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| Cloning and Sequencing of EIN3 gene from Cucumis sativus, Citrullus lanatus and Cucurbita maxima |
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Abstract Ethylene-insensitive3 (EIN3) and EIN3-like (EIL) proteins are essential transcription factors in the nucleus under the downstream of the ethylene gas signaling transduction. In this paper two oligonucleotide primers were designed from conserved domain of EIN3 and EIL gene family. The forward primer sequence of the primer was 5’ —ttg gag agg agg atg tgg ag—3’ and The reverse primer 5’ —ata ata gca agc cag gta gc—3’. PCR amplifications were performed on Cucumis sativus, Citrullus lanatus and Cucurbita maxima DNA template and produced three fragments of 725 bp Separately. The three sequences that highly matched with other 151 sequences of EIN3 gene were all EIN3 genes by using the program of BLASTn on NCBI GenBank database. The three 725 bp fragments of EIN3 gene in Cucumis sativus, Citrullus lanatus and Cucurbita maxima were deduced 241 amino acid sequences via using the program of ORF Finder (Open Reading Frame Finder). The three sequences had been submitted to the GenBank database and the accession number is AY973275、DQ023225、DQ￥023224 separately. By using the program of BLASTp on NCBI GenBank database, The results which were searched out in MMDB (Molecular Modelling Database) indicated that putative conserved domain 3D structure of 241 amino acid sequences in Cucumis sativus, Citrullus lanatus and Cucurbita maxima had the same 3D structure which are very similar to structure of the DNA-binding domain of ethylene-insensitive3-like3 of the 1WIJA in Arabidopsis thaliana. The results of phylogenetic analysis showed that the EIN3 family from the dicot of cucumber, watermelon, pumpkin, melon, mungbean, barrel medic, tobacco, tomato, Arabidopsis thaliana and the monocot of rice, phalaenopsis equestris had a closer phylogenetic affinity.
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Received: 15 September 2006
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