Cansu BÜLBÜL, İnanç SOYLU, Selcen DOĞAN, Sevilay Münire GİRGİN, Nedim MUTLU

Domateste Agrobacterium Aracılı Geçici Gen İfadesi için Vakum Destekli Düzenek Tasarımı

Hedef genleri bitkilere aktarmak için çeşitli yöntemler kullanılmaktadır. Bunlardan, Agrobacterium aracılı transformasyonda, bitkiye genler kalıcı olarak ikili vektör sistemi ile aktarılabilmektedir. Ancak bu yöntem ile transgenik bir bitki elde etmek için birkaç aylık bir zamana ihtiyaç duyulduğu düşünüldüğünde, süreç oldukça yoğun iş gücü, emek ve zaman gerektirmektedir. Geçici gen ekspresyonu (örn., vakum-infiltrasyon) sistemleri, özellikle zamanla ilgili dezavantajların üstesinden gelmek için, kalıcı transformasyona alternatif olarak kullanılabilmektedir. Ancak bu yöntemde, vakum odaları gibi pahalı ekipmanlara ihtiyaç duyulmaktadır. Bu çalışmada, domates fidelerinde bir raportör gen olan modifiye yeşil floresan proteinin (mGFP) geçici ifadesini gösteren bir vakum-infiltrasyon protokolü rapor edilmektedir. Çalışma sonucunda, uygun fiyatlı ekipmana sahip temel bir deney düzeneği (özel yapım bir büyüme odası dahil) ile 6 dakika boyunca ve sadece 200 milibarlık bir vakum uygulanarak tüm yaprağa ilgili geni taşıyan Agrobacterium solüsyonunun infiltre edilebileceği, böylece domateste geçici gen ifadesinin sağlanabileceği gösterilmiştir. Sonuç olarak, T-DNA, PCR ile tespit edilmiş, mGFP'nin varlığı hem SDS-page analizi ile analitik olarak hem de floresan mikroskobu ile görsel olarak belirlenmiştir.

Anahtar Kelimeler:

Bitkide geçici gen ifadesi, vakum infiltrasyonu, Agrobacterium aracılığıyla gen aktarımı, Solanum lycopersicum

The Basic Experimental Setup for the Vacuum-assisted, Agrobacterium-mediated Transient Expression in Tomato

Several methods are available for use, to deliver the gene of interest into plants. Among these, Agrobacterium-mediated transformation utilizes binary vector systems to achieve the stable transformation of plants. Alas, this process is labor-intensive and time-consuming as several months are needed to obtain a true transgenic plant. Transient gene expression (e.g., vacuum-infiltration) systems were offered as an alternative over stable transformation, specifically to overcome time-related drawbacks. However, this method requires expensive equipment such as vacuum chambers. In this study, we report a vacuum-infiltration protocol for the transient expression of a reporter gene, modified green fluorescent protein (mGFP), in tomato seedling. With a basic experimental setup (including a custom-built growth chamber) with affordable equipment, we showed that the entire leaf can be infiltrated by applying a mere 200 millibar vacuum for 6 minutes, and thus the transient expression can be achieved in tomato plant, evidenced by PCR-based detection of the T-DNA, detection of mGFP both analytically with SDS-page analysis, and visually by the images acquired by fluorescence microscopy. Furthermore, different Agrobacterium tumefaciens strains were tested for their transformation efficiency, and we showed that LBA4404 was the most effective strain to use in the vacuum-assisted transient expression.

Keywords:

Solanum lycopersicum, Plant transient expression, vacuum infiltration, Agrobacterium-mediated gene delivery,

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