博碩士論文 102224026 詳細資訊




以作者查詢圖書館館藏 以作者查詢臺灣博碩士 以作者查詢全國書目 勘誤回報 、線上人數:11 、訪客IP:3.238.184.78
姓名 黃文杉(Wen-Shan Huang)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 水稻中受冷誘導的DEAD-box RNA helicase 42 參與 mRNA 的修飾作用
(A cold-induced DEAD-box RNA helicase 42 participates in mRNA splicing in rice)
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摘要(中) 低溫和寒害產生的冷逆境影響全球農作物的產量,所以很多植物演化出抵抗冷逆境
之分子機制包括維持RNA 分子的正常功能。RNA 解旋酶是一種以ATP 水解作為能量將
RNA 雙股解旋的酵素,它參與在與RNA 有關之機制,包括轉錄、核醣體生合成、
pre-mRNA 修飾、RNA 在細胞核與細胞質間運輸、轉譯、mRNA 降解和胞器中基因的表
現。水稻為世界上重要糧食作物,至少含有62 種不同的基因編碼DEAD-box 蛋白,其
中已知功能的水稻DEAD-box 蛋白質很少,本論文主要目的在探討水稻中受冷逆境誘導
的DEAD-box 蛋白質OsRH42 基因為之生理功能,利用RNAi 轉殖株策略,發現三個
OsRH42 弱化表現之獨立轉殖株呈現對冷逆境極為敏感的性狀。OsRH42 位在細胞核中
的splicing speckle。進一步的研究結果顯示OsRH42 功能為維持Pre-mRNA 的正確
splicing, 一些對冷反應的基因, (OsSK12 (shaggy-related protein kinase) 、OsCIR1
(Circadian 1)、OsPRR95 (PSEUDO-RESPONSE REGULATOR)、OsPUB45 (PLANT U-BOX )、
OsEBF1 (EIN3-binding F-box protein 1 ) 、OsSPFH (SPFH/ PHB domain-containing
membrane-associated protein)),在冷逆境下OsRH42-Ri 轉殖株會發生錯誤splicing 的現
象。此外,在OsRH42 基因靜默轉殖株中CBF 基因表現增加。這些結果說明OsRH42
為水稻中表現受冷反應基因的正常splicing 不可或缺的RNA helicase,並且有助於水稻
對抗冷逆境。
摘要(英) Chilling and freezing temperatures negatively affect global crop production. Plants have
evolved several physiological and molecular adaptations to minimize damage from cold stress
programs. RNA helicases are enzymes that are able to unwind RNA duplex in an
ATP-dependent manner and play important roles in various aspects of RNA metabolism,
including transcription, pre-mRNA splicing, ribosome biogenesis, mRNA transport,
translation, and RNA decay. Rice, one of the most important crops in the world, contains at
least 62 different genes encode DEAD-box proteins. Herein, we identified a rice DEAD-box
RNA helicase gene, OsRH42, which expression was induced by cold stress. The OsRH42
knockdown transgenic plants were generated by RNAi approach and three independent lines
of seedlings exhibited cold hypersensitive phenotype, regardless of whether or not they were
cold acclimated. Subcellular localization indicated that the OsRH42 localized into splicing
speckle. Cold-responsive genes, OsSK12 (shaggy-related protein kinase), OsCIR1 (Circadian
1), OsPRR95 (PSEUDO-RESPONSE REGULATOR), OsPUB45 (PLANT U-BOX ), OsEBF1
(EIN3-binding F-box protein 1 ), OsSPFH (SPFH/PHB domain-containing
membrane-associated protein) were misspliced in OsRH42 knockdown transgenic plants
under cold stress. Moreover, expression of cold response key regulators, CBF genes, were
increased in these OsRH42 knowndown lines, as compared to wildtype. There results
demonstrate that the cold-induced RNA helicase, OsRH42, is essential for splicing of
cold-responsive gene thereby contribute to cold stress tolerance in rice.
關鍵字(中) ★ 冷逆境
★ 水稻
★ mRNA修飾
關鍵字(英) ★ DEAD-box RNA helicase
★ cold stress
★ rice (Oryza sativa)
★ mRNA splicing
論文目次 中文摘要 ..................................................................................................................................... I
Abstract ...................................................................................................................................... II
致謝 .......................................................................................................................................... III
目錄 .......................................................................................................................................... IV
表目錄 ....................................................................................................................................... V
圖目錄 ....................................................................................................................................... V
附錄 .......................................................................................................................................... VI
前言 ............................................................................................................................................ 1
環境對農作物的影響 ........................................................................................................ 1
DEAD-box 簡介及生理影響 ............................................................................................. 2
植物對冷逆境的反應 ........................................................................................................ 4
Splicing Speckles 介紹 ...................................................................................................... 5
結果 ............................................................................................................................................ 7
OsRH42 為DEAD box RNA helicases ............................................................................. 7
OsRH42 在不同組織部位以及非生物性逆境(abiotic stress)和荷爾蒙反應(hormone
response)的表現模式 ......................................................................................................... 7
OsRH42 在細胞內的表現位置 ......................................................................................... 8
水稻中OsRH42 RNAi 轉殖株外表型態 .......................................................................... 8
OsRH42-Ri 轉殖株對冷 (4℃)極為敏感,並為重要的耐冷基因之一 .......................... 9
OsRH42 調控水稻冷逆境中的基因表現 ......................................................................... 9
討論 .......................................................................................................................................... 11
OsRH42 的同源性蛋白質及功能和在不同環境中的表現 ........................................... 11
OsRH42 RNAi 轉殖株的影響 ......................................................................................... 12
參考文獻..................................... 14
表1 WT 和Ri 轉殖株的穗長及節間 .................................... 22
表2 WT 和Ri 轉殖株的特性 .................................... 22
表3 各個用途的Primer .......................................................................................... 23
表4 親緣關係蛋白質相關資訊 ............................................................................. 25
圖1. OsRH42 蛋白質的結構與親緣關係 .............................................................. 29
圖2. OsRH42 在各部位組織的表現 ...................................................................... 30
圖3.OsRH42 在不同的非生物性脅迫(abiotic stress)和荷爾蒙反應(hormone
response)的表達 ............................................................................................... 31
圖4.OsRH42 在冷逆境下的表現 ........................................................................... 32
圖5. OsRH42-GFP 蛋白在水稻原生質體螢光觀察 ............................................. 33
圖6. 建構的OsRH42 RNAi 轉殖株的特性 .......................................................... 34
圖7. OsRH42 RNAi 轉殖株對冷的影響 ................................................................ 36
圖8. OsRH42 在冷逆境下參與在Pre-mRNA 修飾 .............................................. 38
圖9.同源性基因的比較 .......................................................................................... 39
附錄一 OsRH42 Promoter ...................................................................................... 40
附錄二 OsRH42 全長及蛋白質序列 .................................................................... 42
附錄三 洋蔥表皮細胞觀察OsRH42 的表現 ........................................................ 45
附錄四 OsRH42 Promoter::GUS T0 代植株 .......................................................... 46
附錄五 阿拉伯芥的Microarray 的數據 ................................................................ 47
附錄六 Prp5 同源性蛋白 ........................................................................................ 48
附錄七 培養基配方 ................................................................................................ 50
附錄八 建構質體 .................................................................................................... 55
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指導教授 陸重安(Chung-An Lu) 審核日期 2016-7-26
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