Domain And Its Function Analysis Of PeaT1 From Alternaria Tenuissima

PeaT1 is an Activator protein from Alternaria tenuissima, which can promote plant growth and plant disease resistance. To study PeaT1 domains function, which were analysed by bioinformatics. PeaT1 protein and its three deletion mutant proteins were expressed and purified.Their biological activities on plants were studied. The results were as follows:1. Expression vectors construction of peaT1 and three deletion mutantspeaT1 full-length gene was cloned, and bioinformatics analysis showed that PeaT1 had 2 conservative domains which were NAC(49-108AA)and UBA/TS-N(162-206AA). The three truncated fragments peaT1-△CD99,peaT1-△ND49 and peaT1-△ND108 were amplified by PCR reaction, and sub-cloned to pET-28a expression vectors .2. Expression and purification of PeaT1 and its mutant proteinsThe recombinant plasmids were induced by IPTG in E.coli BL21(DE3). The target proteins were purified and desalted by AKTA Explorer10.0. The standard MW of PeaT1 was determined by MS.3. Biological functions analysis and comparison of PeaT1 and the mutant proteinsThe results indicated that three mutant proteins could promote plants growth and plant disease resistance as PeaT1. There was no significant difference between PeaT1 and its mutant proteins in biological functions.Bioinformatics analysis predicted the NAC domain may act as molecular chaperone or combine TBP by OB configuration, which act as a transcription co-elicitor to excite the lower gene expression. The UBA/TS-N domain act as a translation co-elicitor as EF-Ts to promote some physiology related proteins expression. Two domains of PeaT1 may act similar biological functions.4. Analysis of proteins up-regulated from Oryza sativa by Stable isotope labeling strategyUnder the same conditions, PeaT1 and its mutant proteins treated Oryza sativa labeled by 15N (Oryza sativa labeled by 14N as CK). The proteomics expression level was analysed by MS. The results showed that PeaT1 and its mutant proteins could promote proteins expression of Oryza sativa. The further analysis confirmed that the up-regulated proteins of Oryza sativa were mainly related with photosynthesis proteins and translation factors.5. Thermal stability analysis of PeaT1 and its mutant proteinsThermal stability of PeaT1 and its mutant proteins were confirmed. This characteristic was attributed to their amino acids composition, molecular force in three-tier structure, and their rigid structure. The common features of PeaT1 and its mutant proteins as follows: there were more charged and hydrophobic amino acids, respectively formed salt bridge and hydrophobic force, which were both very important for proteins stability.A large number of Ala and Glu made proteins easy to formα-helix. On the other hand, all these proteins had less Gln,Asn,Trp and Cys, which were all harmful for protein,s stability because of ammonialyasion, oxidation and the fracture of disulfide bonds, respectively.