Preparation And Application Of Wheat Rubisco And Rubisco Activase Antibodies

Based on the traditional method, the fast anion exchange (DEAE-TOYOPEARL-650M) liquid chromatography was used to purify Rubisco activase from wheat leaves, which showed two predominant bands at 41 000 and 45 000 by SDS-PAGE. The activity of Rubisco activase was measured by 14C-marking method, which suggested the purified Rubisco activase can increase Rubisco activity obviously, but need the existence of ATP regenerating system.Monospecific polyclonal antiserums against purified Rubisco activase and Rubisco were produced commercially in New Zealand rabbits respectively by using in-skin, multi-spot, interval injection. Through cross titration method, we selected the optimal working concentrations and titers of Rubisco activase and Rubisco. The titers of the two antibodies against Rubisco and Rubisco activase were 1:25600 and 1:51200. The optimal antigen working concentrations of Rubisco and Rubisco activase were 0.95 μ g · ml-1 and 0.88μ g · ml-1. The optimal antibody working concentrations against Rubisco and Rubisco activase were 1:800 and 1:1600.We designed several treatments according to different illumination intervals after 72h darkness, light intensity, and nitrogen fertilizer on two wheat species, and an enzyme linked immunosorbtion assay(ELISA) was developed for the measurement of Rubisco and Rubisco activase in horsebean, corn and rape leaves. The results were followed as: Light and nitrogen fertilizae can enhance the synthesis of Rubisco and Rubisco activase. The concentrations of two enzymes from corn leaves were less than the concentrations of two enzymes from bean and rape leaves. The variation trend of chlorophyll content was the same as the variation trend of Rubisco and Rubisco activase content in the correspondent treatments.