Anaylsis of AsDA1-2D gene in response to heat stress in oat (Avena sativa L.) = 귀리유전자 AsDA1-2D의 고온스트레스 저항성 분석

국문 초록 (Abstract)

귀리는 사람들의 식단에서 높은 영양학적 가치와 건강상의 이점을 가지고 있기 때문에 점점 그 중요성이 증가하고 있다. 그러나 생식생장기 동안의 고온 스트레스는 종자 저장 단백질의 구조와 함량 변화시켜 최종산물에 나쁜 영향을 미친다. 유비퀴틴-프로테아좀 중 하나인 DA1은 등숙기 동안 세포의 증식을 제어함으로써 종자의 크기를 조절하는데 중요한 역할을 한다. 그러나 현재까지 귀리 DA1 유전자에 대한 보고서나 연구는 거의 전무하였다. 본 연구에서는 게놈 분석을 통하여 3개의 DA1 유전자 패밀리 (AsDA1-2D, AsDA1-5A, AsDA1-1D)를 확인했다. 그 중 AsDA1-2D는 thermo tolerance assay를 통해 고온 스트레스에 대한 내성을 가지고 있는 것이 확인되었다. Yeast two-hybrid screening을 통하여 AsDA1-2D와 귀리의 저장 단백질인 글로불린 (AsGL-4D) 및 단백질분해효소 억제제 (AsGL-4D) 들과의 물리적 상호작용이 관찰되었다. Subcellular localization 분석 결과 AsDA1-2D와 상호 작용 단백질은 세포기질과 원형질막에 위치하는 것으로 밝혀졌다. In vitro pull-down assay결과 AsDA1-2D는 AsPI-4D와 AsGL-4D 모두와 상호작용을 하는 것으로 나타났다. In vitro cell-free degradation assay 결과, AsGL-4D는 고온에서 AsDA1-2D에 의해 분해되었으며, AsPI-4D는 AsDA1-2D의 기능을 억제하였다. 이러한 결과는 AsDA1-2D가 시스테인 프로테아제의 역할을 하며 고온 스트레스 하에서 귀리 저장단백질인 글로불린을 음성조절 한다는 것을 시사한다.

다국어 초록 (Multilingual Abstract)

The importance of oats has increased because of their high nutritional value and health benefits in the human diet. High-temperature stress during the reproductive growth period has a detrimental effect on grain morphology by changing the structure and concentration of several seed-storage proteins. DA1, a conserved ubiquitin-proteasome pathway component, plays an important role in regulating grain size by controlling cell proliferation in maternal integuments during the grain-filling stage. However, there have been no reports or studies on oat DA1 genes. In this study, we identified three DA1-like genes (AsDA1-2D, AsDA1-5A, and AsDA1-1D) using genome-wide analysis. Among these, AsDA1-2D was found to be responsible for high-temperature stress tolerance via a yeast thermotolerance assay. The physical interaction of AsDA1-2D with oat-storage-globulin (AsGL-4D) and a protease inhibitor (AsPI-4D) was observed using yeast two-hybrid screening. A subcellular localization assay revealed that AsDA1-2D and its interacting proteins are localized in the cytosol and plasma membrane. An in vitro pull-down assay showed that AsDA1-2D forms a complex with both AsPI-4D and AsGL-4D. An in vitro cell-free degradation assay showed that AsGL-4D was degraded by AsDA1-2D under high-temperature conditions and that AsPI-4D inhibited the function of AsDA1-2D. These results suggest that AsDA1-2D acts as a cysteine protease and negatively regulates oat-grain-storage-globulin under heat stress.revealed that AsDA1-2D and its interacting proteins are localized in the cytosol and plasma membrane. An in vitro pull-down assay showed that AsDA1-2D forms a complex with both AsPI-4D and AsGL-4D. An in vitro cell-free degradation assay showed that AsGL-4D was degraded by AsDA1-2D under high-temperature conditions and that AsPI-4D inhibited the function of AsDA1-2D. These results suggest that AsDA1-2D acts as a cysteine protease and negatively regulates oat-grain-storage-globulin under heat stress.

목차 (Table of Contents)

• 1. INTRODUCTION 1
• 2. MATERIAL AND METHODS 5
• 2.1 DA1-like family gene sequence identification 5
• 2.2 Plants growth conditions 6
• 2.3 Measurement of total globulin contents of the harvested grain 7
• 2.4 RNA extraction and quantitative real-time PCR 7
• 2.5 Yeast thermotolerance assay 8
• 2.6 Yeast two-hybrid screening and interaction assay 8
• 2.7 Subcellular localization and bimolecular fluorescence complementation 9
• 2.8 Protein purification 10
• 2.9 In vitro pull-down assay 10
• 2.10 Protein degradation assay 11
• 3. RESULTS 12
• 3.1 Analysis of phenotype and grain-related parameters in different temperature conditions 12
• 3.2 Identification of the DA1-like gene family in oat 14
• 3.3 AsDA1-2D improves heat stress tolerance in transgenic yeast 16
• 3.4 Proteins interacting with AsDA1-2D were identified by yeast two-hybrid library screening 19
• 3.5 AsDA1-2D interacts with AsGL-4D and AsPI-4D 22
• 3.6 AsDA1-2D, which was inhibited by AsPI-4D, negatively regulates AsGL-4D protein levels 26
• 4. DISCUSSION 28
• 5. CONCLUSION 32
• REFERENCES 33
• SUPPLEMENTARY MATERIALS 40