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

Yıl 2017, Cilt: 18 Sayı: 2, 137 - 142, 20.10.2017

İsmail Poyraz , Merve Sağlam

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

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.

Öz

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.

Anahtar Kelimeler

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

Kaynakça

• 1. Aligiannis, N., Kalpoutzakis, E., Mitaku, S. & Chinou, B.I. 2001. Composition and antimicrobial activity of the essential oils of two Origanum species. Journal of Agricultural and Food Chemistry, 49: 4168-4170.
• 2. Attaran, E., Rostás, M. & Zeier, J. 2008. Pseudomonas syringae Elicits Emission of the Terpenoid (E,E)-4,8,12-Trimethyl-1,3,7,11-Tridecatetraene in Arabidopsis Leaves Via Jasmonate Signaling and Expression of the Terpene Synthase TPS4. Molecular Plant-Microbe Interactions, 21/11: 1482-1497.
• 3. Başer, K.H.C., Özek, T., Tümen, G. & Sezik, E. 1993. Composition of the essential oils of Turkish Origanum species with commercial importance. Journal of Essential Oil Research, 5: 619-623.
• 4. Baydar, H. 2005. The effects of different harvest dates on essential oil content and essential oil composition in Origanum minutiflorum O. Schwarz et. P.H. Davis. Akdeniz University Journal of the Faculty of Agriculture, 18: 175-178.
• 5. Baytop, T. 1999. Türkiye’de Bitkiler ile Tedavi, Geçmişte ve Bugün, ilaveli 2. baskı, Nobel Tıp Kitabevleri, İstanbul.
• 6. Bayrak, A., 2006. Gıda aromaları, Gıda Teknolojisi Derneği, Yayın no: 32, Ankara
• 7. Bolhman, J., Gilbert, M.G. & Rodney, C., 1998. Plant terpenoid synthases: Molecular biology and phylogenetic analysis. Proceedings of the National Academy of Sciences,95: 4126-4133.
• 8. Chen, F., Dae-Kyun, R., Petri, J., Gershenzon, J., Bohlmann, J., Pichersky, E. & Tholl, D. 2004. Characterization of a root-specific Arabidopsis terpene synthase responsible for the formation of the volatile monoterpene 1,8-cineole1. Plant Physiology, 135: 1956-1966.
• 9. Degenhardt, J., Köllner, T.G. & Gershenzon, J. 2009. Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry, 70(15-16): 1621-1637.
• 10. Davis, P.H., Mill, R.R. & Tan, K. 1988. Flora of Turkey and the Aegean Islands, Edinburgh Univ. Press, 10, 209, Edinburgh.
• 11. Fakıllı, O. 2010. Türkiye’de Kekik Adı ile Anılan Bitkiler Konusunda Yapılan Çalışmaların Envanteri. Master Thesis, Graduate School of Sciences, Çukurova University.
• 12. Huber, D.P. Philippe, R.R., Godard, K.A., Sturrock, R.N. & Bohlmann, J. 2005. Characterization of four terpene synthase cDNAs from methyl jasmonate-induced Douglas-fir, Pseudotsuga menziesii. Phytochemistry, 66: 1427-1439.
• 13. Jiang, Z., Kempinski, C. & Chappeil, J. 2016. Extraction and Analysis of Terpenes/Terpenoids. Current Protocols in Plant Biology, Doi:10.1002/cppb.20024.
• 14. Keszei, A., Brubaker, C.L., Carter, R., Köllner, T., Degenhardt, J. & Foley, W.J. 2010. Functional and evolutionary relationships between terpene synthases from Australian Myrtacea. Phytochemistry, 71: 844-852.
• 15. Kaloustian, J., El-Moselhy, T.F. & Portugal, H. 2003. Chemical and thermal analysis of the biopolymers in thyme (Thymus vulgaris). Thermochimica Acta, 401/2 77-86.
