Application and delivery of double-stranded RNAs in Arabidopsis thaliana = 애기장대에서 double-stranded RNA의 적용과 이동에 관한 연구

다국어 초록 (Multilingual Abstract)

RNA interference (RNAi) is an RNA-dependent gene silencing process regulated by interaction of RNA-induced silencing complex (RISC) and double-stranded RNA (dsRNA). Exogenous dsRNA is imported directly into the cytoplasm and cleaved to short dsRNA fragments of 20-25 base pairs by Dicer. These short dsRNA fragments, called small interfering RNAs (siRNAs) have sequence specific interaction with target genes. Guide strand of which siRNAs incorporated in RISC interacts with target mRNA sequence, induces the cleavage and thus degrades target messenger RNAs (mRNAs) by ribonucleases in cells. Recent studies show that dsRNA treatment on plants can induce RNAi. However, the diverse application methods and delivery system of dsRNA are poorly explored. In this study, dsRNA is applicated in Arabidopsis thaliana by two kinds of methods, dipping and spray. I synthesized diverse dsRNAs which designed to target enhanced green fluorescent protein (EGFP) and plastid transcriptionally active 10 (pTAC10) in Arabidopsis thaliana genome. After application of dsRNA that targets EGFP, I found the obvious reduction of GFP expression through fluorescence microscope and mRNA expression level using quantitative reverse transcription PCR (qRT-PCR) in auxin-sensitive reporter DR5-eGFP Arabidopsis thaliana. In addition, after dsRNA application that targets pTAC10, I observed growth repression of Columbia-0 (Col-0) wild type Arabidopsis thaliana. The data revealed that application of target gene specific exogenous dsRNA with dipping and spray methods can induce suppression of target genes of interest. This study might provide a foundation of understanding the processes of dsRNA application and delivery system in plants that can be contributed to RNAi-based technology.

국문 초록 (Abstract)

RNA 간섭은 RNA-induced silencing complex (RISC) 및 double-stranded RNA(dsRNA)의 상호 작용에 의해 조절되는 RNA 의존적 유전자 침묵과정이 다. 외인성 dsRNA는 세포질 내로 들어가 Dicer에 의해 small interferingRNA로 절단되어, 이 중 guide strand가 RISC와 상호 작용하여 특정 mRNA sequence를 절단한다. 최근 연구에 따르면 식물에서의 dsRNA 처리는 RNAi를 유도할 수 있음이 알려졌으나, dsRNA의 다양한 적용 방법 및 전달 시스템 연구는 부족하다. 이에 본 연구에서는 enhanced green fluorescent protein (EGFP)와 plastid transcriptionally active 10 (pTAC10) 유전자를 표적하는 다양한 dsRNA를 디자인하고 합성하여 애기장대 식물체에 침지와 분사 방법으로 적용하였다. 각각의 유전자를 표적하는 dsRNA를 처리한 후, 형광 현미경, 실체 현미경 그리고 quantitative reverse transcription PCR (qRT-PCR)을 이용하여 EGFP 유전자와 pTAC10 유전자가 저해됨을 확인하였다. 이러한 결과들을 종합하여, 외인성 dsRNA가 침지와 분사 방법으로 식물체 내에 적용되며, 이는 RNA 간섭을 활성화하여 표적 유전자의 발현을 억제 시킬 수 있음을 알 수 있었다. 이는 또한 RNA 간섭 기반 기술에 기초를 제공 할 수 있을 것이다.

목차 (Table of Contents)

• Introduction 1
• Materials and Methods 4
• Plant materials and growth conditions 4
• DNA template synthesis 4
• dsRNA synthesis 5
• Dipping and spray methods for dsRNA application 6
• Fluorescence microscopy 6
• Stereo microscopy 7
• Total RNA extraction 7
• Reverse transcription and qRT-PCR 7
• Results 9
• Analysis of EGFP fluorescence and EGFP mRNA expression levels in DR5-EGFP transgenic Arabidopsis thaliana 9
• Design and synthesis of dsRNAs targeting EGFP and pTAC10 9
• Determine the effective amounts of dsRNAs to apply dsRNAs into Arabidopsis thaliana using dipping and spray methods 10
• Suppression of EGFP fluorescence and EGFP mRNA expression levels using dipping and spray methods with dsRNA_EGFP_5 and dsRNA_EGFP_3 11
• Using dipping and spray methods with dsRNA_pTAC10_5 and dsRNA_pTAC10_m showed repression of plant growth and pTAC10 mRNA expression levels 14
• Discussion 17
• Figures and Tables 21
• References 54
• Supplementary Materials 57
• Abstract in Korean 68
• Acknowledgements 69