跳到主要內容

臺灣博碩士論文加值系統

(3.235.60.144) 您好!臺灣時間:2021/07/27 01:00
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

: 
twitterline
研究生:李岳峯
研究生(外文):Yueh-FengLee
論文名稱:蝴蝶蘭光合作用循環式電子傳遞系統之研究
論文名稱(外文):The study of photosynthetic cyclic electron transport chain in Phalaenopsis aphrodite
指導教授:張清俊張清俊引用關係
指導教授(外文):Ching-Chun Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:96
中文關鍵詞:循環性電子傳遞鏈NdhPgr5病毒誘導基因靜默
外文關鍵詞:Cyclic electron transport chainNdhPgr5VIGS
相關次數:
  • 被引用被引用:0
  • 點閱點閱:167
  • 評分評分:
  • 下載下載:6
  • 收藏至我的研究室書目清單書目收藏:0
在阿拉伯芥的光合作用研究中循環性電子傳遞系統(Cyclic electron transport )有兩條獨立的路徑,分別為Ndh-dependent pathway和Pgr5-mediated pathway,由於一般種子植物的葉綠體基因組具有11個Ndh基因,但是在台灣阿嬤蝴蝶蘭(Phalaenopsis aphrodite subsp. formosana)葉綠體基因體內卻缺少3個Ndh (NdhA、F和H)基因,且其他8個Ndh基因為偽基因。由於演化過程,大部分葉綠體基因已轉移至細胞核,因此本研究探討蝴蝶蘭葉綠體基因體所遺失的功能性Ndh基因是否轉移至核中,藉由細胞核來表現其基因的功能。在研究發現,雖然蝴蝶蘭核基因體具有Ndh DNA片段,但無法偵測到Ndh基因的表現,也無蛋白質的活性存在。因而推測蝴蝶蘭中並無Ndh-dependent的循環式電子傳遞鏈之存在維持。為探討Pgr5-mediated pathway的存在,已由蝴蝶蘭選殖並定序Pgr5序列全長,並得知蝴蝶蘭細胞核基因體中具有2~3個copies的Pgr5基因存在,另外,觀察到Pgr5基因在葉片組織的表現較高。進一步利用病毒誘導靜默抑制蝴蝶蘭中Pgr5基因的表現,發現蝴蝶蘭生長及外表性狀並無顯著差異。將再進一步探討循環式電子傳遞的狀況。
The photosynthetic cyclic electron transport chains in Arabidopsis are consist of two pathways, one is Ndh-dependent, and the other is Pgr5-mediated pathways. The chloroplast genomes of angiosperm usually encode 11 subunits of NADH dehydrogenase complex. However, the chloroplast genome of Phalaenopsis aphrodite lost 3 Ndh (NdhA, F and H ) genes, and the other 8 Ndh genes are non-functional pseudogenes. In this study, we investigate the cyclic electron transport chains in P. aphrodite. Since chloroplast genes were frequently transfer to nucleus during evolution, we hypothesized that the functional copy of lost plastid Ndh genes might already have been transferred to nuclear genome, and after transcription and translation, the polypeptides were imported back to chloroplast. Although the Ndh gene fragments could be identified by Southern blot and PCR in the nuclear genomes, the Ndh transcripts could not be detected by Northern blot and RT-PCR. In addition, the activity of NADH dehydrogenase could not be identified in the chloroplasts of P. aphrodite. Therefore, the Ndh-dependent cyclic electron transport chain probably is not functional in P. aphrodite. In contrast, the Pgr5 gene of P. aphrodite was cloned, and 2~3 gene copies were present in the nuclear genome as revealed by Southern blot. Moreover, the leaves of P. aphrodite have higher level of Pgr5 transcripts than roots and flower as demonstrated by Northern blot and RT-PCR. This study suggests that only PGR5-mediated pathway is involved in the cyclic electron transport chain in P. aphrodite. The expression of endogenous Pgr5 gene was inhibited by virus-induced gene silencing (VIGS) method. However, no significant growth or phenotypic effects was observed. The cyclic electron transport chain of PGR5-mediated pathway will be further studied by chlorophyll fluorescence in the near future.
目錄
第一章、研究背景 1
1)蝴蝶蘭特性 1
2)植物葉綠體的起源: 2
3)葉綠體基因體的構造 2
4)植物葉綠體所扮演的角色: 3
5)葉綠體基因組中參與光合作用的基因 3
6)葉綠體基因體遺傳特性 5
7) 循環性電子傳遞系統 6
第二章、目的 13
1. 實驗材料 14
1.1台灣阿嬤蝴蝶蘭(Phalaenopsis aphrodite subsp. formosana): 14
1.2 本論文所使用的引子序列:見附表一 14
2. 實驗方法 14
2.1 純化蝴蝶蘭基因體DNA 14
2.2 純化蝴蝶蘭RNA 15
2.3 反轉錄步驟 16
2.4 聚合酶連鎖反應步驟 17
2.5 南方墨點轉印法 18
2.6 北方墨點轉印法 22
2.7 葉綠體的純化 25
2.8 測量NADH dehydrogenase的酵素活性(Zymogram) 27
2.9 巢式PCR(Nested PCR) 27
2.10 快速放大3’端cDNA 反應 (3’-RACE) 29
2.11 快速放大5’端cDNA 反應 (5’-RACE) 33
2.12 北方墨點轉印法(檢測Pgr5的基因表現) 37
2.13 病毒誘導Pgr5基因靜默(VIGS)的質體構築 39
2.14 病毒誘導Pgr5基因靜默的(VIGS)質體藉電穿孔法至農桿菌 41
2.15 利用農桿菌滲入法感染阿嬤蝴蝶蘭 43
2.16 萃取蝴蝶蘭葉綠體蛋白質 44
2.17 西方墨點轉印法(檢測PGR5的蛋白表現) 45
第四章、結果: 47
1. 阿嬤蝴蝶蘭葉綠體中Ndh-dependent循環電子傳遞鏈不存在: 47
2. 阿嬤蝴蝶蘭中具有PGR5-mediated的循環性電子傳遞: 48
第五章、討論: 52
1. 阿嬤蝴蝶蘭所存有的循環性電子傳遞鏈之探討: 52
2. 阿嬤蝴蝶蘭pgr5基因特性之探討: 53
3. PGR5對植物抵抗逆境的影響: 53
4. 利用VIGS將阿嬤蝴蝶蘭的pgr5基因表現抑制後的影響: 54
第六章、參考文獻 57