• 16. Köllner, T.G., Schnee, C., Gershenzon, J. & Degenhardt, J. 2004. The variability of sesquiterpenes emitted from two Zea mays cultivars is controlled by allelic variation of two terpene synthase genes encoding stereoselective multiple product enzymes. The Plant Cell, 16: 1115-1131.
• 17. Lambert, R.J.W., Skandamis, P.N., Coote, P.J. & Nychas, G.J.E. 2001. A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol. Journal of Applied Microbiology, 91: 453-462.
• 18. Lange, B.M. 2015. The evolution of plant secretory structures and emergence of terpenoid chemical diversity. Annual Review of Plant Biology, 66: 139-159.
• 19. López, N L, Nair V., Bang W.Y., Zevallos L.C. & Heredia, J.B. 2016. Protective role of terpenes and polyphenols from three species of Oregano (Lippia graveolens, Lippia palmeri and Hedeoma patens) on the suppression of lipopolysaccharide-induced inflammation in RAW264.7 macrophage cells. Journal of Ethnopharmacology, 187: 302-312.
• 20. Mewalal, R., Rai, D.K., Kainer, D.K., Chen, F., Külheim, C., Peter, G.F. & Tuskan, G.A. 2016. Plant-Derived Terpenes: A Feedstock for Specialty Biofuels. Trends in Biotechnology, Doi: http://dx.doi.org/10.1016/j.tibtech.2016.08.003.
• 21. Pichersky, E. 2006. Biosynthesis of plant volatiles: nature's diversity and ingenuity. Science, 311(5762): 808-811.
• 22. Poyraz, I., Sozen, E. & Arslanyolu, M. 2010. Isolation of quality total RNA from the aromatic plant Origanum onites. Zeitschrift für Naturforschung C, 65: 266-270.
• 23. Poyraz, I. 2007. Origanum onites’de Mitojenler Tarafından Aktive Edilen Protein Kinaz Kinaz (MAPKK) Enzim Ailesi Üyelerinden Birinin Klonlanması ve Enzimatik Olarak Tanımlanması. PhD. Thesis, Graduate School of Science, Anadolu University.
• 24. Poyraz, I. 2015. Molecular cloning and characterization of a mitogen-activated protein kinase kinase (OoMAPKK1) in Origanum onites L. (Lamiaceae). Journal of Plant Biochemistry and Biotechnology, 24(1): 75- 83.
• 25. Sarıhan, E.O., İpek, A., Arslan, N. & Gürbüz, B. 2006. Farklı sıra arası ve sıra üzeri mesafelerinin kekik (Origanum vulgare var. hirtum)’de verim ve verim öğeleri üzerine etkisi. Ankara Üniversitesi Ziraat Fakültesi Tarım Bilimleri Dergisi, 12(3): 246-251.
• 26. Tholl, D. 2006. Terpene synthases and the regulation, diversity and biological roles of terpene metabolism. Current Opinion in Plant Biology, 9: 297-304.
• 27. Tokul, H.E. 2015. İzmir kekiği (Origanum onites L.)’nde farklı su ve azot uygulamalarının verim ve kalite üzerine etkileri ile bunların fizyolojik denetimi. PhD. Thesis, Graduate School of Science, Ege University.
• 28. Wallaart, T.E., Bouwmeester, H.J., Hille, J., Poppinga, L. & Maijers, N.C.A. 2001. Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin. Planta, 212/3: 460-465.
• 29. Yahyaa, M., Tholl, D., Cormier, G., Jensen, R., Simon, P.W. & Ibdah, M., 2015. Identification and Characterization of Terpene Synthases Potentially Involved in the Formation of Volatile Terpenes in Carrot (Daucus carota L.). Roots. Journal of Agricultural and Food Chemistry., 63: 4870−4878.
• 30. Yang, C.Q., Wu, X.M., Ruan, J.X., Hu, W.L., Mao, Y.B., Chen, X.Y. & Wang, L.J. 2013. Isolation and characterization of terpene synthases in cotton (Gossypium hirsutum). Phytochemistry, 96: 46-56.
• 31. Zhang, P., Fuentes, S., Siebert, T., Krstic, M., Herderich, M., Barlow, E.W.R. & Howell, K. 2016. Terpene evolution during the development of Vitis vinifera L. cv. Shiraz grapes. Food Chemistry, 204: 463-474.