Isolation and characterization of a hexose converter from olive

Abstract

Introduction:Hexose sugars are key components of glycolysisand photosynthesis. The genes regulating their conversion intoone another, is therefore, of great importance for the control ofcarbon metabolism. In this study, we report isolation and charac-terization of a cDNA associated with conversion of hexoses inolive. The cDNA putatively named aldolase based on bioinfor-matic and experimental analyses.Material-Methods:Characterization based on nucleotide andamino acids were conducted using bioinformatic tools such asnucleotide and protein BLAST, BioEdit, Primer3, FinchTV, CLCGenomic Workbench, ExPASY, TargetP, SOSUI and Web Pro-moter Scan. Comparison of the genomic and cDNA sequence ofthe gene and detailed bioinformatic analyses including cellularlocation, hydropathy analysis, amino acid-nucleotide compositionand predicted 3D structure were also conducted using the bioin-formatics tools mentioned above. Temporal expression pattern ofthe putative aldolase were conducted using real-time PCR experi-ments. SDS and Western blot analyses were completed while bio-chemical analyses are ongoing. Results:The cDNA clone identified from a cDNA library weconstructed was analyzed and labeled as a putative aldolasebased. This similarity was confirmed with detailed BLAST analy-ses. Real-time PCR analyses revealed the putative gene expressed3–5 fold more than the housekeeping gene (GAPDH) in leaves.Polymorphism analysis revealed olive aldolase had multiple SNPsamong about 30 cultivars while Ayvalık and Cormona cultivarswere the closest to each other based on this sequence. The puta-tive aldoase cDNA was transferred into bacteria to express theprotein it encodes. The protein was displayed on SDS-PAGE andWestern blotting analyses which will be followed by biochemicalcharacterization assays.Acknowledgements: This work was supported by T€UB_ITAKwith grant number 110O108.Keywords:Olea europaeaL., Bioinformatic analyzes, hexosealdolase, Real-time PCR, SDS-PAGE.