The Resistance To Fungal Pathogens And The Subcellular Localization Of Arabidopsis ELHYPRP2

AtELHYPRP2(EARLI1-LIKE HYBRID PROLINE-RICH PROTEIN2, AT4G12500) is a member of EARLI1subfamily in Arabidops is thaliana. It possesses an open reading frame of534base pairs in length and has no intron. The protein encoded by AtELHYPRP2consisits of a N-terminal signal peptide (1-26), a middle hydrophilic proline-rich domain (PRD,27-94) and a C-terminal conservative hydrophobic eight cysteine motif (8CM,95-177). The theoretical isoelectric point and the relative molecular weight of this protein are8.93and18.35kDa, respectively. Mining to the results came from microarray experiments and stored in database indicated that AtELHYPRP2could be induced by biotic and abiotic stresses, suggesting it functioned in defense response of A. thaliana.In the present work, the coding sequence of AtELHYPRP2was isolated by polymerase chain reaction and the genomic DNA of Arabidopsis ecotype Col-0was used as template. Because AtELHYPRP2contains a recognition sequence of Nco Ⅰ, the restriction sites of Pag I (isocaudamer of Nco Ⅰ) and Spe I were added in5’-end of the upstream primer and the downstream primer, respectively. After digestion with Pag I and Spe Ⅰ, the amplified fragment was ligated into Nco Ⅰ/Spe Ⅰ digested pCAMBIA1302to produce the fusion expression vector pCAMBIA1302-AtELHYPRP2-GFP. Then the leaf explants were infected with Agrobacterium tumefaciens strain LBA4404harbouring the recombinant plasmid and transgenic tobacco plants were regenerated on selection medium. RT-PCR and Western blotting analyses confirmed that AtELHYPRP2-GFP fusion construct expressed effectively in these transgenic tobacco plants. Observation under laser confocal microscopy revealed that the green fluorescence of AtELHYPRP2-GFP fusion protein could overlap with the red fluorescence came from counterstain with propidium iodide, indicating AtELHYPRP2is localized to cell surface and mainly exists in cell wall. Inoculation of the leaves from transgenic plants with the conidia of Gibberella fujikuroi showed that the constitutive expression of AtELHYPRP2could enhance the resistance of tobacco to fungal pathogens, and the infection sites could accumulate H2O2obviously. Analysis to the systemic acquired resistance (SAR) revealed that the basal expression levels of PR1and the systemic expression levels of PR1and PR5in transgenic tobacco plants were higher than that of the wild-type plants, suggesting AtELHYPRP2is associated with the systemic immunity of plants.In addition, a prokaryotic expression vector was constructed in this work. The coding sequence of AtELHYPRP2lacking the signal peptide region was amplified from genomic DNA of Arabidopsis ecotype Col-0at first, and was further inserted into pET28a after digestion with restriction enzymes to produce pET-28a-AtELHYPRP2. Escherichia coli BL21(DE3) strain transformed with pET-28a-AtELHYPRP2was identified by colony PCR. These works laid a good foundation for subsequent large-scale preparation and in vitro antimicrobial test of the recombinant protein.