Changes Of Defensive Enzymes Activity And Proteome In Sugarcane Under Leifsonia Xyli Subsp.xyli Stress

Sugarcane is a kind of C4plant, and its light energy conversion efficiency,photosynthetic rate and biomass productivity are significantly higher than other crops. It is the most important sugar crop and most promising energy crop in China. Ratoon stunting disease (RSD) is a bacterial disease spread by seedcanes and it has become one of the most serious diseases worldwide. Sugarcane infected with RSD has no obvious external symptoms, but the causal bacteria accumulate extensively in the basal stem and widely spread. The cane yield and sucrose content are lost due to the disease, especially for ratoon crops. Studies on the pathogenicity of the pathogen and the interaction between the causal bacterium and the host are limited because the pathogen is difficult to isolate from sugarcane. In the present study, RSD bacteria were isolated from RSD-infected sugarcane as antigen for the preparation of RSD polyclonal antibody. The RSD bacteria were injected into rabbits to obtain polyclonal antibody,and then inoculated into healthy sugarcane, the defense enzyme activity in infected and healthy sugarcane were detected, the differential protein expressions were analyzed with proteomics approach, the genes related to RSD stress were analyzed and cloned, Fluorescent quantitative expression analysis. The results are as follows.1. The sugarcane plants with RSD infected were identified by PCR and used as the experimental material. The pathogen bacterium from cane juice was successfully isolated on modified SC medium, and confirmed by morphology, PCR method and rDNA-ITs sequence alignment, respectively to ensure the colonies which are reliable pathogenic bacterium. The isolated pathogen Lxx was cultivated for enlargement, mixed and emulsified with Freund’s complete adjuvant, then injected into rabbits under multiple subcutaneous points for immunization, and the polyclonal antibody was obtained, and its titer was finally evaluated by ELISA. The results showed that the titer of RSD polyclonal antibody is greater than1:256000. Using the TBIAmethod, comparison of the RSD polyclonal antibody and from USA imported RSD polyclonal antibody showed that sugarcane with RSD infected were detected by both antibodies which were not significant different. The results indicated that RSD polyclonal antibody has been successfully made in the present study.2. Sugarcane variety GT11with RSD-infected and RSD-free were used as plant materials to analyze the defense enzymes activities. Under the RSD stress, the activities of CAT, SOD, POD, APX and GR increased in both leaf and stem, but the activities of different enzymes in the same organ or the same enzyme activity in different organs are distinguished.In stem, the activities of CAT, SOD, POD and GR in RSD-infected plants were lower in90days but higher in180days than those in control, while the APX activity showed higher in RSD-infected plants than that in the control. However, these defense enzymes activities in leaf showed the opposite trend as compared to the stem. The POD activity showed higher in the RSD infected plant than that in the control all the time, while the activities of CAT, SOD, APX and GR showed higher in90days and lower in180days in the RSD infected plant than those in the control. The different enzyme activities between leaf and stem may be due to the different time for invasion and colonization of the pathogen in leaf and stem.3. Two-dimensional electrophoresis technology was used to analyze differential protein expression under the RSD pathogenic bacterium stress. Forty differential protein spots have been found and amongy them,16were up-regulated and24were down-regulated. With Tandem Mass Spectrometry (MALDI-TOF-TOF/MS),23proteins were successfully identified which were divided into6categories according to their functions. They are the proteins for participating in defense response, cell growth and division, modification and processing, basic metabolism, signal transduction and unknown function that the ratios are31%,9%,18%,23%,5%and14%, respectively. The results indiacted that the response mechanism for Lxx stress is a integrated process involving multisystem and multilevel.4. Using homology-based cloning method, the full length of4genes associated with RSD infection have been cloned, and they are isoflavone reductase gene (SoIRL), NADP-isoctrate dehydrogenase gene (SoNADP-IDH), ethanol dehydrogenase gene (SoADH) and Caffeic acid,3-O-transferase gene (SoCOMT). Four of the genes were successfully expressed inE. Coli,andtheir expressedfused proteins were also obtained. Besides, fluorescence quantitative PCR analysis showed that the4genes were constitutively expressed in vivo, and the expressions could be induced by Lxx-infection, drought (PEG), low temperature (4℃), hormones (ABA) and high salinity (NaCl).