博碩士論文 992204017 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:3.226.251.81
姓名 陳玉夫(Yu-fu Chen)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 水稻中五個DEAD-box RNA helicase - RH2、RH6、RH22、RH42和RH51基因之探討
(Characterization of five DEAD-box RNA helicase genes, RH2, RH6, RH22, RH42 and RH51 in Oryza sativa)
相關論文
★ 水稻CAF1基因之功能分析-水稻CAF1基因的選殖、定性及表現★ 水稻OsDEADl-1基因的功能性探討
★ 利用水稻細胞之懸浮培養建立蛋白質高效率分泌系統★ 水稻CCR4基因之功能分析- 水稻CCR4基因的選殖、定性及表現
★ 阿拉伯芥 AtMYBS 基因功能性探討★ 水稻OsMYBS2基因的功能性分析
★ 水稻CCR4基因的功能分析- 繁衍大量表現和靜默表現的基因轉殖水稻★ 水稻OsVALs基因的功能性分析- 水稻OsVALs基因的選殖、定性及表現
★ 分析水稻T-DNA插入突變株: M0022150, M0023563, M0023580, M0037352及M0032079★ 以水稻懸浮培養細胞蛋白質生產系統生產mGMCSF
★ 建立表現耐熱澱粉普魯南糖酶基因之轉植甘藷★ 阿拉伯芥AtMYBSs基因參與在糖訊息及離層酸訊息傳遞之研究
★ I. II.★ 探討αAmy3、OsCIN1與Os33KD信號肽在水稻懸浮培養細胞中的功能及特性
★ 水稻CAF1基因在水稻懸浮培養細胞之研究★ 探討阿拉伯芥兩個MYB-related轉錄因子在糖訊息傳遞中所扮演的角色
檔案 [Endnote RIS 格式]    [Bibtex 格式]    [相關文章]   [文章引用]   [完整記錄]   [館藏目錄]   至系統瀏覽論文 ( 永不開放)
摘要(中) 陸生植物因無法移動,當遭受到生物逆境與非生物逆境時,必須透過改變分子、細胞和生理性機制來適應這些不同的逆境。在非生物逆境中,溫度是影響植物發育過程中重要因子之一。低溫會抑制種子發芽、莖部延長和花粉活力,進而使農作物的產量降低。Helicase是一種需要消耗ATP將雙股RNA與DNA解開的motor蛋白質。RNA helicase參與大部分的細胞生理反應的過程,包括RNA合成、修復、轉錄、重組、剪切和降解的表現。DEAD-box RNA helicase屬於 helicase superfamily 2,在motif II保留的區域中有一段胺基酸序列Asp-Glu-Ala-Asp,D-E-A-D,因而命名為DEAD。水稻是世界上重要的糧食作物之一,並已完成序列解序,可做為單子葉植物的模式植物。經由資料料庫比對,在水稻中找到5個和阿拉伯芥中會受低溫誘導的AtRH2、AtRH6、AtRH22、AtRH42和AtRH51相似的基因,分別命名為OsRH2、OsRH6、OsRH22、OsRH42和OsRH51。利用水稻懸浮系統進行初步分析這些基因在逆境下的表現量。結果顯示OsRH42和OsRH51會受溫度與高鹽影響,其餘的3個基因則未受逆境影響。為了更進一步瞭解這6個基因在水稻中的功能與扮演的角色,我分別建構這五個DEAD-box RNA helicase的RNAi knock-down轉殖株,並獲得T0轉殖株。在T0代轉植株生長的過程中發現,OsRH2 RNAi轉殖株株高較野生型水稻矮小,且節點間距也較短,種子的結實率也比野生型水稻低。用顯微鏡觀察可能影響結實率的花粉,發現OsRH2 RNAi轉殖株的花粉不健全率比野生型水稻高。綜合以上的結果,推測將OsRH2 RNAi knock-down後會造成植株成長減緩及花粉發育不健全。
摘要(英) Land plants are immobile and therefore encounter for various biotic stresses and abiotic stresses. Plants have to change in molecular, cellular and physical mechanisms to adapt those stresses. Temperature is the one of important factor for all of developmental stage of plant, form germination to reproduction. Low temperature inhibits seed germination, shoot elongation and overall metabolism and therefore cause to decrease agricultural productivity reduction. RNA helicase is motor protein witch can unwind of duplex RNA/RNA in an ATP-dependent manner. RNA helicases are involved in most of the basic cellular progresses including nuclear transcription, ribosome biogenesis, pre-mRNA splicing, nucleocytoplasmic transport, translation, mRNA decay and organall gene expression. DEAD-box RNA helicases belong to the helicase superfamily 2. The name is derived from the amino-acid sequence D-E-A-D (Asp-Glu-Ala-Asp) in the conserved motif II of RNA helicase family. Rice is one of the most important crops and also is a model plant for monocots. Base on amino acid sequence analysis, found the five homologues DEAD-box RNA helicase genes, OsRH2, 6, 22, 42 and 51, in rice that are similar to cold-stress response genes in Arabidopsis. The expression of five genes, showed that OsRH42 and OsRH51 are induced of temperature and salt stress in WT suspension cell. But the OsRH2, 6, 22 are consistive expression in various stresses treated cells. We further generated the RNAi knock-down transgenic lines for those DEAD-box RNA helicase genes. OsRH2 RNAi transgenic plant present plant height and internode were shorter than WT, and seeds fertility are also less than WT. Microscope analysis showed that pollen aborted rate of OsRH2 RNAi transgenic plants were higher than WT. Our results suggest that OsRH2 may have function in plant growth and seed development.