圖目錄
圖一、 利用PCR偵測到蝴蝶蘭核基因組Ndh基因。 64
圖二、 利用南方墨點法檢測蝴蝶蘭Ndh基因。 65
圖三、 利用RT-PCR去檢測蝴蝶蘭Ndh基因的表現。 66
圖四、 利用北方墨點法檢測蝴蝶蘭Ndh基因的表現。 67
圖五、 蝴蝶蘭葉綠體NADH dehydrogenase的活性測試。 68
圖六、 阿拉伯芥Pgr5基因序列和蝴蝶蘭EST資料庫比對結果。 69
圖七、 蝴蝶蘭EST clone具有和阿拉伯芥PGR5類似的序列。 70
圖八、 三個不同物種(阿拉伯芥、甜瓜和玉米)的Pgr5基因序列比對。 71
圖九、 利用Nested-PCR檢視蝴蝶蘭是否具有Pgr5基因。 72
圖十、 將阿嬤蝴蝶蘭Pgr5的5’RACE clones之基因序列和蝴蝶蘭EST資料庫的Singletons15403做多重序列比對(MegAlign)。 73
圖十一、將阿嬤蝴蝶蘭Pgr5的3’RACE clones之基因序列和蝴蝶蘭EST資料庫的Singletons15403利用DNASTAR做多重序列比對後的結果。 74
圖十二、利用PCR方式偵測到蝴蝶蘭基因體DNA中具有Pgr5 DNA片段之存在。 75
圖十三、阿嬤蝴蝶蘭的Pgr5基因和蝴蝶蘭EST資料庫的clones之核苷酸序列比對。 76
圖十四、將阿嬤蝴蝶蘭的PGR5胺基酸序列和蝴蝶蘭EST資料庫得到的兩條序列做序列分析(Singletons15403和contig01025)。 77
圖十五、台灣阿嬤蝴蝶蘭和其他6個物種的PGR5胺基酸序列之多重排比。 78
圖十六、南方墨點雜交法偵測Pgr5的基因拷貝數。 79
圖十七、北方墨點雜交偵測Pgr5在不同的組織部位表現之情形。 80
圖十八、利用RT-PCR檢測蝴蝶蘭不同組織的Pgr5基因表現的情形。 81
圖十九、利用Orchidstra蝴蝶蘭資料庫顯示Pgr5基因在不同組織的表現情況。 83
圖二十、利用CymMV病毒誘導基因靜默的載體構築示意圖。 84
圖二十一、阿嬤蝴蝶蘭的Pgr5基因受病毒誘導靜默的情形。 85
圖二十二、蝴蝶蘭Pgr5基因受到病毒誘導靜默抑制的情形。 86
圖二十三、蝴蝶蘭PGR5蛋白受到病毒誘導靜默抑制的情形。 88


