| Activator Protein is a type of protein elicitor from pathogen fungi with the properties of inducing plant resistance to pathogen, enhancing resistance to adversity and prompting seedlings growth. There were few reports about the characteristics, mechanism or bioactivity of Activator Protein at present. To understand the mechanism of Activator Protein-plant interaction, it is valuable to obtained purified protein and its functional gene. This is the first report about the purification procedures of Activator Protein from rice blast (Magnaporthe griesea.) and its bioactivities. The cDNA library was constructed successfully with rice leaves treated with/without Activator Protein by suppression subtractive hybridization. The functional gene of Activator Protein was cloned from M. griesea and a strain was acquired, in which the soluble Activator Protein with bioactivity was expressed stably. This research laid the foundation for further study of the structure and property of Activator Protein.A series of purification procedures of Activator Protein was set up. The crude protein extraction was firstly seperated with Hitrap Q FF ion exchange chromatography and then purified with HitrapSuperdex 75 column. The resulting heat-stable protein with the property of 40 KDa and pI 4.7 had no glycosyl. The amino acid sequences of 12 peptides were determined by mass spectrogram analysis. In comparison with the proteins in GenBank, all the sequences had 100% homology with a hypothetical protein originated from M. griesea whose ID was EAA52615.A wide range of bioactivities of Activator Protein was determined by bioassay. Seed germination and young seedling growth could be significantly promoted after treatment with purified Activator Protein. Activator Protein could also enhance the resistance to pathogen and aridity. The control effects to tomato grey mold and rice blast were 44% and 50.68%, respectively. The integrated drought resistance index of rice was increased from 55 to 92. Physiological and biochemical studies revealed that the activities of cellulase and alcohol dehydrogenase, the amount of hydrogen peroxide and proline were dramatically enhanced. These elements played important roles in promoting plant growth and enhancing stress tolerance.The SSH library was constructed and 28 up-regulated genes were selected. Two-dimensional electrophoresis and reversed phase chromatography results showed that Activator Protein could change the protein amount in rice seedling leaves. The amounts of a group of hydrophilic proteins were reduced while the amounts of another group of hydrophobic proteins were increased. A suppression subtractive hybridization (SSH) library was constructed with cDNA from rice leaves treated with/without activator protein. 1756 clones were screened from the library, 264 available clones were further selected by reversal-southern blotting and sequenced. Sequence similarity searches were performed with BLAST in GenBank database. 28 genes were identified as up-regulated ones. The results indicated that these genes were related to various physiochemical functions, such as photosynthesis, transportation and metabolism of nutrient substance.The functional gene of Activator Protein was cloned and expressed in E.coli. Its function ofpromoting plant growth was determined for the first time. Genome DNA sequences of activator protein cloned from Magnaporthe griesea. Northl-24, CH353, CH363, CH369 were the same, but they were not consistent with the whole genome sequences of Magnaporthe griesea in the GenBank database. It was confirmed that the sequence from Magnaporthe griesea Northl-24 was the right one by repetitive sequencing. Total cDNAs of Magnaporthe griesea were amplified by magnetic bead PCR and cDNA of Activator Protein was obtained from them. A new vector pET22b-GST was reconstructed and a strain obtained which could express stable, soluble Activator Protein was obtained. The biological activities of the expression protein had been determined.
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