關鍵字(中) ★ RNA解旋酶 關鍵字(英) ★ RNA helicase
論文目次 中文摘要................................................... I
英文摘要.................................................. II
目錄.................................................... III
圖目錄................................................... VI
表目錄................................................... VII
縮寫檢索表 ............................................. VIII
壹、序論................................................... 1
1. DEAD-box RNA解旋酶...................................... 1
2. RNA helicase在植物中的功能 .............................. 2
2.1. RNA helicase影響植物的發育 ............................ 2
2.2. RNA helicase參與植物的抗環境逆境 ....................... 3
2.3. DEAD-box RNA helicase中translation的關係 ............. 4
2.4. DEAD-box RNA helicase與葉綠體Chloroplast發育 .......... 6
貳、研究動機與目的 .......................................... 8
参、材料與方法 ............................................. 9
第一部分 質體建構............................................ 9
1. 資料庫序列分析........................................... 9
1.1. Database ........................................... 9
1.2. Analysis Tool....................................... 9
2. 製備cDNA .............................................. 9
2.1. 製備DEPC-treated ddH2O (RNase-free)溶液 .............. 9
2.2. RNA抽取 ............................................. 9
2.3. 去除RNA中DNA (DNA-freeTM, Ambion) .................... 9
2.4. 合成cDNA (The SuperScriptTMIII First-Strand Synthesis System for RT-PCR, Invitrogen) ........................... 9
3.以PCR分別合成OsRs基因全長片段 ............................. 10
3.1.引子(primer)的設計 ................................... 10
3.2. 以PCR合成DNA片段 .................................... 10
3.3. 瓊脂膠體回收限制酵素切過的DNA(使用Gel/PCR DNA Fragments Extraction Kit DF300, Geneaid)........................... 11
4. 接合反應............................................... 12
4.1. 線性載體DNA的去磷作用(Dephosphorylation of linearized plasmid DNA) ............................................ 12
4.2. 黏頭端載體DNA與插入DNA片段的接合( Ligation of cohesive-ended DNA fragment) ..................................... 12
IV
4.3. 齊頭端DNA片段的接合(Ligation of blunt-ended DNA fragment)................................................ 12
5. 細菌的轉殖作用.......................................... 13
5.1. 製備E.coli competent cell (JM109 strain) ........... 13
5.2. 細菌的轉殖(Transformation) .......................... 13
5.3. 小量純化細菌質體DNA方法(使用Quick Mini-prep. of plasmid)................................................. 13
5.4. DNA定序分析 ......................................... 14
第二部分 水稻基因轉殖....................................... 15
6. 水稻細胞利用農桿菌 (Agrobacterium) 轉殖的方法 ............. 15
6.1. 水稻未成熟胚之癒傷組織的誘導............................. 15
6.2. 製作Agrobacterium competent cell (EHA105 strain).... 15
6.3. 農桿菌的轉殖方........................................ 15
6.4. 農桿菌的生化檢測 (Ketolactose test) .................. 16
6.5. 水稻的轉殖作用........................................ 16
第三部分 水稻轉殖株分析方法.................................. 17
7. 轉殖株GUS assay ....................................... 17
8. 基因組DNA (gnomic DNA)的PCR分析 ........................ 17
8.1. genomic DNA抽取 .................................... 17
8.2. 基因組DNA PCR....................................... 17
9. 轉殖植物RNA的分析 ...................................... 17
9.1. 轉殖植物RNA的抽取 .................................... 17
9.2. RT-PCR分析轉植水稻中RNA的表現 ......................... 17
10. 轉殖植物花粉的染色分析................................... 18
肆、結果.................................................. 19
1. OsRHs基因的序列分析 .................................... 19
1.1. 資料庫比對........................................... 19
2. RT-PCR分析野生型水稻中OsRH2、6、22、42、51在逆境下的表現 .... 19
2.1. 在4oC低溫逆境下OsRH2、6、22、42、51的表現 .............. 19
