博碩士論文 101224027 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:3 、訪客IP:35.172.233.215
姓名 謝易軒(Yi-syuan Sie)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 探討水稻OsRH2 DEAD-box RNA解旋酶之功能
(Characterization of DEAD-box RNA helicase genes, OsRH2, in Oryza sativa L.)
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摘要(中) 核糖核酸解旋酶是一種機動蛋白,以三磷酸腺苷水解來產生能量將雙股核糖核酸解開的酵素。DEAD-box核糖核酸解旋酶依照其N端和C端所延伸的氨基酸具有和蛋白質結合的功能,藉著與蛋白質結合的特性,決定其受質特異性、在細胞中的位置和參與生理的功能,因此在基礎細胞中的各個過程裡包括核醣體生成、信使核糖核酸接合、核糖核酸運輸、核糖核酸轉譯和核糖核酸降解等等中扮演著相當重要的角色。目前的研究發現水稻中有62個不同的DEAD-box 核糖核酸解旋酶。此論文是研究水稻中DEAD-box 核糖核酸解旋酶,OsRH2。OsRH2為真核起始因子4AIII蛋白質,與OsRH34同為真核起始因子4AIII蛋白質且與OsRH2為高相似度蛋白。OsRH2是持續表現的一個基因且先前的研究中發現受到冷逆境所誘導。我們利用核糖核酸干擾的方式來產生轉植株並做其生理分析,經由分析我們現已獲得三個獨立植株分別命名為OsRH2Ri -2b, -4及-14b。我們從這些植株的性狀中觀察到植株矮化、較短的穗長和節間、繁殖期延後、胚胎缺失、不正常的花粉和低捻實率。這些性狀類似於吉貝素相關的賀爾蒙發育缺失,在吉貝素生合成或吉貝素感應相關蛋白質的突變株上可以觀察到。此外,OsRH2核糖核酸干擾轉植株顯示在吉貝素20氧化酶2酵素表現中發現未切除乾淨的內含子片段的信使核糖核酸有所累積。從目前的研究中我們可以發現OsRH2參與在信使核糖核酸的接合外同時影響水稻的生長發育。
摘要(英) RNA helicases are motor proteins that catalyze the unwinding of duplex RNA/RNA in an ATP-dependent manner and thereby play important roles in most of the basic cellular processes, including ribosome biogenesis, mRNA splicing, RNA export, mRNA translation, and RNA decay. Rice (Oryza sativa) genome contains at least 62 different genes for DEAD-box RNA helicase proteins. This study investigated two DEAD box RNA helicase, OsRH2 and OsRH34, in rice. Amino acid sequence analysis indicated that the OsRH2 and OsRH34 shared 99% identity and 100% similarity. From the BiFC results, OsRH2 exhibited physical interaction with OsMAGOa and OsY14a, indicated that OsRH2 is core components of the EJC in rice. The OsRH2 were constitutively expression and activated by cold stress. RNA interference transgenic plants were generated for studying biological roles of OsRH2 and OsRH34 in rice. The phenotypes of three independent OsRH2 RNAi transgenic lines, OsRH2 RNAi -2b, -4 and -14b, indicated dwarf, short length of internode, reproduction stage delaying, embryonic development defect, abnormal pollen development, and low seed setting rate. These phenotypes resembled gibberellin-related developmental defects which were exhibited in several GA biosynthesis and GA signaling protein mutants. Besides, the OsRH2 RNAi transgenic lines exhibited unsplicing GA20ox2 mRNA accumulation. My study showed that OsRH2 and OsRH34 participate in mRNA splicing and are also involved in plant development in rice.
關鍵字(中) ★ Oryza sativa
★ DEAD-box RNA helicase
★ dwarf
★ gibberellin
關鍵字(英)
論文目次 Table of Contents
中文摘要 I
Abstract II
致謝 III
Table of Contents IV
Figure List VI
Table List IX
Appendix list IX
Introduction 1
Materials and methods 6
Plant Materials and growth condition 6
Primers 6
Plasmids 6
Plasmid constructions 7
Hormone and stress treatments 7
RT-PCR and qRT-PCR analyse 8
Plant transformation 8
Subcellular localization analysis and bimolecular fluorescence complementation (BiFC) assay 9
GA induced sheath elongation analysis 9
α-amylase activity assay 10
Result 11
OsRH2 and OsRH34 are putative DEAD box RNA heliecases 11
Expression pattern of OsRH2 and OsRH34 11
OsRH2 and OsRH34 co-localize to nucleus and cytoplasm 12
OsRH2 is one partner of EJC core complex 12
Characterization of the rice OsRH2 RNAi transgenic lines 13
The dwarf phenotype of OsRH2 RNAi transgenic plants can be partially rescued when treating GA 15
OsRH2 RNAi transgenic plants showed influence on GA biosynthesis and GA signaling genes 16
OsRH2 RNAi transgenic plants exhibit unspliced GA20ox2 mRNA accumulation 17
No significance different phenotype between OsRH2 RNAi transgenic plants and wild-type plants under cold stress 17
Discussion 18
Reference 23
Appendix 70
Appendix for pharmaceuticals prescription 91



Figure List
Figure 1. Protein sequence alignment of eIF4A proteins 29
Figure 2. OsRH2 and OsRH34 are belong to eIF4AIII protein 30
Figure 3. Expression pattern of exon junction complex core genes 31
Figure 4. Expression patterns of OsRH2 and OsRH34 genes under various plant hormone and abiotic stress treatments 32
Figure 4 (cont.) Expression patterns of OsRH2 and OsRH34 genes under various plant hormone and abiotic stress treatments 33
Figure 5. Subcellular localization of OsRH2 and OsRH34 fusion protein 34
