The Characteristics Of Leifsonia Xyli Subsp. Xyli Hypothetical Membrane Protein (Lxx18460)

The cultivation, preparation and detection of polyclonal antibody for the ratoon stunting diseases (RSD) has been studyed, but there has not been seen any report on monoclonal antibody preparation, detection and genetic transformation of anti-sigma K (aK) factor (RskA) gene of the hypothesized membrane protein (MP), in Leifsonia xyli subsp. xyli (Lxx) in sugarcane yet. Based on the result of our previous research, we induced and purified the open reading frame fragment located in 349-717 which transmembrane domain was removed in Lxx 18460 gene. The protein induced in E. coli was used as antigen to produce monoclonal antibody by immuning pure bred BALB/C mice for many times. We detected the total bacterial proteins and the proteins from the infected and healthy sugarcane plants with the monoclonal antibody. The eukaryotic expression vector of Lxx 18460 gene was obtained and transformed into wild type tobacco, and detected by PCR. The results of this study are useful for further investigating the function of anti-sigma K factor and its role in Lxx. The main results were as follows.1. The bacterium suspenction containing the Lxx18460 gene pET-30 a plasmid prepared in our laboratory was used to induce the Lxx18460 gene protein expression with 0.1 mM IPTG. The induced bacteria suspention around was collected and put into a 50 mL of centrifuge tube, and centrifuged at 5000 rpm and 4℃ (pre-cooled) with for 20 min, and all supernatant was abandoned. The protein was purified by using two different protein purification methods which were compared for selection. The results show that the protein can be purified by both methods, but the improved method produced purer and more amount of the protein. The purified protein were further dialysised and enriched, and the protein concentration was detected with ultraviolet spectrophotometry before being used as antigens.2. The protein induced in E. coli was used as antigen to produce monoclonal antibody by immuning pure bred BALB/C mice for many times. ELISA test results showed that its titer was higher than 1:512000. Western blot test showed that the gene expressed in bacteria and Lxx infected sugarcane samples, but no expression in the healthy sugarcane samples.3. The eukaryotic expression vector of Lxx 18460 genes was obtained in this study and the corresponding verification was conducted. The results showed that carrier can be used for the next step.To obtain transgenic plants by further transforming tobacco.The phenotype of transgenic plants was observed. It was found that transgenic tobacco plants showed obvious dwarf symptoms compared with the control, indicating that the transgene inhibit the growth of tobacco in a certain extent.