跳到主要內容

臺灣博碩士論文加值系統

(3.236.84.188) 您好!臺灣時間:2021/08/03 16:58
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:周曉君
研究生(外文):Hsiao-Chun Chou
論文名稱:植物中CIPK6、MYB34及MYB51基因的上游開放讀序框(uORF)之探討
論文名稱(外文):Analysis of CIPK6, MYB34 and MYB51 upstream open reading frames in plants
指導教授:施明哲施明哲引用關係
指導教授(外文):Ming-Che Shih
口試委員:郭志鴻劉少倫
口試委員(外文):Chih-Horng KuoShao-Lun Liu
口試日期:2015-07-21
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:98
中文關鍵詞:阿拉伯芥十字花科上游開放讀序框RNA降解體核糖體滯留CIPK6MYB硫代葡萄糖苷演化
外文關鍵詞:Arabidopsis thalianaBrassicacaeasPAREribosome stallinguORFCIPK6MYBGlucosinolates (GSL)evolution
相關次數:
  • 被引用被引用:0
  • 點閱點閱:107
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
上游開放讀序框(uORF)為位於5''端非轉譯區中之開放讀序框。過去研究顯示,植物中有些高度保守的上游開放讀序框在高濃度的胺基酸、糖、抗壞血酸或多胺的環境下會導致下游主讀序框 (main ORF)蛋白質轉譯受到抑制。其中有些上游開放讀序框會轉譯出短胜肽鍊並造成核醣體滯留在上游開放讀序框區域,甚至導致此mRNA降解。藉由分析大量RNA降解中間產物,我們發現產生保守胜肽鍊的上游開放讀序框會累積受停滯核糖體保護的RNA降解片段,利用此特徵,我們透過uORF的預測得知在蛋白激酶CIPK6和轉錄因子MYB34及MYB51的5''端非轉譯區具有過去尚未發現之有調控功能的上游開放讀序框。CIPK6參與在植物生長素傳輸和鹽害、乾旱及離層酸反應當中。序列分析顯示CIPK6之上游開放讀序框之胺基酸序列和ATG起始點的周邊序列在不同維管束植物中具高度保守性。且破壞CIPK6 上游開放讀序框會造成下游主讀序框的表現增加。而CIPK6上游開放讀序框對於下游主讀序框的調節,主要藉由影響蛋白轉譯效率,而少部分經由影響RNA量的多寡。MYB34及MYB51與 MYB28、MYB29、MYB122共同參與調控硫代葡萄糖苷生合成途徑。除MYB122上游開放讀序框外,其餘4個MYB皆具有只在十字花科出現,轉譯保守胜肽序列的上游開放讀序框。MYB34及MYB51的上游開放讀序框會抑制下游報導基因活性。在此研究當中,我們證實了由分析RNA降解中間產物找到的上游開放讀序框的調節功能,並闡明一個種系特異性的上游開放讀序框的演化。

Highly conserved upstream open reading frame (uORF) in the 5′ untranslated region (UTR) can regulate downstream main ORF translation in response to metabolites. Few uORFs have been shown to stall ribosomes and induce mRNA decay. The genome-wide analysis of RNA degradation intermediates has revealed footprints of stalled ribosomes in conserved peptide uORFs and identified novel regulatory uORFs in genes encoding CBL-interacting protein kinase 6 (CIPK6) and two transcription factors, MYB34 and MYB51. CIPK6 is involved in auxin transport, salt, drought and abscisic acid response. We show that both peptide sequence and ATG context of CIPK6 uORFs are highly conserved in vascular plants and the disruption of CIPK6 uORF enhanced the expression of downstream main ORF. The CIPK6 uORF-mediated regulation is mainly in translation efficiency and lesser in RNA level. MYB34 and MYB51 regulate glucosinolate biosynthesis along with MYB28, MYB29 and MYB122. Except MYB122, they all contain a conserved peptide uORF specific in the Brassicacaeas family. Both MYB51 and MYB34 uORFs repress the downstream reporter activity. Our results validate the regulatory function of uORFs predicted by the analysis of mRNA degradation fragments and unravel the evolution of a lineage-specific uORF.

