Expression And Interacting Protein Identification Of Rice Pi-Hit-1 Gene

Pi-hit-1 gene was identified by differential display PCR (DD-PCR), which expressed higher in space flight induced blast sensitive mutant 972-4 and on-ground control, 972ck. Sequence analysis revealed that pi-hit-1 gene encodes a member of ATP-dependent Clp protease family. To better understand the function of Pi-HIT-1, GST pull down technique was used to investigate its interacting proteins.The recombinant vector pGEX-Pi-HIT-1 was transformed into E.coil BL21 to express GST-Pi-HIT-1 fussion protein. Fusion protein was purified by Glutathione Sepharose 4 Fast Flow. Purified fussion protein was incubated with rice total protein extracts and complex of fussion protein and Pi-HIT-1 interacting proteins was obtained by using Glutathione Sepharose 4 Fast Flow. The complex was eluted and analysed on 12% SDS-PAGE. The interacting proteins were identified by mass spectrometry. Results showed that seven proteins interact with Pi-HIT-1, including Elongation factor 1-α, Nonspecific lipid-transfer protein 2 precursor, Zinc knuckle family protein, Fip1 motif family protein, and three hypothetical proteins containing ring Zn-finger, BTB/POZ domain or TPR-like domain respectively. Those proteins involve in DNA replication, transcription and translation control, cell apoptosis and signal transduction, cell cycle, plant defense, photosynthesis and respiration, plant growth and development. Taken together, our results indicate that these seven protens have potential functions in Pi-HIT-1 mediated blast resistnce change. In addition, Pi-HIT-1 may also functions in those biological processes.