Bir Aromatik Bitki Origanum onites L.’de Terpen Sentaz-6 (TPS-6) Geninin Kısmi Klonlanması ve Tanımlanması

Öz

Origanum onites L. (kekik), tıp kozmetik ve farklı endüstri alanlarında kullanılan aromatik bir bitkidir. Uçucu yağ içerir ve dünyada aromatik bir bitki olarak yaygın kültüre edilir. Bu yüzden yiyeceklerde bir baharat olarak da tercih edilir. Terpen sentaz-6 (TPS-6), aromatik bitkilerde sesqui-terpenlerin sentezinden sorumlu bir enzimdir. O. onites’de bu enzimin gen dizi bilgisi ve ifade karakteristiğinin incelenmesi, tıp ve kozmetik vb. endüstrilerde ham madde olarak terpenlerin sentezinin arttırılmasına katkı sağlayabilir. Bu çalışmada, O. onites’de TPS-6 geninin kısmi olarak klonlanması ve dizilenmesi amaçlandı. O. onites’den toplam RNA izolasyonu TRI Reagent solüsyonu ile gerçekleştirildi. O. onites mRNA’ları Ters Transkripsiyon Yöntemi ile cDNA’lara dönüştürüldü. TPS-6 geninin orta bölgesi, TPS-6 genine özel dizayn edilmiş dejeneratif primerler kullanarak PCR yöntemiyle çoğaltıldı. 481 bç büyüklüğünde çoğaltılmış DNA parçaları elde edildi ve PGEM T Easy plazmit vektörüne T4 DNA ligaz enzimiyle eklendi. Rekombinant plazmit vektör XLI-Blue konakçı hücresine (E. coli) transfer edildi. Konakçı bakterilerden rekombinant plazmit vektör saflaştırıldı ve DNA dizi analizi gerçekleştirildi. Elde edilen DNA dizisi, NCBI gen bankası veri tabanında TPS-6 geni için doğrulandı ve EBI veri tabanındaki InterProScan ve Clustal Omega programları ile analiz edildi. Diğer bitki TPS-6 genler ile karşılaştırmak için filogenetik dendrogram çizildi. Elde edilen gen dizi bilgisi, ifade analizleri kullanılarak bu genin verimliliğinin arttırılmasında biyoteknolojik araştırmalara rehberlik edecektir. 

Anahtar Kelimeler

• Origanum onites,TPS-6,Gene klonlama,DNA dizileme

PARTIAL CLONING AND IDENTIFICATION OF TERPENE SYNTHASE-6 GENE (TPS-6) IN AN AROMATIC PLANT Origanum onites L.

Abstract

Origanum onites L. (oregano) is an aromatic plant used in medicine, cosmetics and different industrial fields. It includes essential oil and is extensively cultivated as aromatic plant in the world. Therefore, it is also preferred as a spice in foods. Terpene synthase-6 (TPS-6) is an enzyme responsible for synthesis of sesqui-terpenes in aromatic plants. The investigation of gene sequence information and expression characteristics of this enzyme in O. onites may contribute to increase of synthesis of terpenes as raw materials in medicine and cosmetic etc. industry. In this study, partial cloning and sequencing of the TPS-6 gene in the O. onites was aimed. Total RNA isolation from O. onites was performed with TRI Reagent solution. The mRNAs were transformed to cDNAs by Revers Transcription Method. The middle region of TPS-6 gene was amplified by PCR method using degenerative primers designed for TPS-6. Amplified DNA fragments were obtained at 481bp. size and ligated to PGEM T Easy plasmid vector with T4 DNA ligase. The recombinant plasmid vector was transferred to the XL1-Blue host cells (E. coli) and the cloned DNA was sequenced. The partial TPS-6 DNA sequence was confirmed in NCBI GenBank database and registered with an accession number (MF983853). The obtained DNA sequence was analyzed with InterProScan and Clustal Omega software in EBI database. The analysis results showed that this DNA sequence has a specific motif (DXXD) for TPS-6 catalyzing cyclization reactions. The phylogenetic dendrogram was drawn for comparison with other plant TPS-6 genes. O. onites TPS-6 sequence was determined to be very close to O. vulgare TPS-6 on the dendrogram than other plant TPS-6s. The obtained gene sequence information will guide biotechnological researches in the productivity increase of this gene using expression analysis.

Keywords

• Origanum onites,TPS-6,Gene cloning,DNA sequencing

References

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