CONTENTS
誌謝.......................................... 2
中文摘要....................................... 3
ABSTRACT ..................................... 4
INTRODUCTION ............................... 5
1. Regulation of gene expression by leader sequences and upstream open reading frames .................................... 8
1.1 The regulation of gene expression by leader sequences on prokaryotic mRNA.................................... 8
1.2 The regulation of gene expression by upstream open reading frames (uORFs) on eukaryotic mRNA ............................................... 9
2. Approaches of large-scale analysis with uORFs ..................................... 11
3. Application of PARE data on the analysis of uORFs ............................. 12
4. CIPK6 plays multiple functions in plants ................................................ 14
5. Two clades of MYBs regulate glucosinolate biosynthesis in Brassicaceae ............................................. 15
6. Objective ............................................ 16
MATERIALS AND METHODS ................................................................................. 18
1. Plant materials and growth conditions .................................................... 18
2. Analysis of PARE reads and reads of ribosome protected fragments .. 19
3. Sequence alignments of CIPK6 and GSLMYBs DNA and amino acids... 20
4. Total RNA isolation .......................... 21
5. Reverse transcription, polymerase chain reaction (PCR) and Quantitative Real-time PCR(qPCR) .................................................... 21
6. Genotyping.................................... 23
7. Constructs .......................................... 24
8. Transient assays of uORF regulation by particle bombardment .......... 27
9. Protoplast preparation and Transfection ................................................ 28
10. Luciferase activity assay ................................. 29
11. GUS ( β-glucuronidase) activity assay ................................................. 30
12. Histochemical GUS staining.................................................................. 31
13. Phylogenetic analysis of GSL MYBs ..................................................... 31
14. Statistical Analysis .......................................................
RESULTS ..................................................... 33
Part1:CIPK6 uORF .......................................... 33
1. Analysis of uncapped 5’ ends and ribosome-protected fragments in CIPK6 5’ UTR .......................................... 33
1.1 Distribution of uncapped 5’ ends in CIPK6 5’ UTR in thewild-type Arabidopsis .............................................. 33
1.2 Distribution of uncapped 5’ ends in CIPK6 5’ UTR in xrn4 Arabidopsis mutant .................................. 33
1.3 Distribution of uncapped 5’ ends in the 5’ UTR of CIPK6 in other plant species ...................... 34
1.4 Distribution of ribosome protected mRNA fragements in CIPK6 5’ UTR ............................ 35
2. Sequence analysis of CIPK6 uORF ................................................ 36
2.1 Sequence alignment of CIPK6 uORF encoded peptides ........... 36
2.2 Sequence alignment of ATG context of CIPK6 uORF and main ORF ........................................... 37
3. Functional analysis of CIPK6 uORF .................................................. 38
3.1 Transient assay of CIPK6 uORF function by particle bombardment ...................................... 38
3.2 Functional assay of CIPK6 uORF fused with GUS reporter lines .................................................. 38
3.3 Analysis of secondary RNA structure of normal and mutated CIPK6 5’UTR ........................... 41
Part2:MYB34 and MYB51 uORFs ........................................... 42
1. Analysis of uncapped 5’ ends in MYB34 and MYB51 5’UTR ................ 42
