Cloning And Expression Of Several Genes Encoding Ethylene Signal Transduction Elements In Peach Fruit

Ethylene is a gaseous plant hormone that plays a key role in many processes. To explore the roles of ethylene in plant functions, it is important to know the molecular dissection of signal transduction. Peach (Prunus persica'Feicheng') is a special local product and typically climacteric fruit, which is sensitively affected by ethylene. In this experiment, four genes encoding ethylene signal transduction elements in peach fruit were cloned. Steady-state transcript levels of PpEILs and PpEBF1 were assessed by quantitative real-time PCR. The main results are as follows:1. Two cDNA fragments, named as PpEIL1 and PpEIL2, were isolated from mature peach fruit by RT-PCR using degenerate primers corresponding to the EIN3/EIL conserved region sequence, Both 5′and 3′end sequences of the two cDNAs were cloned using RACE technique. PpEIL1 and PpEIL2 were 2 086 and 2 557 bp in length, respectively, while the predicted open reading frames (ORF) of their cDNAs were both 1 803 bp. The similarity between PpEIL1 and PpEIL2 was only 42% at amino acid level, but 86% between DNA-binding domains of the two cDNAs. The results of phylogenic analysis indicated that PpEIL1 and PpEIL2 could be classified into two different groups of EIN3/EIL protein family.2. According to the nucleic acid sequence from EST database, specific primers were designed and a 3 057 bp cDNA fragment containing 3′and 5′untranslated region was amplified by RT-PCR from ripening peach fruit. The cDNA, named PpEBF1, contains an ORF which is 1 941 bp in length. Based on the nucleic acid sequence from EST database, specific primers were designed and a 765 bp cDNA fragment containing 3′untranslated region was amplified by RT-PCR from ripening peach fruit. The cDNA, named PpERF1, contains an ORF which is 708 bp in length. PpERF1 contains a highly conserved, plant-specific DNA-binding domain, which is composed of threeβ-sheets and oneα-helix. Multiple alignment of the ERF conserved domains shows that PpERF1 has 68.4%-100% identity to ERF homolog in other plants. PpERF1 shared high similarity at both nucleotide and polypeptide level with corresponding sequences from Arabidopsis, grape and pea.3. Quantitative real-time PCR analysis revealed that PpEILs and PpEBF1 gene expressed at different levels in peach flowers and different ripening stage of fruits. Accumulation of PpEILs and PpEBF1 transcripts clearly increased after 1ul/L of exogenous ethylene treatment for 12 h and 24 h at 20℃. PpEILs and PpEBF1 mRNAs accumulation were affected by 1ul/L of 1-MCP in peach fruits at 20℃. A marked reduction of specific transcript accumulation were detected in 1-MCP treated peaches.