI sites respectively and cloned in pET26b(+).The new construct was confirmed and transferred to E. coli BL21 (DE3) pLysS. The optimized conditions for expression of PGIP1 was IPTG = 0.5mM, OD = 0.3 and 37ºC for culture temperature. The expression of PGIP1 was confirmed using Anti-His antibody due to the presence of His-tag in expressed protein. The antibody against the expressed protein (PGIP1) was produced and the obtaining serum tested by Elisa. This antibody used for Western blotting to confirm the presence of the expressed PGIP1 in protein profile of transgenic canola plants. Western blot analysis of transgenic lines compared to non-transgenic line of canola plants confirmed the expression of PGIP1 in transgenic lines.