1.1 Distribution of uncapped 5’ ends in MYB34 5’ UTR in the wild-type Arabidopsis ....................... 42
1.2 Distribution of uncapped 5’ ends in MYB51 5’ UTR in the wild-type Arabidopsis ......................................... 42
2. Functional analysis of MYB34 and MYB51 uORFs ................................ 43
2.1 Transient assays of MYB34 and MYB51 uORF function by protoplast transfection ............................................ 43
2.2 Transient assays of the regulation of MYB34 and MYB51 uORFs with tryptophan treatment ................................................... 43
2.3 Analysisof secondary RNA structure of normal and mutated MYB34 and MYB51 5’UTR ............................................ 45
3. Sequence analysis of GSL MYB uORFs ................................................... 45
3.1 Sequence alignment of peptides encoded by GSL MYB uORFs ........................................... 45
4. Phylogenetic analysis of GSL biosynthesis MYBs ................................... 46
4.1 Phylogenetic character mapping of MYB51 uORF ................... 46
4.2 Phylogenetic character mapping of MYB122 uORF ................. 48
4.3 Phylogenetic trees of GSL MYBs and their uORFs .................. 49
7
DISCUSSION .............................. 51
1. The novel application of uncapped 5’ end high throughput sequencing data in the identification of regulatory uORFs .............................................. 51
2. Mechanisms underlying the regulation by CIPK6 uORFs ..................... 52
3. Mechanism underlying the regulation mediated by GSL MYB uORFs 54
4. The origin/evolution of GSL MYB uORFs: a lineage specific uORF ... 56
FIGURES AND TABLES ................................. 59
REFERENCE ...................................................... 81
APPENDIX ...................................................... 87

REFERENCE
Andrews, S.J., and Rothnagel, J.A. (2014). Emerging evidence for functional peptides encoded by short open reading frames. Nat. Rev. Genet. 15, 193-204.
Barbosa, C., Peixeiro, I., and Romao, L. (2013). Gene expression regulation by upstream open reading frames and human disease. PLoS Genet. 9, e1003529.
Bazzini, A.A., Johnstone, T.G., Christiano, R., Mackowiak, S.D., Obermayer, B., Fleming, E.S., Vejnar, C.E., Lee, M.T., Rajewsky, N., Walther, T.C., and Giraldez, A.J. (2014). Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation. EMBO J. 33, 981-993.
Bekaert, M., Edger, P.P., Hudson, C.M., Pires, J.C., and Conant, G.C. (2012). Metabolic and evolutionary costs of herbivory defense: systems biology of glucosinolate synthesis. New Phytol. 196, 596-605.
Celenza, J.L., Quiel, J.A., Smolen, G.A., Merrikh, H., Silvestro, A.R., Normanly, J., and Bender, J. (2005). The Arabidopsis ATR1 Myb transcription factor controls indolic glucosinolate homeostasis. Plant Physiol. 137, 253-262.
Chabregas, S.M., Luche, D.D., Sluys, M.-A.V., Menck, C.F.M., and Silva-Filho, M.C. (2002). Differential usage of two in-frame translational start codons regulates subcellular localization of Arabidopsis thaliana THI1. J. Cell Sci. 116, 285-291.
Chen, C.H., Liao, B.Y., and Chen, F.C. (2011). Exploring the selective constraint on the sizes of insertions and deletions in 5'' untranslated regions in mammals. BMC Evol. Biol. 11, 192.
Chen, L., Ren, F., Zhou, L., Wang, Q.-Q., Zhong, H., and Li, X.-B. (2012). The Brassica napus Calcineurin B-Like 1/CBL-interacting protein kinase 6 (CBL1/CIPK6) component is involved in the plant response to abiotic stress and ABA signalling. J. Exp. Bot. 63, 6211-6222.
Chen, L., Wang, Q.Q., Zhou, L., Ren, F., Li, D.D., and Li, X.B. (2013). Arabidopsis CBL-interacting protein kinase (CIPK6) is involved in plant response to salt/osmotic stress and ABA. Mol. Biol. Rep. 40, 4759-4767.
Dubos, C., Stracke, R., Grotewold, E., Weisshaar, B., Martin, C., and Lepiniec, L. (2010). MYB transcription factors in Arabidopsis. Trends Plant Sci. 15, 573-581.
