探討阿拉伯芥中DEAD-box RNA 7解旋酶之功能

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沈育年(Yu-lien Shen) 查詢紙本館藏

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論文名稱

探討阿拉伯芥中DEAD-box RNA 7解旋酶之功能
(Functional characterization of DEAD-box RNA helicase 7, AtRH7, in Arabidopsis)

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摘要(中)

在阿拉伯芥中DEAD-box RNA解旋酶依其功能及胺酸序列組成一個蛋白質家族，每個解璇酶蛋白質在RNA代謝過程中扮演著不同的功能，例如: 核醣體生合成、mRNA剪輯、RNA運輸、mRNA轉譯和RNA降解。於此，我們進一步分析一個在阿拉伯芥中被命名為AtRH7/PRH75 RNA解旋酶，首先AtRH7/ PRH75 RNA解旋酶這個基因持續表現於各部位組織，特別是在細胞分化旺盛的組織，同時AtRH7基因亦受到冷逆境所誘導大量表現。在T-DNA突變AtRH7基因的轉植株atrh7-2和atrh7-3具有與突變在核醣體蛋白質相關基因所造成的生長素相關發育(Auxin-relative development)缺陷的相同外表型，此外表型在冷逆境中更為嚴重。然而atrh7的突變株對冷逆境具有不耐受性，原因可能來自於降低CBF1、CBF2和CBF3表現量。在我們的進一步發現，未加工的18S rRNA前驅物在突變株中大量的累積，同時藉由cRT-PCR的實驗發現，其前驅物的累積主要是因為在A2位置剪裁上的缺失所造成的，進一步影響atrh7突變株對一些抑致核醣體小單元的抗生素產生不敏感的外表型，也因此推測AtRH7可能也參與在核醣體的組合過程當中。根據以上的結果，我們的研究表示AtRH7參與rRNA的生合成中A2位置的加工過程及核醣體組合並且在阿拉伯芥適應冷逆境的環境的過程中扮演著很重要的角色。

摘要(英)

DEAD box RNA helicases belong to a protein family that plays specific roles in various RNA metabolism processes, including ribosome biogenesis, mRNA splicing, RNA export, mRNA translation, and RNA decay. This study investigated a DEAD box RNA helicase, AtRH7/PRH75, in Arabidopsis. The AtRH7/PRH75 expression was ubiquitous, was activated in the cell-dividing region, and was induced by cold stress. The phenotypes of 2 allelic AtRH7/PRH75-knockout mutants, atrh7-2 and atrh7-3, resembled the auxin-related developmental defects exhibited in several ribosomal protein mutants and were more severe under cold stress. The atrh7 mutants exhibited decreased CBF1, CBF2, and CBF3 expression, which could be responsible for the subsequent cold intolerance; they also exhibited unprocessed 18S pre-rRNA accumulation. By a circular RT-PCR analysis displayed the accumulated 18S pre-rRNA, indicating that rRNA processing ceased at the A2 site. The atrh7 mutants were hyposensitive to the antibiotics that target to ribosomal small subunits, suggesting that AtRH7 was also involved in ribosome assembly. Taken together, our studies indicate that the AtRH7 participates in A2 site processing of rRNA biogenesis and ribosome assembling, and involves in plant development and cold stress tolerance in Arabidopsis.

關鍵字(中)

★ 死盒RNA解旋酶
★ 阿拉伯芥RNA解旋酶7號
★ 核醣體RNA生合成

關鍵字(英)

★ DEAD-box RNA helicase
★ AtRH7
★ rRNA biogenesis

論文目次

Table of Content
中文摘要 I
Abstract II
Table of Content IV
Table List VI
Figure List VII
Introduction 1
Materials and Methods 5
Plant materials and growth conditions 5
Quantitative RT-PCR 5
Circular RT-PCR 6
Primers 7
Plasmid constructions 7
Transformation of plants 7
Characterization of the atrh7-1, atrh7-2, and atrh7-3 alleles and segregation analysis 8
Siliques and embryo development 8
Tissue expression analysis 8
Antibiotic treatments 9
Results 10
AtRH7 expression is ubiquitous and active in the cell-dividing region 10
AtRH7 disruption causes severe defects in plant development 11
AtRH7 involvement in embryo development 12
AtRH7 is necessary for tolerance to prolonged cold stress in Arabidopsis 14
AtRH7 participates in rRNA biogenesis 15
atrh7 mutants exhibit resistance to ribosomal antibiotics 17
Discussion 19
Reference 25

Table List
Table 1 The progeny genotypes from self-crossed wild-type and heterozygous mutant plants 32
Table 2 Synchrony of embryogenesis in various developmental siliques in wild-type Arabidopsis 33
Table 3 Delayed embryogenesis in the globular stage in various developmental siliques in atrh7 heterozygous Arabidopsis 34
Table S1 Accession numbers and identities of AtRH7 homologous proteins 46
Table S2 Primers used in the study 47
Table S3 WT, atrh7-2, and atrh7-3 germination ratios 49

Figure List
Figure 1 AtRH7 expression patterns 35
Figure 1 (Cont.) AtRH7 expression patterns 36
Figure 2 Phenotypes of the Arabidopsis atrh7 mutants 37
Figure 3 Seed development in the developing siliques of the atrh7 (+/−) heterozygous and atrh7-2 homozygous plants. 38
Figure 4 Cold-intolerant phenotype in the atrh7 mutants 39
Figure 5 Accumulated pre-rRNAs containing 5’ETS, ITS1, ITS2, and 3’ETS in the atrh7 mutants 41
Figure 6 AtRH7 participates in the A2 site processing of 35S/45S pre-RNA in Arabidopsis 42
Figure 6 (cont.) AtRH7 participates in the A2 site processing of 35S/45S pre-RNA in Arabidopsis 43
Figure 6 (cont.) AtRH7 participates in the A2 site processing of 35S/45S pre-RNA in Arabidopsis 44
Figure 7 atrh7 mutants exhibit resistance to specific antibiotics 45
Figure S1 Amino acid sequences and domain structure of the AtRH7 protein 50
Figure S2 Phylogenetic tree of the AtRH7 homologous proteins 51
Figure S3 Characterization of the Arabidopsis atrh7 mutants 52
Figure S4 Phenotypes of the Arabidopsis atrh7 and atrh7-complement mutants 53
Figure S5 Comparison of developing embryo morphology between the WT plants and the atrh7-3 homozygous mutants 54

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指導教授

陸重安(Chung-an Lu)

審核日期

2014-7-31

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