Prokaryotic Expression, Antiserum Preparation And Immuno-gold Location Of Wheat Mlo Gene

Wheat powdery mildew, caused by Blumeria graminis f sp. tritici is a severe fungal disease that makes drop in the production and quality of wheat, especially in all the north of china. Because the pathogen has many physiological races and the pathogen can change rapidly, moreover, the race-specific resistance of wheat is easily lost, the disease can't be controlled through traditional methods. Mlo gene. first discovered in barley, is a kind of genes related to the broad-spectrum resistance to most races of powdery mildew. The mutant of Mlo gene can give barley a broad-spectrum resistance to almost all known isolates of powdery mildew pathogen. In order to understand the mechanism of mlo-resistance clearly and take the advantage of the gene in wheat molecular breeding, we study the functions of Mlo gene and MLO protein through molecular cytological methods.- The fragment encoding the polypeptide of wheat MLO protein C terminus located in cytoplast was cloned into the vector pET-30a and expressed in the strain BL21 of E coli. After induction by IPTG at 37 for 5 h, the expressed protein was purified by Ni2+-NTA agrose column. And then the antiserum was raised by immunizing New Zealand rabbit. Western blot and immuno-gold labeling showed that the antiserum gained was specific reaction with the expressed fusion protein and wheat MLO protein.The study of MLO protein's immuno-cytochemical localization in wheat leaf and powder mildew pathogen was based on the antiserum. In the experiment, we found the time that the first antibody binds the sample made a good role in the final results. So we made 120 minute as the first antibody's label time. Then the antiserums, including wheat MLO protein antiserum, barley MLO protein antiserum. Cl protein antiserum, streptavidiu antiserum, cellulose antiserum and rabbit serum. were used to study the immumo-cytochemical localization of wheat MLO protein. The results showed that MLO protein was detected especially in hypha and conidiaof powdery mildew pathogen. Furthermore, the result that much more high density of MLO protein antiserum on choloroplast of wheat leaf than rabbit serum maybe tell us MLO protein is a kind of plant proteins.