阿拉伯芥HIT4及HSP40蛋白質保護植物耐受高溫逆境之研究

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羅予苡(Yu-Yi Lo) 查詢紙本館藏

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生命科學系

論文名稱

阿拉伯芥HIT4及HSP40蛋白質保護植物耐受高溫逆境之研究
(Explores of the protecting mechanisms of Arabidopsis thaliana HIT4 and HSP40 against high temperature stress)

相關論文

★ 阿拉伯芥突變種(hit1)之位址定位	★ 阿拉伯芥之HIT1蛋白質為酵母菌Vps53p之對應物且能影響植物對高溫及水份逆境之耐受性
★ 阿拉伯芥繫鏈同源蛋白質HIT1對頂端生長之影響及熱耐受基因HIT2之遺傳定位	★ 阿拉伯芥hit3遺傳位址定位與HIT1啟動子分析
★ 利用基因功能活化法研究阿拉伯芥乙烯生合成之調控機制	★ 阿拉伯芥突變種hit2之位址定位
★ 利用化學遺傳法研究阿拉伯芥 revert to eto1 41 (ret41) 之功能研究	★ 阿拉伯芥hit3和et突變種之生理定性及其基因定位
★ 阿拉伯芥囊泡繫鏈因子HIT1在逆境下維持內膜完整性之探討與ret8之基因定位	★ 阿拉伯芥HS29之基因定位及ET參與植物耐熱機轉之探究
★ 阿拉伯芥中藉由核運輸接受器HIT2/XPO1A進行核質間運輸以促使植物耐受高溫逆境之專一分子的探索研究	★ 阿拉伯芥hs49與78hs突變株之生理定性及其耐熱基因定位
★ 阿拉伯芥HIT4為不同於MOM1的新調節方式調控熱誘導染色質重組並在各個植物生長發育轉換時期表現	★ 阿拉伯芥熱誘導性狀突變株R45之基因定位及HSP40參與植物耐熱機轉之探究
★ 阿拉伯芥hit4逆轉株r13及r34之基因定位與r34耐熱機轉之探究	★ 蛋白質法尼脂化修飾參與植株耐熱反應

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摘要(中)

植物為固著型生物，是以植物演化出許多適應及抵禦環境逆境的策略，避免受其傷害。由於溫室效應，全球暖化的關係，高溫逆境成為近年來嚴重影響作物
產量的重要原因之一，因此如何提高植物耐受逆境已成為重要的課題。本實驗室
先前利用前向式遺傳學研究法 (forward genetic approach)篩選到一棵對持續性熱
逆境及熱休克逆境敏感的突變株 heat intolerant 4 (hit4)。後續研究發現， HIT4所
編碼的產物位在染色質中心上。在植物遭受熱逆境時， HIT4會藉由染色質構
型重塑，誘使染色質中心呈現消散狀態，促使平時靜默不轉錄 (transcriptional gene silencing, TGS)的基因活化，協助植物耐受高溫逆境。為了更進一步了解 HIT4耐
熱的相關機制，本研究從 hit4突變植物篩選出能耐受持續性高溫的逆轉突變株
(hit4 命名為 revertant98 13 (r98 13)。接著以基因定位的方式，欲找
尋使 hit4不耐熱性狀逆轉的突變基因。研究結果顯示， r98 13突變基因位於阿拉
伯芥第二條色體底部。此外，為了瞭解參與 HIT4調節染色質中心凝縮或消散的
其他因子，本研究也從 HIT4 GFP轉植株挑選出染色質中心數目異於野生型植株
的突變株，命名為 18 18 2。 18 18 2具有晚花及雄配子異常的性狀。另一方面，
本實驗室先前有篩到一棵對持續性熱逆境敏感但耐受熱休克逆性的突變株
hit intolerant 5( 其突變位置坐落於蛋白質法尼脂轉移酶 (PFT)之 β次單元基
因上。阿拉伯芥家族 HSP40中的 J2及 J3已經被證實為 PFT的受質蛋白。 J2與
J3的胺基酸序列有高達 90%的相同度 (identity)，但只有 j3剔除突變株與 hit5有
相同的熱反應性狀，且與 J3無法被法尼脂化之轉植株亦同，證實 J3法尼脂化與
否會影響植物對熱逆境的耐受程度。本研究進一步證實法尼脂化對 HSP40與其
他 HSPs間交互作用所產生的影響，及釐清 J2、 J3在熱逆境下分別扮演的角色。