附表目錄
附表1: 本實驗所使用之引子 89
附錄: 藥品與溶液配方 91


第六章、參考文獻
蘇鴻傑.(1974). 台灣的野生蘭. 豐年社

廖仁滄. (2001). 台灣阿嬤. 國立科學博物館電子報2001/040/03第12期

千葉雅亮. (2002). 胡蝶蘭の原種. 「胡蝶蘭の原種」刊行会

林咸嘉. (2005). 蝴蝶蘭葉綠體基因體之分析. 國立成功大學生物科技研究所碩士論文.

李培芬.(2005). 台灣的自然資源與生態資料庫II 綠色大地. 行政院原住民委員會.

曾文宏. (2007). 蝴蝶蘭葉綠體基因RNA編輯之分析. 國立成功大學生物科技研究所碩士論文.

刑少辰. (2008). 葉綠體基因組研究進展. 生物化學與生物物理進展 35: 21-28

鄭丞峰. (2010).兩種台灣原生種蝴蝶蘭(台灣阿嬤與姬蝴蝶蘭)葉綠體基因體之比較分析. 國立成功大學生物科技研究所碩士論文.

Arnon DI, Allen MB, Whatley FR. 1958. Assimilatory power in photosynthesis. Science. 127:1026–34

Arnon DI. 1991. Photosynthetic electron transport: Emergence of a concept, 1949–59. Photosynth. Res. 29:117–31.

Battchikova N, Zhang P, Rudd S, Ogawa T, Aro EM. 2005. Identification of NdhL and Ssl1690 (NdhO) in NDH-1L and NDH-1M complexes of Synechocystis sp. PCC 6803. J Biol Chem. 280(4):2587-2595.

Bendall, D.S., and Manasse, R.S. 1995. Cyclic photophosphorylation and electron transport. Biochim. Biophys. Acta. 1229: 23–38.

Chang CC, Lin HC, Lin IP, Chow TY, Chen HH, Chen WH, Cheng CH, Lin CY, Liu SM, Chang CC, Chaw SM. 2006.The chloroplast genome of Phalaenopsis aphrodite (Orchidaceae): comparative analysis of evolutionary rate with that of grasses and its phylogenetic implications. Mol Biol Evol. 23(2):279-91.

