Inhibition Effects Of The Recombinant EARLI1Protein To The Rowth Of Botrytis Cinerea And Saccharomyces Cerevisiae

Based on the high similarity of its83-amino-acid-long C-terminal8CM to plant nsLTPs, EARLI1has been classified as a putative number of lipid transfer protein family (LTP). Previous researches indicated EARLI1might be involved in maintenance of membrane or cell wall stability, and participated in cutin formation, surface wax formation, embryogenesis, defense reactions against phytopathogens, or in the adaptation of plants to various environmental conditions.To identify the resistance of EARLI1against fungi, its recombinant form was expressed in Escherichia coil. Genomic DNA of Arabidopsis ecotype Col-0was used as template in amplification of the encoding sequence of EARLI1lacking the signal peptide by polymerase chain reaction because it has no intron. The fragment was double-digested with BamH I and Xho I. and ligated into the prokaryotic expression vector pET-28a. After sequencing, the correct recombinant plasmid was introduced into BL21(DE3) strain of E. coli. The expression of EARLI1was induced with IPTG. fractionated by SDS-PAGE, confirmed with Western blotting and purified via affinity chiomatography. SDS-PAGE analysis showed that EARLI1lacking the signal peptide was mainly expressed as inclusion bodies. In in vitro experiments, recombinant EARLI1showed significant antifungal activity to Botrytis cinerea, and addition of the recombinant EARLI1in liquid medium could repress the growth of Saccharomyces cerevisiae obviously. In order to confirm the in vitro antifungal activity of EARLI1, the intact encoding sequence of it was amplified and inserted into pESC-URA under the control of GAL1promoter. Expression of EARLI1in Saccharomyces cerevisiae after induction with galactose also showed remarkable inhibition effect on the growth of yeast cells.In the present work, wild-type Col-0and EARLI1overexpressing Arabidopsis plants were used for infection analyses with Pseudomonas syringae ES4326. Compared to the overexpressing plants, more dead cells could be observed on leaves of wild-type plants after Trypan blue staining. In addition, the infection sites on leaves of overexpressing plants could be stained more intensively by DAB, indicating P. syringae could trigger hypersensitive response which was characterized by accumulation of larger amounts of H2O2in cells surrounding the infection sites on leaves of EARLI1overexpressing plants, and the local programmed cell death could prevent the spread of the pathogens. All these results showed that EARLI1plays important roles in defense system of Arabidopsis to biotic stresses.