2.2. 在38oC熱逆境下OsRH2、6、22、42、51的表現................ 20
2.3. 在高鹽、ABA荷爾蒙逆境下OsRH2、6、22、42、51的表現 ........ 20
3. 建立OsRH2、6、22、42、51靜默表達 (RNAi) 的轉殖水稻 ........ 21
4. 利用GUS活性分析OsRH2, 6, 22, 42, 51 RNAi細胞株 .......... 21
5. OsRH2, 6, 22, 42, 51 RNAi轉殖株genomic DNA的分析 ....... 22
6. OsRH2 RNAi轉殖株的生長發育情形 .......................... 22
7. OsRH2 RNAi轉殖株的種子發育情形 .......................... 23
8. OsRH2 RNAi轉殖株的花粉發育情形 .......................... 24
9. OsRH2 RNAi T0轉殖株RT-PCR分析 ......................... 24
10. OsRH2 RNAi T1轉殖株外表型分析 .......................... 25
11. OsRH2 RNAi T1轉殖株genomic PCR和RT-PCR分析 ............ 25
伍、討論.................................................. 26
1. 水稻OsRH2、6、22、42、51基因的選殖 ....................... 26
2. OsRH2、6、22、42、51 RNAi轉殖水稻的建立與分析 ............. 27
3. OsRH2 RNAi轉殖株的生長發育情形分析 ....................... 28
陸、參考文獻 .............................................. 31
附錄I ................................................... 73
參考文獻 Alexander, M.P. (1969). Differential staining of aborted and nonaborted pollen. Stain Technol 44, 117-122.
Asakura, Y., Galarneau, E., Watkins, K.P., Barkan, A., and van Wijk, K.J. (2012). Chloroplast RH3 DEAD box RNA helicases in maize and Arabidopsis function in splicing of specific group II introns and affect chloroplast ribosome biogenesis. Plant Physiol 159, 961-974.
Asano, T., Hakata, M., Nakamura, H., Aoki, N., Komatsu, S., Ichikawa, H., Hirochika, H., and Ohsugi, R. (2011). Functional characterisation of OsCPK21, a calcium-dependent protein kinase that confers salt tolerance in rice. Plant Mol Biol 75, 179-191.
Chan, C.C., Dostie, J., Diem, M.D., Feng, W., Mann, M., Rappsilber, J., and Dreyfuss, G. (2004). eIF4A3 is a novel component of the exon junction complex. Rna 10, 200-209.
Chi, W., He, B., Mao, J., Li, Q., Ma, J., Ji, D., Zou, M., and Zhang, L. (2012). The function of RH22, a DEAD RNA helicase, in the biogenesis of the 50S ribosomal subunits of Arabidopsis chloroplasts. Plant Physiol 158, 693-707.
Chinnusamy, V., Gong, Z., and Zhu, J.K. (2008). Nuclear RNA export and its importance in abiotic stress responses of plants. Curr Top Microbiol Immunol 326, 235-255.
Cordin, O., Banroques, J., Tanner, N.K., and Linder, P. (2006). The DEAD-box protein family of RNA helicases. Gene 367, 17-37.
de la Cruz, J., Kressler, D., and Linder, P. (1999). Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families. Trends Biochem Sci 24, 192-198.
Dever, T.E. (2002). Gene-specific regulation by general translation factors. Cell 108, 545-556.
Ferraiuolo, M.A., Lee, C.S., Ler, L.W., Hsu, J.L., Costa-Mattioli, M., Luo, M.J., Reed, R., and Sonenberg, N. (2004). A nuclear translation-like factor eIF4AIII is recruited to the mRNA during splicing and functions in nonsense-mediated decay. Proc Natl Acad Sci U S A 101, 4118-4123.
Gong, Z., Dong, C.H., Lee, H., Zhu, J., Xiong, L., Gong, D., Stevenson, B., and Zhu, J.K. (2005). A DEAD box RNA helicase is essential for mRNA export and important for development and stress responses in Arabidopsis. Plant Cell 17, 256-267.
Harris, E.H., Boynton, J.E., and Gillham, N.W. (1994). Chloroplast ribosomes and protein synthesis. Microbiol Rev 58, 700-754.
Huang, C.K., Huang, L.F., Huang, J.J., Wu, S.J., Yeh, C.H., and Lu, C.A. (2010a). A DEAD-box protein, AtRH36, is essential for female gametophyte development and is involved in rRNA biogenesis in Arabidopsis. Plant Cell Physiol 51, 694-706.
Huang, T.S., Wei, T., Laliberte, J.F., and Wang, A. (2010b). A host RNA helicase-like protein, AtRH8, interacts with the potyviral genome-linked protein, VPg, associates with the virus accumulation complex, and is essential for infection. Plant Physiol 152, 255-266.
