Analysis On Protein Elicitor Of Colletotrichum Destructivum

We have isolated the protein elicitor from mycelium of Colletotrichum destructivum by heating, centtrifugation, precipitation,dialysis and gel chromatography column.To functionally investigate the certain biological activity, mechanisms of disease resistance and defense response induced by the protein elicitor, we measured the prevention and protection by the spray and friction inoculation methods and we measured the dynamic activities of hydrogen peroxidase(POD), polyphenol oxidase(PPO), penylalanin ammonialyase(PAL), proline(PRO) and the contents of gene expression by RT-PCR technique. Our results not only purify a new protein elicitor from mycelium of Colletotrichum destructivum, and provide a supplemental method for controlling tabacco disease, but also provide a theoretical foundation for plant-elicitor interactions and for elucidatation of the mechanisms of the protein elicitor inducing plant resistance.1. We have isolated the crude protein elicitor from mycelium of Colletotrichum destructivum by heating, centtrifugation, precipitation and dialysis. The results showed that the molecular weight of the crude protein elicitor was approximately 38 kD on silver-stained SDS- PAGE. Through tobacco seed germination test, it concluded that the induced resistance protein treatment group had a higher rate of sprouting, growing overall neat, stem significantly longer than the control group. So germination treatment group was significantly better than the control group, so the induced resistance protein has a certain biological activity.2. According to induce anti crude protein molecular weight distribution, using Sephacryl S200(separation range 5-250 kD) to induce anti-gel column to separate and purify the crude protein and in accordance with a certain chromatographic purification conditions, we get four peaks. According four component of peaks, named Pro-P1 TX, Pro-P2 TX, Pro-P3 TX and Pro-P4 TX. The four fractions were concentrated and washed out a lot of salt, supernatant desalted salting part dialyzed through Sephadex G10. Finally, the purified protein fractions were isolated electrophoresis by using SDS- polyacrylamide gel electrophoresis.The results showed that the protein bands found in Pro-P2 TX, the other components were not found protein bands, so the target protein present in the Pro-P2-TX component.3. The crude protein elicitor and the pure protein elicitor can induce disease resistance in tabacco. The results showed that the resistance effect to tobacco anthracnose were 58.00%, 48.99% and 49.65% after 3, 5 and 7 days, respectively, and theresistance effect to tobacco powdery mildew were 83.26%, 80.76% and 78.60%, respectively. And the crude protein elicitor had some effects of induction resistance on Tobacco mosaic virus. The inhibitory effect to tobacco mosaic virus(TMV) was 73.79% respectively. And the pure protein elicitor could remarkably accelerate the individual plant height of 72.43%, leaf width of 61.06% and stem circumference of 29.05% respectively. Taken together, the protein elicitor has the role of inducing tabacco resistance and facilitating the growth.4. The activities of POD, PPO, PAL and PRO were increased significantly in the crude protein elcitor treatment of tabacco cell suspensions. The maximal activity of POD, PPO, PAL and PRO appeared after 8h, 12 h, 6h and 12 h treatment. Transcription of acid pathogenesis-related gene 1(PR1-a), basic pathogenesis-related gene 1(PR1-b) and non-expresser of pathogenesis-related gene 1(NPR1) was upregulated after the protein elicitor treatment as well.