Figure 5 (cont.) Subcellular localization of OsRH2 and OsRH34 fusion protein 35
Figure 6. Co-localization of fusion protein GFP-OsRH2 and fusion protein mCherry-OsRH34 in nucleus and cytoplasm 36
Figure 7 BiFC analysis of rice MAGO, Y14, and eIF4AIII 37
Figure 7 (cont.) BiFC analysis of rice MAGO, Y14 and eIF4AIII 38
Figure 8. Characterization of OsRH2 binary construct RNAi transgenic lines 39
Figure 9. Phenotypes of the OsRH2 RNAi transgenic 40
Figure 10. Comparison of the internode between WT and OsRH2 RNAi transgenic lines 41
Figure 11. OsRH2 RNAi transgenic lines had low seed setting rates 42
Figure 12. OsRH2 RNAi transgenic lines were defect in embryonic development 43
Figure 13. The dwarf phenotype of OsRH2 RNAi transgenic plants can be partially rescued upon GA treatment 44
Figure 14. GA induced α-amylase activity in aleurone layer was not affected in seeds 45
Figure 15. Comparison of the relationship between WT and OsRH2 RNAi transgenic lines in GA biosynthesis and GA signaling pathway 46
Figure 16. OsRH2 RNAi transgenic lines exhibited accumulation of unsplicing GA20ox2 mRNAs 47
Figure 17. Cold responses of OsRH2 and OsRH34 48
Figure S1. Amino acid sequences and domain structure of the OsRH2 and OsRH34 protein 56
Figure S2. OsRH2 and OsRH34 have 97% identity in nucleotide sequence, 99% in amino acid sequences 57
Figure S3. Expression pattern of OsRH2 and OsRH34 in every spike, internode and node 58
Figure S4. The stigma and anther comparisons of WT and OsRH2 RNAi transgenic plants 59
Figure S5. The pollen and anther staining of WT and OsRH2 RNAi transgenic plants 60
Figure S6. The different phenomena embryo defect of OsRH2 RNAi transgenic plants 61
Figure S7. The phenotypes of OsRH2 RNAi transgenic mature and immature seeds 61
Figure S8. Comparison of the seeds length between OsRH2 RNAi transgenic plants and WT 62
Figure S9. OsRH2 RNAi transgenic plants seeds had defect in germination 63
Figure S10. Expression patterns of GA signaling genes 64
Figure S11. Expression patterns of GA biosynthesis genes. 65
Figure S12. Expression patterns of GA biosynthesis genes. 66
Figure S13. GA biosynthesis genes intron accumulation expression patterns 67
Figure S14. Expression patterns of OsUDT1. 68
Figure S15. Characterization of OsRH2 overexpression transgenic plants 69


Table List
Table S1. Sequences of all primers used in this study are listed 49
Table S2. Accession numbers and identities of eIF4A homologous proteins 51
Table S3. Accession numbers and identities mention in this study are listed 52
Table S4. Internode length and spikelet of the OsRH2 RNAi transgenic plants 53
Table S5. Panicle and grains analysis of the OsRH2 RNAi transgenic plants 53
Table S6. The grains weight analysis of the OsRH2 RNAi transgenic plants 53
A 1000 grains weight of the OsRH2 RNAi transgenic plants. 53
Table S7. The ratio of different stages after seed mature between WT and OsRH2 RNAi transgenic plant. 54
Appendix list
1. Construction strategy of yTA-OsRH2 71
2. Construction strategy of pENTR-TOPO-OsRH2 72
3. Construction strategy of PMDC84-OsRH2 C-terminal nonstop coden 73
4. Construction strategy of yTA-OsRH34 74
5. Construction strategy of pENTR-TOPO-OsRH34 75
6. Construction strategy of pMDC84-OsRH34 C-terminal nonstop coden 76
7. Construction strategy of yTA-OsRH2 promoter 77
8. Construction strategy of pCAMBIA1305-OsRH2 promoter 78
9. Construction strategy of yTA-OsRH34 promoter 79
10. Construction strategy of pCAMBIA1305-OsRH34 promoter 80
11. Construction strategy of pUbi-ER8-OsRH2 RNAi 81
12. Construction strategy of pUbi-ER8-OsRH34 RNAi 82
13. Construction strategy of pDonor221-OsRH2 83
14. Construction strategy of pSAT4-DEST-nEYFP-OsRH2 and pSAT5-DEST-cEYFP-OsRH2 84
15. Construction strategy of pDonor221-OsMago 85
16. Construction strategy of pSAT4-DEST-nEYFP-OsMago and pSAT5-DEST-cEYFP-OsMago 86
17. Construction strategy of pDonor221-OsY14 87
18. Construction strategy of pSAT4-DEST-nEYFP-OsY14b and pSAT5-DEST-cEYFP-OsY14b 88
19. Construction strategy of pDonor221-OsNOM1 89
20. Construction strategy of pSAT4-DEST-nEYFP-OsNOM1 and pSAT5-DEST-cEYFP-OsNOM1 90
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指導教授 陸重安(Chung-an Lu) 審核日期 2015-7-27
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