阿拉伯芥HS29之基因定位及ET參與植物耐熱機轉之探究

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吳家榮(Jia-Rung Wu) 查詢紙本館藏

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生命科學系

論文名稱

阿拉伯芥HS29之基因定位及ET參與植物耐熱機轉之探究
(Gene mapping of Arabidopsis thaliana HS29 mutant and the study of ET participating in heat-tolerance mechanism)

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摘要(中)

植物因無法自由移動來逃避外在的不利環境，而時時刻刻都面臨環境中的各種影響與刺激。高溫對於植物而言是主要的非生物性逆境之一，高溫逆境會影響植物正常的生長發育，嚴重的話會導致植物的死亡，因此植物必需發展出一套有效的機制來因應高溫所帶來的傷害，提高在高溫逆境下的存活率。為了瞭解植物如何抵禦高溫逆境，我們利用正向式遺傳研究法(forward genetic approach)找尋植物中抵禦高溫相關的耐熱基因。本實驗是以化學突變劑甲基磺酸乙酯(ethylmethane sulfonate, EMS)處理阿拉伯芥 (Arabidopsis thaliana)野生型種子，以高溫處理，挑選出兩株對熱不具耐受性的突變株，分別命名為hs 29 (heat sensitive 29)及et (essential for thermotolerance)。實驗結果顯示hs 29突變基因位於阿拉伯芥第三條染色體底部；et突變點則是阿拉伯芥第五條染色體上一個基因內的一個核苷酸由C變成A，導致其所編碼之胺基酸由絲胺酸(serine)變成酪胺酸(tyrosine)。ET為一個含434個胺基酸、帶有細胞核定位訊號(nuclear localization signal sequence, NLS)，但功能未知的蛋白質。實驗結果得知ET位於細胞核中由異染色質(heterochromatin)所聚集形成的染色質中心(chromocenter)上，推測當植物遭遇高溫逆境時ET可能是藉由調控某些平時表現被抑制但對植物抵禦高溫必需的基因的活化。

摘要(英)

Plants, being immobile and cannot escape from unfavorable environmental conditions, must develop effective protecting mechanisms for survive when exposed to high temperature. To identify the genetic determinants that are essential for plant to cope with heat stress, we have used a forward genetic approach and isolated the et (for essential for thermotolerance) mutant of EMS-mutagenized Arabidopsis. Incubation at 37℃ for 4 days was lethal for et but not for wild-type plants. Gene mapping showed that the mutated locus of et is a single nucleotide change from C to A within the 6th exon of ET gene, leading to a Ser to Tyr substitution at amino acid 227. BLASTp analysis showed that ET is a novel protein whose biological function has never been characterized. Nevertheless, a typical nuclear localization sequence (NLS) was identified, and fluorescent tagged ET was subnuclearly localized to chromocenters. Chromocenters are compact region of chromosome rich in genes under control of transcriptional gene silencing (TGS). Furthermore, we found that heat can mediate chromocenter decondensation and TGS release in wild-type but not in et mutant. Therefore, we conclude that ET may be involved in the epigenetic regulation which is essential for theromotolerance of plants.

關鍵字(中)

★ 耐熱基因
★ 阿拉伯芥

關鍵字(英)

