Bayram ÇEVİK, Richard F. LEE, Charles L. NIBLETT

Genetic transformation of Citrus paradisi with antiense and untranslatable RNA-depent RNA polymerase genes of Citrus tristeza closterovirus

Bir hastalık etmeninden elde edilen dayanıklığın (EED) protein ve RNA'ya dayalı şekli farklı bitkilerde bir çok virüse karşı geliştirilmiştir. Ancak citrus tristeza virüsünün (CTV) kılıf proteinini kodlayan yapısal genler ve diğer genleri ile transform edilen turunçgil türlerinde henüz bir dayanıklılık geliştirilememiştir. Replikasyonla ilgili genlerin farklı bitkilerde bir çok virüse karşı RNA'ya dayalı EED geliştirmede başarılı bir şekilde kullanıldığı için bu çalışmada CTV'ün RNA-bağımlı RNA polimeraz (RdRp) geni turuçgillerde CTV ye karşı RNA'ya dayalı dayanıklık geliştirmek amacıyla kullanıldı. RdRp geni CTV'nün DP3800 izolatının genomundan polimeraz zincir reksiyon yöntemiyle çoğaltılarak klonlandı ve bitki transformasyonu için tersine çevrilmiş (RdRp-AS) ve 5' ucunda ardarda beş tane translasyonu durdur kodonu içerdiği için protein sentezleyemeyen (untranslatable, RdRp-UT) formları oluşturuldu. Hazırlanan RdRp-AS ve RdRp-UT genleri Agrobacterium tumefaciens'e dayalı transformasyon yöntemi kullanılarak Duncan greyfurt (Citrus paradisi Macf. cv. Duncan) çeşidinden elde edilen toplam 3120 epikotil parçasına aktarıldı. Bunlardan 1040 tane kanamicine dayanıklı sürgün çoğaltıldı ve elde edilen sürgünlerden floresan mikroskop ve p-glukronidaz (GUS) testiyle toplam 131 potansiyel transgenik sürgün belirlendi. GUS pozitif sürgünlerin 100 tanesi köklendirilerek bitki oluşturuldu. Bu bitkilerden sağlıklı kalarak yaşamını sürdüren 66 tanesi toprak ile doldurulmuş saksılara dikilip seraya aktarılarak büyütüldü. Toplam 41 bitki PCR yöntemiyle test edilerek raportör GUS genini ve CTV'un RdRp genlerini taşıyıp taşımadıkları araştırıldı. Sonuçta GUS geni ve RdRp genlerinden birini taşıyan 18 transgenik greyfurt bitkisi (8 GUS ve RdRp-AS pozitif, 10 GUS ve RdRp-UT pozitif) belirlendi.

Citrus triteza closterovirüsü'nün tersine çevrilmiş ve protein sentezleyemeyen RNA-bağımlı RNA polimeraz Citrus paradisi'in genetik transformasyonu

Protein and RNA-mediated forms of pathogen-derived resistance (PDR) have been developed against many viruses in different plants. However, no resistance has been reported against Citrus tristeza virus (CTV), a closterovirus, in Citrus species transformed with coat protein genes or other sequences of CTV. The successful use of replication-associated genes in RNA-mediated resistance in other crops prompted the use of the RNA-dependent RNA poiymerase (RdRp) gene of CTV for the development of RNA-mediated PDR in Citrus. The RdRP gene was amplified from CTV isolate DP13800 from Florida and used to generate antisense (RdRp-AS) and untranslatable (RdRp-UT) constructs with point mutation consecutive stop codons in the 5' end of the RdRp gene for use in plant transformation. A total of 3120 etiolated epicotyl segments of Duncan grapefruit (Citrus paradisi Macf. cv. Duncan) were transformed with these constructs using Agrobacterium tumefaciens-medlsteâ transformation. From these segments 1040 kanamycin-resistant shoots were regenerated, and a total of 131 putative transgenic shoots were identified by fluorescent microscopy and histochemical (3-glucuronidase (GUS) assays. One hundred GUS positive plants were rooted and 66 plants survived and were established on soil. A total of 41 plants were tested by poiymerase chain reaction (PCR) for the presence of the GUS gene and for the transgenes. Eighteen GUS-positive and transgene-positive plants (8 with RdRp-AS, and 10 with RdRp-UT) were identified.

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