Regulation Patterns Of Ethylene Signal Transcription Factor LeERF1 And LeERF2 In Tomato

Ethylene is an endogenous plant hormone that influences many aspects of plant growth and development. Control of ethylene responses is conducted by ethylene signal transduction pathway. Ethylene Responsive Element Binding Proteins (EREBPs) are transcriptional factors function in the ethylene signaling pathway in plants, helping to regulate ethylene responses by regulating genes transcription and expression. LeERFl and LeERF2 are identified as members of the EREBPs family, characterized by a conserved ERF domain. In order to further examine their regulation patterns, some works have been down as follows.(1) Cloning and identification of LeERF1 and LeERF2 gene: The entire coding sequences of LeERFl and LeERF2 were cloned by RT-PCR from breaking tomato fruit. The PCR products were subcloned into prokaryotic protein expression vectors pET-30a to generate two recombinant plasmids pET-LeERF1 and pET-LeERF2 in E.coli BL21.(2) Expression and purification of LeERFl and LeERF2: LeERF1 and LeERF2 proteins were expressed under 1.0mM IPTG induction for 3 h at 37℃ in E.coli BL21. The expression level of LeERF1 was about 53% of total cellular proteins, and the expression level of LeERF2 was about 58% of total cellular proteins. The purification level of LeERFl was about 96% purified by Ni-NTA agarose column, and the purification level of LeERF2 was about 99% purified by Ni-NTA agarose column. LeERFl and LeERF2 recombinant protein have GCC box binding activity identified by Electrophoretic Mobility Shift Assay.(3) Anti-LeERF1 and Anti-LeERF2 polyclonal antibodies: The anti-LeERF1 and anti-LeERF2 serum were obtained by immunizing rabbits, and titer of either anti-LeERFl serum or anti-LeERF2 serum was above 10000. The purification level of Anti-LeERF1 polyclonal antibody was about 98% purified by DEAE-52 ion-column, and the purification level of Anti-LeERF2 polyclonal antibody was about 99% purified by DEAE-52 ion-column. Both Anti-LeERFl and Anti-LeERF2 polyclonal antibodies had specific immunization function identified by Western blotting.(4) Subcellular localization of LeERFl: LeERFl was subcellular localized by the immunocytochemical localization in vascular tissues of tomato fruit. LeERF1 were mainly localized in the nucleus (N), nucleolus (Nu) and plastid (P), and Little LeERFl signal was detected in the cell wall (CW) and vaculoe (V) . During the phase of immature green stage (IG), there were a few LeERF1 in the cells of tomato tissues. As the development, the signal of LeERFl increased obviously in the nucleus (N) , nucleolus (Nu) and plastid (P). There were larger LeERF1 in breaking tomato fruit than in green tomato fruit identified by Western blotting. The experiments showed that LeERFl had relationship with mature of tomato fruit.(5) Separation and identification of LeERFl binding promoters: TLBP1 and TLBP2 were cloned by the binding method of LeERFl protein-DNA in vitro. The sequence of TLBP1 was 893bp, and TLBP2 was 734bp. Both TLBP1 and TLBP2 all had GCC box, and TLBP1 had TATA box and CAAT box. The alignment showed that LeERFl and TLBP2 were homologous with tomato promoter genes of AY343058/AY343059/AY343060, which encoded chitnase, with above 80% similarity at nucleotide acid sequence level.(6) Relationship between LeERFl and pathogen of tomato fruit: Tomato fruits, at the stage of green, breaking, pink and red fruits, were infected by Penicillium expansum Link. The results showed that LeERF1 was increased with the raise of ethylene, and LeERF1 was regulated by ethylene at the level of protein. There were the same changes trends between LeERFl and mRNA of LECHI17, which encoded chitnase, and there were the same changes trends between mRNA of LECHI17 and chitnase. The experiments showed that LeERFl might regulated Penicillium expansum Link pathogen of tomato fruit by controlling transcription and translation at the level of protein.