Ebina, I., Takemoto-Tsutsumi, M., Watanabe, S., Koyama, H., Endo, Y., Kimata, K., Igarashi, T., Murakami, K., Kudo, R., Ohsumi, A., Noh, A.L., Takahashi, H., Naito, S., and Onouchi, H. (2015). Identification of novel Arabidopsis thaliana upstream open reading frames that control expression of the main coding sequences in a peptide sequence-dependent manner. Nucleic Acids Res. 43, 1562-1576
Fahey, J.W., Zalcmann, A.T., and Talalay, P. (2001). The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 56, 5-51.
Fernando de la Torre, F., Gutierrez-Beltran, E., Pareja-Jaime, Y., Chakravarthy, S., Martin, G.B., and del Pozo, O. (2013). The tomato calcium sensor Cbl10 and its interacting protein kinase Cipk6 define a signaling pathway in plant immunity. The Plant cell 25, 2748-2764.
Franzke, A., Lysak, M.A., Al-Shehbaz, I.A., Koch, M.A., and Mummenhoff, K. (2011). Cabbage family affairs: the evolutionary history of Brassicaceae. Trends Plant Sci. 16, 108-116.
Frerigmann, H., and Gigolashvili, T. (2014). MYB34, MYB51, and MYB122 distinctly regulate indolic glucosinolate biosynthesis in Arabidopsis thaliana. Mol. Plant 7, 814-828.
Frerigmann, H., Berger, B., and Gigolashvili, T. (2014). bHLH05 is an interaction partner of MYB51 and a novel regulator of glucosinolate biosynthesis in Arabidopsis. Plant Physiol. 166, 349-369.
German, M.A., Pillay, M., Jeong, D.H., Hetawal, A., Luo, S., Janardhanan, P., Kannan, V., Rymarquis, L.A., Nobuta, K., German, R., De Paoli, E., Lu, C., Schroth, G., Meyers, B.C., and Green, P.J. (2008). Global identification of microRNA-target RNA pairs by parallel analysis of RNA ends. Nat. Biotechnol. 26, 941-946.
Goodstein, D.M., Shu, S., Howson, R., Neupane, R., Hayes, R.D., Fazo, J., Mitros, T., Dirks, W., Hellsten, U., Putnam, N., and Rokhsar, D.S. (2012). Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res. 40, D1178-1186.
Hayden, C.A., and Jorgensen, R.A. (2007). Identification of novel conserved peptide uORF homology groups in Arabidopsis and rice reveals ancient eukaryotic origin of select groups and preferential association with transcription factor-encoding genes. BMC Biol. 5, 32.
Held, K., Pascaud, F., Eckert, C., Gajdanowicz, P., Hashimoto, K., Corratge-Faillie, C., Offenborn, J.N., Lacombe, B., Dreyer, I., Thibaud, J.B., and Kudla, J. (2011). Calcium-dependent modulation and plasma membrane targeting of the AKT2 potassium channel by the CBL4/CIPK6 calcium sensor/protein kinase complex. Cell Res. 21, 1116-1130.
Hill, J.R., and Morris, D.R. (1992). Cell-specific Translation of S-Adenosylmethionine Decarboxylase mRNA. J. Biol. Chem. 267, 21886-21893.
Hinnebusch, A.G. (1997). Translational Regulation of Yeast GCN4. J. Biol. Chem. 272, 21661–21664.
Hsu, M.K., and Chen, F.C. (2012). Selective constraint on the upstream open reading frames that overlap with coding sequences in animals. PLoS ONE 7, e48413.
Jiao, Y., Wickett, N.J., Ayyampalayam, S., Chanderbali, A.S., Landherr, L., Ralph, P.E., Tomsho, L.P., Hu, Y., Liang, H., Soltis, P.S., Soltis, D.E., Clifton, S.W., Schlarbaum, S.E., Schuster, S.C., Ma, H., Leebens-Mack, J., and dePamphilis, C.W. (2011). Ancestral polyploidy in seed plants and angiosperms. Nature 473, 97-100.
Judith, B., and Gerald, R.F. (1998). A Myb homologue, ATR1, activates tryptophan gene expression in Arabidopsis. Proc. Natl. Acad. Sci. USA 95, 5655-5660.
