Zeytin (Olea europaea) bitkisinden karbonik anhidraz enzimini kodlayan genin klonlanması

Abstract

Carbonic anbydrase (CA) is a zinc-containing enzyme that catalyzes the reversible hydration of carbon dioxide (CO2 + H2O ↔ HCO3 - + H+ ) and is widely distributed in animals, plants, archaebacteria, and eubacteria. There are five evolutionarily unrelated CA families: α, β, γ, δ and ζ. CAs are involved in a variety of biological processes including pH regulation, CO2 transfer, ion exchange, respiration, biosynthesis, and photosynthetic CO2 fixation. Expression of CA transcript is differentially up regulated in response to various abiotic stresses. In this study 392bp fragment of β-CA cDNA was cloned from leaves of olive exposed to cold stres. The sequence of cloned fragment shared significant homology with a region of Oleae europeae contig sequence of β-carbonic anhydrase which is found in NCBI databases. Forward and reverse primers were designed using sequence information of the contig. Firstly 3’RACE reactions performed to clone full length cDNA and a fragment about 1150 bp was obtained. The fragment was cloned and sequenced. The sequence showed more than 75% similarity to tobacco and tomato chloroplast CA and cotton and pea β-CAs in BLASTN and BLASTP analysis. Genomic DNA PCR was performed using forward and reverse primers and a fragment of 900 kb was synthesized. The genomic fragment was cloned and sequenced. The sequence of genomic clones showed 95-96% to the sequence of the cDNA clone.