摘要(英)

Given that plants are immobile, their growth and development will be hampered seriously if environmental conditions are not suitable for their growth and development. High temperature is one of the abiotic stresses. In order to understand how plants to cope with high temperature stress, we used a forward genetic approach to isolate a heat sensitive mutant of the Arabidopsis named heat intolerant 4 (hit4). The hit4 mutant is sensitive to both prolonged heat stress and sudden heat shock treatment. The role of HIT4 was suggested to bring to date unidentified moleculars away from chromocenter thus allowing heat induced chromocenter decondensation to occur. To understand more about which molecular involved in these heat triggered HIT4 dependent responses, we isolated a revertants of hit4 named revertant 98 13 (r98 13), that restored the ability to tolerate prolonged heat stress like wild type. By map based cloning method, r98 13 mutated locus was shown to locate at a region between AGI map 19,628 kb~19,674 kb of the chromosome II. Meanwhile, we isolated a mutant, named as 18 18 2, whose number of chromocenter is different from that of wild type. The phenotype of 18 18 2 included delayed flowering and abnormal stamen. In addition, we have previously isolated the hit intolerant 5 (hit5) mutant. Incubation at 37°C for 4 days was lethal for hit5 but not for wild type plants. HIT5 encodes the β subunit of protein farnesyl transferase (PFT). Two of the Arabidopsis HSP40 homologs, AtJ2 (J2) and AtJ3 (J3), are PFT substrates. Although J2 and J3 have 90% amino acid sequences identitly, only j3 show the same heat stress phenotypes as hit5. Blocking the farnesylation of J3 in plants also show the same heat stress phenotypes as hit5. These results indicated that J3 involve in the PFT regulated heat stress response. In this study, the roles of J3 in protecting plants against prolong heat stress was also explored.

關鍵字(中)

★ 高溫逆境
★ 表觀遺傳
★ 染色質重塑
★ 染色質中心
★ 不完全顯性
★ 熱休克蛋白

關鍵字(英)

★ Heat stress
★ Epigenetic
★ Chromatin remodeling
★ Chromocenter
★ incompletely-dominant
★ Heat shock protien