Juntawong, P., Girke, T., Bazin, J., and Bailey-Serres, J. (2014). Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis. Proc. Natl. Acad. Sci. USA 111, E203-212.
Kim, Y.B., Li, X., Kim, S.J., Kim, H.H., Lee, J., Kim, H., and Park, S.U. (2013). MYB transcription factors regulate glucosinolate biosynthesis in different organs of Chinese cabbage (Brassica rapa ssp. pekinensis). Molecules 18, 8682-8695.
Kozak, M. (1986). Point Mutations Define a Sequence Flanking the AUG Initiator Codon That Modulates Translation by Eukaryotic Ribosomes. Cell 44, 283-292.
Kozak, M. (2005). Regulation of translation via mRNA structure in prokaryotes and eukaryotes. Gene 361, 13-37.
Laing, W.A., Martinez-Sanchez, M., Wright, M.A., Bulley, S.M., Brewster, D., Dare, A.P., Rassam, M., Wang, D., Storey, R., Macknight, R.C., and Hellens, R.P. (2015). An upstream open reading frame is essential for feedback regulation of ascorbate biosynthesis in Arabidopsis. The Plant cell 27, 772-786.
Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., and Higgins, D.G. (2007). Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947-2948.
Liu, M.J., Wu, S.H., Wu, J.F., Lin, W.D., Wu, Y.C., Tsai, T.Y., Tsai, H.L., and Wu, S.H. (2013). Translational landscape of photomorphogenic Arabidopsis. The Plant cell 25, 3699-3710.
Menschaert, G., Criekinge, W.V., Notelaers, T., Koch, A., Crappe, J., Gevaert, K., and Damme, P.V. (2013). Deep Proteome Coverage Based on Ribosome Profiling Aids Mass Spectrometry-based Protein and Peptide Discovery and Provides Evidence of Alternative Translation Products and Near-cognate Translation Initiation Events. Mol. Cell Proteomics 12, 1780-1790.
Pelechano, V., Wei, W., and Steinmetz, L.M. (2015). Widespread Co-translational RNA Decay Reveals Ribosome Dynamics. Cell 161, 1400-1412.
Rahmani, F., Hummel, M., Schuurmans, J., Wiese-Klinkenberg, A., Smeekens, S., and Hanson, J. (2009). Sucrose control of translation mediated by an upstream open reading frame-encoded peptide. Plant Physiol. 150, 1356-1367.
Reichelt, M., Brown1, P.D., Schneider, B., Oldham, N.J., Stauber, E., Tokuhisa, J., Kliebenstein, D.J., Mitchell-Olds, T., and Gershenzon, J. (2002). Benzoic acid glucosinolate esters and other glucosinolates from Arabidopsis thaliana. Phytochem. 59, 663-671.
Rogozin, I.B., Kochetov, A.V., Kondrashov, F.A., Koonin, E.V., and Milanesi, L. (2001). Presence of ATG triplets in 5'' untranslated regions of eukaryotic cDNAs correlates with a''weak''context of the start codon. Bioinformatics 17, 890-900.
Ruan, H., Shantz, L.M., Pegg, A.E., and Morris, D.R. (1996). The Upstream Open Reading Frame of the mRNA Encoding S-Adenosylmethionine Decarboxylase Is a Polyamine-responsive Translational Control Element. J. Biol. Chem. 271, 29576-29582.
Ruberti, I., Sessa, G., and Morelli, G. (2006). Functional Analysis of Transcription Factors by Microparticle Bombardment. Arabidopsis Protocols, 2nd Edition, Methods in Molecular Biology 323, 231-236.
Rymarquis, L.A., Souret, F.F., and Green, P.J. (2011). Evidence that XRN4, an Arabidopsis homolog of exoribonuclease XRN1, preferentially impacts transcripts with certain sequences or in particular functional categories. RNA 17, 501-511.
Sachs, M.S., and Geballe, A.P. (2006). Downstream control of upstream open reading frames. Genes Dev. 20, 915-921.