Chang, S. B., Chen, W. H., Chen, H. H., Fu, Y. M., and Lin Y.S. 2000. RFLP and inheritance patterns of chloroplast DNA in intergeneric hybrids of Phalaenopsis and Doritis. Bot. Bull. Acad. Sin. 41:219-223.

Corrieau, J. a. A. C. 1988. Rapid screening method to dectect potential biparental inheritance of plasti DNA and results for over 200 angiosperm species. Am. J. Bot. 75:1443-1458.

DalCorso G, Pesaresi P, Masiero S, Aseeva E, Schünemann D, Finazzi G, Joliot P, Barbato R, Leister D. 2008. A complex containing PGRL1 and PGR5 is involved in the switch between linear and cyclic electron flow in Arabidopsis. Cell. 132(2):273-85.

Endo T, Kawase D, Sato F. 2005. Stromal over-reduction by high-light stress as measured by decreases in P700 oxidation by far-red light and its physiological relevance. Plant Cell Physiol. 46:775–81

Fu CH, Chen YW, Hsiao YY, Pan ZJ, Liu ZJ, Huang YM, Tsai WC, Chen HH. 2011. OrchidBase: A collection of sequences of the transcriptome derived from orchids. Plant Cell Physiol. 52(2):238-43.

Gamboa J, Mun˜oz R, Quiles MJ. 2009. Effects of antimycin A and n-propyl gallate on photosynthesis in sun and shade plants. Plant Sci. 177: 643–647.
Gruissem, W. 1989. Chloroplast gene expression: how plants turn their plastids on. Cell. 56:161-170.

Gunning, B. E. S., Steer, M. W. 1996. Plant Cell Biology: Structure and Function. Jones and Bartlett, Sudbury, MA.

Heber, U., and Walker, D. 1992. Concerning a dual function of coupled cyclic electron transport in leaves. Plant Physiol. 100, 1621–1626.

Henry, R.J. 2005. Plant Diversity and Evolution: Genotypic and Phenotypic Variation in Higher Plants. CABI. 45-68.

Hung CH, Huang JY, Chiu YF, Chu HA. 2007. Site-directed mutagenesis on the heme axial-ligands of cytochrome b559 in photosystem II by using cyanobacteria Synechocystis PCC 6803. Biochim Biophys Acta. 1767(6):686-93.

Ibáñez H, Ballester A, Muñoz R, Quiles MJ. 2010. Chlororespiration and tolerance to drought, heat and high illumination. J Plant Physiol. 167(9):732-8.

Jheng CF, Chen TC, Lin JY, Chen TC, Wu WL, Chang CC. 2012. The comparative chloroplast genomic analysis of photosynthetic orchids and developing DNA markers to distinguish Phalaenopsis orchids. Plant Sci. 190:62-73.

Joliot P, Johnson GN. 2011. Regulation of cyclic and linear electron flow in higher plants. Proc Natl Acad Sci U S A. 108(32):13317-22.

Lehtimäki N, Lintala M, Allahverdiyeva Y, Aro EM, Mulo P. 2010. Drought stress-induced upregulation of components involved in ferredoxin-dependent cyclic electron transfer. J Plant Physiol. 167(12):1018-1022.

Lianwei Peng, Hideyuki Shimizu, and Toshiharu Shikanai.2008. The Chloroplast NAD(P)H Dehydrogenase Complex Interacts with Photosystem I in Arabidopsis. J. Biol. Chem. 283:34873–34879.

Lin MJ, Hsu BD. 2004. Photosynthetic plasticity of Phalaenopsis in response to different light environments. J Plant Physiol. 161(11):1259-68.
Liu, Y., Q. Zhang, Y. Hu, and Sodmergen. 2004. Heterogeneous pollen in Chlorophytum comosum, a species with a unique mode of plastid inheritance intermediate between the maternal and biparental modes. Plant Physiol. 135:193-200.

