Purification,Antibody Preparation And Quantitation Of Rubisco In Rice Leaves

Rubisco is the key enzyme in photosynthesis in green leaves. The level of Rubisco in a leaf is usually clearly related to photosynthetic activity. The studies of Rubisco in rice leaves have signification to the improvement of rice production. Rubisco is a protein encoded by nuclear genes and chloroplast genes. It can be used as a genetic marker in hybrid analysis, species identification, and so on. The purpose of this study was to set up suitable techniques for the purification and quantitation of rice Rubisco in our lab. The results showed that:A new method for rice Rubisco purification was introduced in this paper. Extracts were made in a medium containing 50mmol/L Tris-HCl, pH8.0, 20mmol/L MgCl2, 1mmol/L EDTA, 5mmol/L DTT, 10mmol/L Vc and 0.5% Gly. Then the crude extracts were purified by 30-50% fraction precipitation of (NH4)2SO4 and preparative polyacrylamide slab gel electrophoresis. Purity of the enzyme preparations was established by polyacrylamide gel electrophoresis and SDS dissociating electrophoresis.Antisera against Rubisco were raised in two white rabbits using rice Rubisco. Before injection, Rubisco were emulsified with equal volume of Freund's complete(or incomplete) adjuvant. One rabbit was injected 3 times with 1.7mg antigen. The other was injected 5 times with 2.9mg antigen. At last, antibody was detected in both of them.Rocket immunoelectrophoresis was chosen to quantitate Rubisco. Some improvements to this technique were involved: (a) the gels were prepared in Tris-HCl buffer(pH8.6). (b) Tris-Gly buffer(pH8.3) was used as electrophoresis buffer.(c) 20-24 hours at 4'Cwith 12v/cm constant voltage was the suitable electrophoresis condition, linea correlation between The height of rocket peak and the content of Rubisco was found in the range of 1-2.5 u g Rubisco content.