Molecular analysis of lipid transfer gene selected from olive (Olea europaea) ESTs (expressed sequence tags) collection

According to the classification systems; the olive is a species in the Oleaceae family comprises 600 species in 24 genus [1]. Previously, in our department two cDNA libraries were constructed from young olive leaves and fruits for generation of ESTs (Expressed Sequence Tags), 3840 colonies which were randomly selected from the libraries were sequenced for obtained olive ESTs collection. The sequences were assembled in to contig with the Phred / Phrap, CAP3 programs. For this study, the presence of genes related to stress in EST collection, which prepared for the fruit and the olive leaves, were investigated. A major stress genes, lipid-transfer gene's sequence informations were found in EST's and contigs. This important stress gene, lipid transfer gene, was intended to clone. The purpose of gene cloning, full-length lipid-transfer gene was tried to obtain by using RACE (Rapid Amplification of cDNA Ends), is a method of molecular-based PCR (Polymerase Chain Reaction). Specific primers were designed as described in the user manual of the RACE kit to the 3 'and 5' ends which was obtained from the sequence information of EST and the contig which are related to lipid transfer gene. Total RNA was extracted from olive and used for obtaining cDNA by reverse transcription. As following Invitrogen RACE kit user manual instructions, specific primer was initially designed and the gene specific primer used for elongation of lipid transfer protein gene's 3' end. After ligation to the pGEM-T vector and transformation to E. coli compotent cells of the obtained DNA, plasmid was isolated and the plasmid was sent for sequencing which to find out if it was containing our interested gene. The sequence information was compared with the results in NCBI database and determined that it was a partial lipid transfer protein gene. The 5' end gene specific primer was designed using the sequence information and the primer was used for elongation of the cDNA's 5' end. After ligation and transformation steps, plasmid was isolated. The plasmid was sent for sequence analysis. The obtained sequence information was compared with the results in NCBI database and confirmed that it was lipid transfer protein gene. In this study, the preliminary studies were done to clone full length lipid transfer protein gene which is induced by stress and pariticipating to biological roles such as facilitate transfer of the lipids, defense reactions against pathogens, cutin formation, embryogenesis, symbiosis in Gemlik plant.