Transgenik patateste farklı promotörlerin aktivitesinin karşılaştırılması
Yükleniyor...
Tarih
2019
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Niğde Ömer Halisdemir Üniversitesi / Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu tez çalışmasında patatesten elde edilen yeşil dokuya özgü pRCA promotörü ile Arabidopsis thaliana bitkisinden elde edilen ve stres koşullarında uyarılabilen pRD29A promotörünün Lady Olympia patates (Solanum tuberosum L.) çeşidinde gen ifade seviyelerine bakılmıştır. Çalışmada konstitütif -35S CaMV promotörü kontrol olarak kullanılmıştır. Seçilen promotörler GUS-INT genini kontrol edecek şekilde pCAMBIA-1301 plazmidine yerleştirilmiştir. Bitki transformasyonu Agrobacterium tumefasiens bakterisinin LBA4404 suşu aracılığıyla sağlanmıştır. Eksplant olarak yaprak ve boğum araları kullanılmıştır. Elde edilen transgenik hatlar histokimyasal GUS analizi, PCR, gen ifadesini belirlemek için qRT-PCR ve fluorogenik assay analizi kullanılarak doğrulanmıştır. Laboratuvar koşullarında transgenik bitkilerin taranması için higromisin fosfotransferaz (hptII), en uygun konsantrasyonda (5 mg/l) kullanılmıştır. Transformasyon etkinliği % 0,98 olarak hesaplanmıştır. pRD29A içeren transgenik patateslerde GUS gen ifadesinin kontrol koşullara göre farklı abiyotik stresler altında 10 kattan fazla arttığı gözlemlenmiştir. pRCA promotöründe kök/yumru kısımlarına göre yapraklarda GUS gen ifadesi 4 kat artmıştır. Bu promotörlerin aktivitelerine bakılarak, ileride farklı genler ile beraber kullanılmaları durumunda patateste transgenik teknolojinin risklerini minimuma indirmek ve olumsuz çevre koşullarına dayanıklı patates hatları geliştirmek mümkün olabilir.
In this thesis, the efficiency of green tissue specific (pRCA, isolated from potato) and stress inducible promoter (pRD29A, isolated from Arabidopsis thaliana) was compared in transgenic potato (Solanum tuberosum L.) plants in comparison with constitutive promoter (35S CaMV). The promoter fragments were cloned upstream to a reporter gene (GUS-INT) in pCAMBIA-1301 binary vector. Agrobacterium tumefaciens strain LBA4404 harboring recombinant binary vector was used to infect leaf discs and internodal explant of Lady Olympia cultivar. Transgenic lines were verified by GUS histochemical analysis, PCR, gene expression via RT-qPCR and fluorogenic GUS assay techniques. Hygromycin phosphotransferase (hptII) was used at optimal concentration (5 mg/l) for the screening of in vitro cultured putative transgenic plants. Overall transformation efficiacy was calculated as 0,98%. As a result of the validation methods, pRD29A transgenic potatoes were observed to increase GUS gene expression more than 10 times in different abiotic conditions compared to controls. In pRCA promoter, GUS gene expression was increased 4-fold in leaves compared to root / tuber parts. The results of this study encourage the appropriate use of promoter to drive the expression of foreign gene(s) for the development of potato lines resistant/tolerant against biotic and abiotic stresses. Thus, minimizing the risks of transgenic technology can be possible in potatoes using different genes under suitable promoters in the future.
In this thesis, the efficiency of green tissue specific (pRCA, isolated from potato) and stress inducible promoter (pRD29A, isolated from Arabidopsis thaliana) was compared in transgenic potato (Solanum tuberosum L.) plants in comparison with constitutive promoter (35S CaMV). The promoter fragments were cloned upstream to a reporter gene (GUS-INT) in pCAMBIA-1301 binary vector. Agrobacterium tumefaciens strain LBA4404 harboring recombinant binary vector was used to infect leaf discs and internodal explant of Lady Olympia cultivar. Transgenic lines were verified by GUS histochemical analysis, PCR, gene expression via RT-qPCR and fluorogenic GUS assay techniques. Hygromycin phosphotransferase (hptII) was used at optimal concentration (5 mg/l) for the screening of in vitro cultured putative transgenic plants. Overall transformation efficiacy was calculated as 0,98%. As a result of the validation methods, pRD29A transgenic potatoes were observed to increase GUS gene expression more than 10 times in different abiotic conditions compared to controls. In pRCA promoter, GUS gene expression was increased 4-fold in leaves compared to root / tuber parts. The results of this study encourage the appropriate use of promoter to drive the expression of foreign gene(s) for the development of potato lines resistant/tolerant against biotic and abiotic stresses. Thus, minimizing the risks of transgenic technology can be possible in potatoes using different genes under suitable promoters in the future.
Açıklama
Anahtar Kelimeler
Patates, Dokuya özgü ve uyarılabilir promotörler, Transformasyon, 35S, pRCA ve RD29A, Potato, Tissue specific and inducible promoters, Gene transformation, 35S, pRCA and RD29A
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Rahamkulov, İ. (2019). Transgenik patateste farklı promotörlerin aktivitesinin karşılaştırılması. (Yüksek Lisans Tezi) Niğde Ömer Halisdemir Üniversitesi, Fen Bilimleri Enstitüsü, Niğde
