생태와 환경 Vol. 52 No. 4 (p.394-402)

|보 문|
Investigation of Agrobacterium-mediated Transient dsRNA Expression in Tobacco

키워드 :
dsRNA,transient expression,tobacco,real-time PCR,digital PCR

목차

Abstract
Introduction
Materials and Methods
   1. Plant material and syringe agro-infiltration
   2. Construction of dsRNA expression
   3. RNA extraction and cDNA synthesis
   4. Quantitative real time PCR
   5. Droplet digital PCR
Results and Discussion
   1. Agro-infiltration into N. benthamiana
   2. Real-time PCR analysis
   3. Estimation of transgene copy number by ddPCR
Conclusion
References

초록

The Agrobacterium tumefaciens mediated gene transfer is widely used to generate genetic transformation of plants and transient assay of temporal exogenous gene expression. Syringe infiltration system into tobacco (Nicotiana benthamiana) leaves is a powerful tool for transient expression of target protein to study protein localization, protein-protein binding and protein production. However, the protocol and technical information of transient gene expression, especially double strand RNA (dsRNA), in tobacco using Agrobacterium is not well known. Recently, dsRNA is crucial for insecticidal effect on destructive agronomic pest such as Corn rootworm. In this study, we investigated the factor influencing the dsRNA expression efficiency of syringe agro-infiltration in tobacco. To search the best combination for dsRNA transient expression in tobacco, applied two Agrobacterium cell lines and three plant vector systems. The efficiency of dsRNA expression has estimated by real-time PCR and digital PCR. As a result, pHellsgate12 vector constructs showed the most effective accumulation of dsRNA in the cell. These results indicated that the efficiency of dsRNA expression was depending on the kind of vector rather than Agrobacterium cells. In summary, the optimized combination of transient dsRNA expression system in tobacco might be useful to in vivo dsRNA expression for functional study and risk assessment of dsRNA.