Lu HC, Hsieh MH, Chen CE, Chen HH, Wang HI, Yeh HH. 2012. A high-throughput virus-induced gene-silencing vector for screening transcription factors in virus-induced plant defense response in orchid. Mol Plant Microbe Interact. 25(6):738-46.

Lascano HR, Casano LM, Martín M, Sabater B. 2003. The activity of the chloroplastic Ndh complex is regulated by phosphorylation of the NDH-F subunit. Plant Physiol. 132(1):256-262.

Mogenen, H. L. 1996. The hows and whys of cytoplasmic inheritance in seed plants. Am. J. Bot. 83: 383-404

Munekage, Y., Honjo, M., Meuer, J., Endo, T., Tasaka, M., and Shikanai, T. 2002. PGR5 is involved in cyclic electron flow around photosystem I and is essential for photoprotection in Arabidopsis. Cell. 110: 361–371 .

Munekage Y, Hashimoto M, Miyake C, Tomizawa K, Endo T, Tasaka M, Shikanai T. 2004. Cyclic electron flow around photosystem I is essential for photosynthesis. Nature. 429(6991):579-82.

Munekage YN, Eymery F, Rumeau D, Cuiné S, Oguri M, Nakamura N, Yokota A, Genty B, Peltier G. 2010. Elevated expression of PGR5 and NDH-H in bundle sheath chloroplasts in C4 flaveria species. Plant Cell Physiol. 51(4):664-8.

Ogawa T. 1991. A gene homologous to the subunit-2 gene of NADH dehydrogenase is essential to inorganic carbon transport of Synechocystis PCC6803. Proc Natl Acad Sci U.S.A. 88(10):4275-4279.

Okegawa Y, Tsuyama M, Kobayashi Y, Shikanai T. 2005. The pgr1 mutation in the Rieske subunit of the cytochrome b6f complex does not affect PGR5-dependent cyclic electron transport around photosystem I. J Biol Chem. 280(31):28332-28336.

Okegawa Y, Long TA, Iwano M, Takayama S, Kobayashi Y, Covert SF, Shikanai T. 2007. A balanced PGR5 level is required for chloroplast development and optimum operation of cyclic electron transport around photosystem I. Plant Cell Physiol. 48(10):1462-1471.

Paul A.Burrows, Leonid A.Sazanov, Zora Svab, Pal Maliga and Peter J.Nixon. 1998. Identification of a functional respiratory complex in chloroplasts through analysis of tobacco mutants containing disrupted plastid ndh genes. EMBO J. 17(4):868-876.

Prommeenate P, Lennon AM, Markert C, Hippler M, Nixon PJ. 2004. Subunit composition of NDH-1 complexes of Synechocystis sp. PCC 6803: identification of two new ndh gene products with nuclear-encoded homologues in the chloroplast Ndh complex. J Biol Chem. 279(27):28165-28173.

Reith, M. E. a. J. M. 1995. Complete nucleotide sequence of the Porphyra Purpurea chloroplast genome. Plant Mol. Biol. Rep. 13:333-335.

Shikanai T, Munekage Y, Shimizu K, Endo T, Hashimoto T. 1999. Identification and characterization of Arabidopsis mutants with reduced quenching of chlorophyll fluorescence. Plant Cell Physiol. 40:1134–42.

Shikanai T. 2007. Cyclic electron transport around photosystem I: genetic approaches. Annu Rev Plant Biol. 58:199-217.

Su CL, Chao YT, Alex Chang YC, Chen WC, Chen CY, Lee AY, Hwa KT, Shih MC. 2011. De novo assembly of expressed transcripts and global analysis of the Phalaenopsis aphrodite transcriptome. Plant Cell Physiol. 52(9):1501-14.

Sugiura, C., Y. Kobayashi, S. Aoki, C. Sugita, and M. Sugita. 2003. Complete chloroplast DNA sequence of the moss Physcomitrella patens: evidence for the loss and relocation of rpoA from the chloroplast to the nucleus. Nucleic Acids Res. 31:5324-5331.