Shashikanth, M., Krishna, A.R., Ramya, G., Devi, G., and Ulaganathan, K. (2008). Genome-wide comparative analysis of Oryza sativa (japonica) and Arabidopsis thaliana 5''UTR sequences for translational regulatory signals. Plant Biotech. J. 25, 553-563.
Sonderby, I.E., Geu-Flores, F., and Halkier, B.A. (2010). Biosynthesis of glucosinolates--gene discovery and beyond. Trends Plant. Sci. 15, 283-290.
Sonderby, I.E., Hansen, B.G., Bjarnholt, N., Ticconi, C., Halkier, B.A., and Kliebenstein, D.J. (2007). A systems biology approach identifies a R2R3 MYB gene subfamily with distinct and overlapping functions in regulation of aliphatic glucosinolates. PLoS ONE 2, e1322.
Subtelny, A.O., Eichhorn, S.W., Chen, G.R., Sive, H., and Bartel, D.P. (2014). Poly(A)-tail profiling reveals an embryonic switch in translational control. Nature 508, 66-71.
Takahashi, H., Takahashi, A., Naito, S., and Onouchi, H. (2012). BAIUCAS: a novel BLAST-based algorithm for the identification of upstream open reading frames with conserved amino acid sequences and its application to the Arabidopsis thaliana genome. Bioinformatics 28, 2231-2241.
Tanaka, M., Takano, J., Chiba, Y., Lombardo, F., Ogasawara, Y., Onouchi, H., Naito, S., and Fujiwara, T. (2011). Boron-dependent degradation of NIP5;1 mRNA for acclimation to excess boron conditions in Arabidopsis. The Plant cell 23, 3547-3559.
Tatematsu, K.-i., Uchino, K., Sezutsu, H., and Tamura, T. (2014). Effect of ATG initiation codon context motifs on the efficiency of translation of mRNA derived from exogenous genes in the transgenic silkworm, Bombyx mori. SpringerPlus 3, 136.
Tripathi, V., Syeda, N., Laxmi, A., and Chattopadhyay, D. (2009a). Role of CIPK6 in root growth and auxin transport. Plant J. 58, 778-790.
Tripathi, V., Parasuraman, B., Laxmi, A., and Chattopadhyay, D. (2009b). CIPK6, a CBL-interacting protein kinase is required for development and salt tolerance in plants. Plant J. 58, 778-790.
Uchiyama-Kadokura, N., Murakami, K., Takemoto, M., Koyanagi, N., Murota, K., Naito, S., and Onouchi, H. (2014). Polyamine-Responsive Ribosomal Arrest at the Stop Codon of an Upstream Open Reading Frame of the AdoMetDC1 Gene Triggers Nonsense-Mediated mRNA Decay in Arabidopsis thaliana. Plant Cell Physiol. 0, 1-12.
Wang, Z., and Sachs, M.S. (1997). Ribosome Stalling Is Responsible for Arginine-Specific Translational Attenuation in Neurospora crassa. Mol. Cell. Biol. 17, 4904-4913.
Wiese, A., Elzinga, N., Wobbes, B., and Smeekens, S. (2004). A conserved upstream open reading frame mediates sucrose-induced repression of translation. The Plant cell 16, 1717-1729.
Willmann, M.R., Berkowitz, N.D., and Gregory, B.D. (2014). Improved genome-wide mapping of uncapped and cleaved transcripts in eukaryotes--GMUCT 2.0. Methods 67, 64-73.
Yanofsky, C. (1981). Attenuation in the control of expression of becterial operons. Nature 289, 090751-090758.
Ye, C.-Y., Xia, X., and Yin, W. (2013). Evolutionary analysis of CBL-interacting protein kinase gene family in plants. Plant Growth Regulation 71, 49-56.
Zur, H., and Tuller, T. (2013). New Universal Rules of Eukaryotic Translation Initiation Fidelity. PLoS Compu. Biol. 9, e1003136.


QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top