Kant, P., Kant, S., Gordon, M., Shaked, R., and Barak, S. (2007). STRESS RESPONSE SUPPRESSOR1 and STRESS RESPONSE SUPPRESSOR2, two DEAD-box RNA helicases that attenuate Arabidopsis responses to multiple abiotic stresses. Plant Physiol 145, 814-830.
Kim, J.S., Kim, K.A., Oh, T.R., Park, C.M., and Kang, H. (2008). Functional characterization of DEAD-box RNA helicases in Arabidopsis thaliana under abiotic stress conditions. Plant Cell Physiol 49, 1563-1571.
Koroleva, O.A., Calder, G., Pendle, A.F., Kim, S.H., Lewandowska, D., Simpson, C.G., Jones, I.M., Brown, J.W., and Shaw, P.J. (2009). Dynamic behavior of Arabidopsis eIF4A-III, putative core protein of exon junction complex: fast relocation to nucleolus and splicing speckles under hypoxia. Plant Cell 21, 1592-1606.
Linder, P., and Jankowsky, E. (2011). From unwinding to clamping - the DEAD box RNA helicase family. Nat Rev Mol Cell Biol 12, 505-516.
Lorkovic, Z.J., Herrmann, R.G., and Oelmuller, R. (1997). PRH75, a new nucleus-localized member of the DEAD-box protein family from higher plants. Mol Cell Biol 17, 2257-2265.
Nielsen, P.J., and Trachsel, H. (1988). The mouse protein synthesis initiation factor 4A gene family includes two related functional genes which are differentially expressed. Embo J 7, 2097-2105.
Nishimura, K., Ashida, H., Ogawa, T., and Yokota, A. (2010). A DEAD box protein is required for formation of a hidden break in Arabidopsis chloroplast 23S rRNA. Plant J 63, 766-777.
Rocak, S., and Linder, P. (2004a). DEAD-box proteins: the driving forces behind RNA metabolism. Nat Rev Mol Cell Biol 5, 232-241.
Rocak, S., and Linder, P. (2004b). DEAD-box proteins: the driving forces behind RNA metabolism. Nature reviews. Molecular cell biology 5, 232-241.
Shibuya, T., Tange, T.O., Sonenberg, N., and Moore, M.J. (2004). eIF4AIII binds spliced mRNA in the exon junction complex and is essential for nonsense-mediated decay. Nat Struct Mol Biol 11, 346-351.
Shimizu, K.K., Ito, T., Ishiguro, S., and Okada, K. (2008). MAA3 (MAGATAMA3) helicase gene is required for female gametophyte development and pollen tube guidance in Arabidopsis thaliana. Plant Cell Physiol 49, 1478-1483.
Stern, D.B., Goldschmidt-Clermont, M., and Hanson, M.R. (2010). Chloroplast RNA metabolism. Annu Rev Plant Biol 61, 125-155.
Vain, P., Thole, V., Worland, B., Opanowicz, M., Bush, M.S., and Doonan, J.H. (2011). A T-DNA mutation in the RNA helicase eIF4A confers a dose-dependent dwarfing phenotype in Brachypodium distachyon. Plant J 66, 929-940.
Vashisht, A.A., and Tuteja, N. (2005). Cold stress-induced pea DNA helicase 47 is homologous to eIF4A and inhibited by DNA-interacting ligands. Arch Biochem Biophys 440, 79-90.
Vashisht, A.A., Pradhan, A., Tuteja, R., and Tuteja, N. (2005). Cold- and salinity stress-induced bipolar pea DNA helicase 47 is involved in protein synthesis and stimulated by phosphorylation with protein kinase C. Plant J 44, 76-87.
Wang, Y., Duby, G., Purnelle, B., and Boutry, M. (2000). Tobacco VDL gene encodes a plastid DEAD box RNA helicase and is involved in chloroplast differentiation and plant morphogenesis. Plant Cell 12, 2129-2142.
Williams-Hill, D.M., Duncan, R.F., Nielsen, P.J., and Tahara, S.M. (1997). Differential expression of the murine eukaryotic translation initiation factor isogenes eIF4A(I) and eIF4A(II) is dependent upon cellular growth status. Arch Biochem Biophys 338, 111-120.
指導教授 陸重安 審核日期 2013-1-22
推文 facebook   plurk   twitter   funp   google   live   udn   HD   myshare   reddit   netvibes   friend   youpush   delicious   baidu   
網路書籤 Google bookmarks   del.icio.us   hemidemi   myshare   

若有論文相關問題,請聯絡國立中央大學圖書館推廣服務組 TEL:(03)422-7151轉57407,或E-mail聯絡  - 隱私權政策聲明