Functional Study Of Eliciting Plant Response Protein Gene Epl1from Trichoderma Aspereullm

Fungi are the most common plant pathogens and fungal disease is accounting for80%of plant disease. Biological control of plant fungal phytopathogen is safe to human and other living beings, environmentally sound, and socially acceptable control.Based on the overwhelmingly positive features of biological control, it is the prime candidate in the search for reducing dependency on chemical pesticides.In order to investigate the function of eliciting plant response protein1gene(Epl1) from biocontrol fungus Trichoderma asperellum ACCC30536, the series of primers were designed based on the transcriptomics sequence of Epll and the total RNA and genome DNA extracted from T. asperellum ACCC30536as the template. Using PCR method, the full length cDNA and DNA sequence of Epl1gene finally were obtained, with accession numbers of HM572232and JF970203, respectively. The cDNA sequence of Epl1was690bp in length, encoding139amino acids. BlastP search indicated that Epl1amino acid sequence shared the highest similarity of92%with Epl1of T. atroviride (CAL80754). Furthermore. signalP prediction showed that the Epl1amino acid sequence was cleaved by signal peptidase between positions18and19(VSA//DT). Pfam protein family prediction showed that Epl1protein belonged to cerato-platanin family. To obtain homologous gene sequences of Epl1from the genome databases of four species Trichoderma, the amino acids sequences of Epl1from T. asperellum ACCC30536was used to as queries, resulting that3eliciting plant response protein sequences with high similarity were obtained from the T. virens Gv29-8, T. atroviride ATCC74058and T. harzianum CBS226.95, respectively, and5similarity sequences from T. asperellum CBS433.97. Epl1amino acid sequence shared the highest similarity of91%with EPL1-Tas, located on scaffold7of T. asperellum CBS433.97.Real-time RT-PCR was conducted to analyze the expression of Epl1in Trichoderma asperellum after treated by eight different culture conditions. The results indicated that the Epl1gene is differentially regulated by treatments of different culture condition. The expression of the Epl1gene was down-regulated during the MM(0.5%glucose),N starvation,1%Populus dividiana×P. bolleana root powder culture and showed lowest expression at4,72,4h and12.97,10.82,31.91times lower than pretreatment. Gene Epl1is mainly up-regulated under C starvation,1%stem powder,1%leaves powder,1%Alternaria alternata cell wall,5%A. alternata fermentation liquor culture condition and showed peak expression level at72.12,8,72,72h, respectively. The peak expression was higher7.15,373.56,45.00,75.57,149.92times than before treatment, respectively.The cloned Epl1gene was inserted into Prokaryotic expression vector pGEX-4T-2 and recombinant expression vector was named pGEX-Epl1. The recombinant pGEX-Epl1was transformed into competent Escherichia coli BL21and gene of Epl1was expressed in E.coli BL21induced by IPTG, the protein bands of40.5kDa was observed by SDS-PAGE. The result conformed to expectation, indicating that Epl1gene has been expressed in E. coli, also suggesting that the gene from T. asperellum ACCC30536could express well in E. coli.In conclusion, the cloning of Epll gene has laid the foundation for the research on the three dimensional structure, function and protein property, provides technical support for further study on plant systemic resistance of poplar eliciting by recombinant protein Epl1, and provides a material basis for developing a biological pesticide of recombinant protein Epl1.