★ heat-tolerance mechanism
★ arabidospis
★ gene mapping

論文目次

中文摘要……………………………………………………………………………….I
英文摘要…………………………………………………………………………….. II
致謝……………………………………………………………………………..…….III
總目錄………………………………………………………………………………...IV
圖表目錄……………………………………………………………………………...VI
緒論……………………………………………………………………………………1
材料及方法……………………………………………………………………………5
1. 突變種之位置定位……………………………………………………………..5
2. 突變種顯隱性之鑑定…………………………………………………………10
3. 含ET及et基因之載體構築…………………………………………………11
4. ET在植物原生質體中表現…………………………………………………...16
5. DAPI(4’’,6-diamidino-2-phenylindole)染色……………………………..19
6. 螢光原位雜合(fluorescent in situ hybridization, FISH)……........19
7. 及時定量聚合酶鏈鎖反應(quantitative real time polymerase chain reaction, qRT PCR)............................................................................................23
8. 互交試驗(reciprocal cross test)………………………………………..24
9. 基因型之鑑定(genotyping)………………………………………………….25
三、實驗結果
1. 突變種之表現型……………………………………………………………....27
2. 突變種顯、隱性突變鑑定..............................................................................27
3. 突變種突變點之定位......................................................................................27
4. ET胺基酸序列分析.........................................................................................28
5. ET的T-DNA品系測試.....................................................................................28
6. ET與et於細胞中之表現位置.......................................................................29
7. 熱誘導染色質中心(chromocenter)結構變化..............................................30
8. 熱誘導TGS之減緩……………………………………………………………31
9. HSP在et突變株中的表現情況……………………………………………...32
10. 與ET交互作用之因子………………………………………………………32
四、討論
1. hs29突變株分析及突變基因定位之結果…………………………………...34
2. 植物面對高溫逆境時細胞內所產生之反應………………………………....34
3. 染色質中心所扮演之角色……………………………………………………34
4. TGS減緩及染色質中心結構改變的分子機制……………………………….37
5. 與ET相關之因子……………………………………………………………..38
6. 熱誘導表現的熱休克蛋白在et突變株中的表現量………………………..39
7. ET可能參與在其它功能……………………………………………………...39
8. ET為獨立演化存在於植物中………………………………………………...40
9. 總結…………………………………………………………………………….40
五、參考文獻……………………………………………………………………......41
圖表目錄
圖一、突變種的表現型……………………………………………………………..46
圖二、突變株染色體上各分子標記之交換率……………………………………..47
圖三、hs29突變株基因定位之結果……………………………………………….48
圖四、ET演化樹分析……………………………………………………………….49
圖五、ET於細胞中表現位置……………………………………………………….50
圖六、熱誘導染色質中心結構變化………………………………………………..51
圖七、熱誘導染色質中心結構變化比例上之統計………………………………..52
圖八、qPCR分析染色質中心區域上基因之表現………………………………….54
圖九、熱休克蛋白基因表現………………………………………………………..55
圖十、胺基酸序列比對……………………………………………………………..57
圖十一、ET與CENH3在細胞中表現之位置……………………………………….58
圖十二、野生型植株與et突變株熱處理前後根部DAPI染色…………………..59
圖十三、熱休克處理後回復(recovery)期間染色質中心結構型態之觀察……..61
圖十四、預測與ET有交互作用之蛋白……………………………………………62
圖十五、ET與HDT4在細胞中表現之位置………………………………………63
圖十六、Bimolecular fluorescence complementation (BiFC)測試…………………64
圖十七、ET會影響HDT4在高溫處理後細胞內分佈位置………………………...65
圖十八、野生型植株之ET與突變株之et在高溫處理前後表現位置型態
的改變………………………………………………………………………………..66
圖十九、ET及et基因之載體構築所用之載體………………………………......67
表一、突變種顯隱性之鑑定………………………………………………………..68
表二、突變株與染色體間之分子標記……………………………………………..69
表三、突變株定位之分子標記……………………………………………………..70
表四、T-DNA insertion line genotyping分析結果…………………………...71
表五、互交試驗(reciprocal cross test)結果…………………………………71
表六、ET及et之cDNA片段以PCR方式放大所用之引子……………………….72
表七、基因型鑑定所用之引子……………………………………………………..72
表八、ET胺基酸序列在阿拉伯芥與其它物種間之一致度(identity)及相似度(similarity) …………………………………………………………………………...73
表九、預測與ET有交互作用之蛋白………………………………………………74
附錄一、et核苷酸定序結果……….......................................................................75
附錄二、ET基因結構與突變點所在位置………………………………………….76
附錄三、ET基因功能上游序列之特性分析……………………………………….77

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指導教授

吳少傑(Shaw-Jye Wu)

審核日期

2012-7-20

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