Sugiura, M. 1992. The chloroplast genome. Plant Mol Biol. 19:149-168.

Tagawa K, Tsujimoto HY, Arnon DI. 1963. Role of chloroplast ferredoxin in the energy conversion process of photosynthesis. Proc. Natl. Acad. Sci. USA. 49:567–72.

Takabayashi A, Kishine M, Asada K, Endo T, Sato F. 2005. Differential use of two cyclic electron flows around photosystem I for driving CO2-concentration mechanism in C4 photosynthesis. Proc Natl Acad Sci U S A. 102(46): 16898-903.

Thierry Joet, Laurent Cournac, Eva M. Horvath, Peter Medgyesy, and Gilles Peltier. 2001. Increased sensitivity of photosynthesis to antimycin A induced by inactivation of the chloroplast ndhB gene. Evidence for a participation of the NADH-Dehydrogenase complex to cyclic electron flow around Photosystem I. Plant Physiol. 125(4):1919-1929.

Thierry Joet, Laurent Cournac, Gilles Peltier, and Michel Havaux. 2002. Cyclic electron flow around Photosystem I in C(3) plants. In vivo control by the redox state of chloroplasts and involvement of the NADH- Dehydrogenase complex. Plant Physiol. 128(2):760–769.

Timmis JN, Ayliffe MA, Huang CY, Martin W. 2004. Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes. Nat Rev Genet. 5(2):123-35.

Wakasugi T, Tsudzuki J, Ito S, Nakashima K, Tsudzuki T, Sugiura M. 1994. Loss of all ndh genes as determined by sequencing the entire chloroplast genome of the black pine Pinus thunbergii. Proc Natl Acad Sci U S A. 91(21):9794-8.

Wang Peng, YE Jiyu, Shen Yungang & MI Hualing. 2006. The role of chloroplast NAD(P)H dehydrogenase in protection of tobacco plant against heat stress. Sci China C Life Sci. 49(4):311-321.

Winter K, Smith JAC.1996. An introduction to crassulacean acid metabolism. Biochemical principles and ecological diversity. In: Winter K, Smith JAC, editors. Crassulacean acid metabolism. Biochemistry, ecophysiology and evolution. Berlin: Springer. p. 1–13.

Wolfe, K. H., C. W. Morden, and J. D. Palmer. 1992. Small single-copy region of plastid DNA in the non-photosynthetic angiosperm Epifagus virginiana contains only two genes. Differences among dicots, monocots and bryophytes in gene organization at a non-bioenergetic locus. J Mol Biol. 223:95-104.

Wu FH, Chan MT, Liao DC, Hsu CT, Lee YW, Daniell H, Duvall MR, Lin CS.2010. Complete chloroplast genome of Oncidium Gower Ramsey and evaluation of molecular markers for identification and breeding in Oncidiinae.
BMC Plant Biol. 10:68.

Zhang, Q., Liy, Y., Sodmergen. 2003. Examination of cytoplasmic DNA in male reproductive cells to determine the potential for cytoplasmic inheritance in 295 angiosperm species. Plant Cell Physiol. 44:941-951.

Zhang P, Battchikova N, Jansen T, Appel J, Ogawa T, Aro EM. 2004. Expression and functional roles of the two distinct NDH-1 complexes and the carbon acquisition complex NdhD3/NdhF3/CupA/Sll1735 in Synechocystis sp PCC 6803. Plant Cell. 16(12):3326-3340.

Zhang P, Battchikova N, Paakkarinen V, Katoh H, Iwai M, Ikeuchi M, Pakrasi HB, Ogawa T, Aro EM. 2005. Isolation, subunit composition and interaction of the NDH-1 complexes from Thermosynechococcus elongatus BP-1. Biochem J. 390(Pt 2):513-20

連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top