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

Year 2017, Volume: 18 Issue: 2, 137 - 142, 20.10.2017

İsmail Poyraz , Merve Sağlam

https://doi.org/10.23902/trkjnat.291972

Abstract

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. 

Keywords

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

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