論文目次

中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
圖表目錄 vii
一、緒論 1
二、材料與方法 8
1. 突變株位址定位 8
1-1 植物材料 8
1-2挑選定位之突變株 8
1-3以分子標記定位出突變基因所在位置 9
1-3-1簡單序列長度多型性(simple sequence length polymorphism, SSLP) 9
1-3-2限制酵素切割擴增片段多型性(cleaved amplified polymorphic sequence, CAPS) 10
1-4 阿拉伯芥基因體萃取 11
1-4突變基因顯隱性之鑑定 12
1-5 DAPI(4′,6-diamidino-2-phenylindole)染色 13
2. AtJ3與AtJ2法尼脂化之耐熱機轉 13
2-1 植物材料 13
2-2 AtJ3與HSP70-4 BiFC assay(bimolecular fluorescence complementation assay)質體建構 13
2-3 純化Plasmid DNA 14
2-4 構築pE3130-J3(J3C417S)及pE3136-HSP70-4質體 15
2-5. AtJ3與HSP70-4之雙分子螢光互補分析(BiFC assay) 16
2-6.螢光蛋白表現實驗之載體構築 18
2-7. 比例式雙分子螢光互補分析(ratiomertric Bimolecular fluorescence)之平均明度計算 20
三、實驗結果 22
1.r98-13突變株具有回復hit4突變株對持續性高溫(Prolonged Heat Stress, PHS)敏感之表現型 22
2. r98-13突變株之顯、隱性鑑定 22
3. r98-13突變株之突變點位址定位 22
4. HIT4-GFP轉植株之突變株18-18-2螢光表現及表現位置之改變 23
5. 18-18-2突變株之顯、隱性鑑定 24
6. 18-18-2突變株之表現型 24
6-1. 18-18-2具有晚花的現象 24
6-2.18-18-2種子大小較野生型Col大 24
6-3 .18-18-2突變株雄配子體異常 25
7. J3蛋白質與HSP70-4蛋白質具有交互作用 25
8. J3的法尼脂化修飾與否不影響其在熱逆境下與HSP70-4之交互作用程度 25
9. J3法尼脂化修飾與否會影響其在熱逆境下與HSP70-4交互作用之位置 26
10. PABP2(Stress Granules, SGs)與HSP70-4/J3WT在熱逆境下所產生的聚集點位置有高比例重疊 26
11.J3法尼脂化修飾與否會影響其熱逆境下是否會形成螢光聚集點 27
12. HSP70-4/J2WT、HSP70-4/J2C417S經熱處理後較HSP70-4/J3 WT具有較少的比例形成螢光聚集點 27
13.量化J2、J3、HSP70-4之間的雙分子螢光互補實驗比較分析 28
14. J2蛋白不會對HSP70-4與J3之間的交互作用有直接影響 29
15.GFP-70-4分別在j2、j3突變株中加熱後皆有螢光質點的形成 29
16. GFP-J3 WT和GFP-J2 WT在hsp70-4突變株中加熱前後細胞中分佈變化之比較 30
17. J3 法尼脂化修飾與否會影響熱逆境下GFP-HSP70-4的細胞分布 30
五、討論 31
1.植物面對熱逆境時細胞內的反應染色質中心點數異常探討 31
1.1 r98-13突變株分析及突變基因定位結果 32
1.2. 18-18-2染色質中心點數異常探討 32
2.J3法尼脂化修飾與熱反應性狀 34
3.J2熱反應性狀與HSP70-4交互作用 36
4. PABP2(Stress Granules, SGs)與HSP70-4/J3WT加熱後之位置探討 37
六、參考文獻 39
圖一、r98-13突變株具有回復hit4突變株對持續性高溫(Prolonged Heat Stress, PHS)敏感之表現型 45
圖二、r98-13突變株之染色體分子標記交換率計算 46
圖三、r98-13突變株基因定位結果 47
圖四、HIT4-GFP轉植株之突變株18-18-2螢光表現及表現位置之改變 48
圖五、18-18-2突變株之表現型 49
圖六、J3蛋白質與HSP70-4蛋白質之雙分子螢光互補 51
圖七、 J3的法尼脂化修飾與否不影響其在熱逆境下與HSP70-4之交互作用程度 52
圖八、HSP70-4與J3WT之雙分子螢光互補實驗、HSP70-4與J3 C417S之雙分子螢光互補實驗加熱前後螢光比較 53
圖九、PABP2(Stress Granules, SGs)作為標記與HSP70-4/J3WT所產生的聚集點位置之比較 54
圖十、J3法尼脂化修飾與否影響加熱後螢光聚集點形成 55
圖十一、HSP70-4與J2之雙分子螢光互補實驗螢光圖 56
圖十二、J2與HSP70-4之雙分子螢光互補實驗加熱前後之螢光圖 57
圖十三、GFP-J2在野生型植株原生質體螢光表現 58
圖十四、量化雙分子螢光互補實驗比較分析 59
圖十五、J2不會對HSP70-4與J3之間的交互作用有直接影響 60
圖十六、GFP-70-4分別在j2、j3突變株中加熱前後細胞中分佈變化 61
圖十七、加熱處理前後GFP- J3WT與GFP- J2WT於細胞中的分布情況 63
圖十八、J3法尼脂化影響HSP70-4螢光聚集點之程度 64
圖十九、J3法尼脂化修飾與HSP70-4共同在熱逆境下形成cytoplasmic foci修護機制之模型 66

表一、r98-13突變株之顯、隱性鑑定 67
表二、阿拉伯芥染色體之分子標記 68
表三、r98-13阿拉伯芥染色體之分子標記 69
表四、r98-13突變株基因位址定位之基因型 70
表五、18-18-2突變株之顯、隱性鑑定 71

附錄一、辨認hit4突變點 73
附錄二、引子序列 74
附錄三、J2蛋白和J3蛋白在常溫及加熱後皆交互作用的產生 76
附錄四、hsp70-4不耐受持續性高溫處理之表現型 77

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指導教授

吳少傑(Shaw-Jye Wu)

審核日期

2020-11-30

推文