The Biological Function And Structure Analysis Of Microbial Protein Elicitor Pea T1

Protein elicitors are the type of proteins which can induce defense responses in plants. Apart from the advantages like non pollutant nature and lack of resistance development in target pests, protein elicitors meet the requirement of agricultural sustainable development and become a new approach for modern plant protection. PeaT1 is a protein elicitor derived from Alternaria tenuissima. The preliminary research indicated that it can promote crop vigour, yield and improve product quality. To further clarify its function, the biological function, mechanism of heat resistance, tertiary structure model of PeaT1 were deeply studied and the relationship between structure and function was preliminarily analyzed in this study. The main results provided scientific foundation for the function mechanism research of PeaT1:(1) Construction of engineered strain of PeaT1The peaT1 gene was ligated to modified expression vector pET28a. The expression protein was simplified, which only added 6×His tag to the N-terminus of PeaT1. Then the plasmid pET28a-peaT1 was transformed into E.coli BL21 (DE3) and got engineered strain for high cell density fermentation.(2) Expression and purification of PeaT1Through high density fermentation, PeaT1 was expressed in high yield and high efficiency. The purification of PeaT1 was done by affinity chromatography (Ni-NTA Resin), anion-exchange chromatography (Mono Q column) and size-exclusion chromatography (Superdex 200 column). The purity of PeaT1 protein reached 90%. The technical route for the preparation and purification of PeaT1 was established.(3) Biological function of PeaT1A series of bioassays were designed to detect the biological function of purified PeaT1. The results showed that PeaT1 could promote drought tolerance, cold resistance of rice, and induce the resistance of tobacco against TMV. This research demonstrated PeaT1 was a protein elicitor with comprehensive functions.(4) Structure research of PeaT11) The secondary structure of PeaT1 during temperature variation process was detected by synchrotron radiation circular dichroism. With the change of temperature, the ratio ofβstand kept stable and the ratio ofαhelix exhibited recoverable changes. This revealed thatβstand stabilizing the protein skeleton conformation and the recovery ofαhelix both contributed the heat resistance of PeaT1.2) The crystallization of PeaT1 was implemented by the hanging-drop vapor-diffusion method. Through optimization, crystals suitable for data collection were obtained at the condition: 20% PEG 3350, 0.2M MgCl2, 0.1M Mes pH6.0. The crystals were diffracted on a Rigaku X-ray diffractometer and a set of data with resolution at 2.4? was collected. The structure was solved by molecular replacement method using 1tr8A as template. Finally, the overall structure of NAC domain was constructed. In this model, NAC domain formed a homodimer from reverse direction. This structure outline was similar with a barrel, with a hole at the center and 6 intercrossedβstrands surrounded.3) The scattering information of PeaT1 at small angle region was determined by small angle scattering to gain the low resolution structure of PeaT1. The homodimer model of PeaT1 was constructed by integrating the structure information of PeaT1. In this model, the NAC dimmer localized at the center; The first part (1-62aa) and the third part (114-162aa) surrounded this center and formed heterogenetic association; The UBA domain distributed at two ends.(5) Relationship between structure and function of PeaT1PeaT1 was divided to four fragments according to the domain information. The four fragments were respectively expressed and purified. None of the four fragments had significant effect on inducing the resistance of tobacco against TMV. This revealed that the function of PeaT1 was executed by its full length peptides and the complete homodimer of PeaT1 may be the premise for its function.