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

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陳宣吟(Hsuan-Ying Chen) 查詢紙本館藏

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論文名稱

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
(Gene mapping of Arabidopsis hit4 revertants r13 and r34, and the study of their roles in the heat stress response in plants)

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摘要(中)

植物因無法自由移動，在面對環境逆境時，必須調整其生理反應，以求生存。近來因溫室氣體排放引起的全球暖化，對農作物形成高溫逆境。因此找尋植物的耐熱相關基因，並進一步瞭解其調控植物耐熱機轉，進而改善作物耐熱能力，便有其重要性與迫切性。本實驗室先前利用前向式遺傳研究法(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是一個坐落在染色質中心，藉由調控染色質中心去凝縮(chromocenter decon-densation)現象，來提升植物耐熱力的蛋白質。為了更進一步瞭解HIT4耐熱調控機轉，本研究再次利用前向式遺傳研究法，從hit4植物挑選出能耐受持續性高溫的逆轉突變株(hit4 revertants)，再以基因定位的方式，尋找致使hit4不耐熱性狀逆轉的突變基因。這些基因的產物，將很有可能是與HIT4共同調控植物耐熱能力之分子。本研究共篩選出兩株hit4逆轉突變株，分別命名為revertant 13 (r13)和revertant 34 (r34)。基因定位顯示，r13突變基因位於阿拉伯芥第二條色體底部；而r34突變株突變點則是位於編號At2g03050，名為SINGLET OXYGEN-LINKED DEATH ACTIVATOR 10(SOLDAT10)基因上，一個DNA鹼基由G變成A，使得相對應的胺基酸由Gly變成Arg的點突變。已知SOLDAT10基因表現出的蛋白質屬於mitochondrial transcription termination factor(mTERF)家族，有調控葉綠體轉錄作用之功能，並被推測參與在葉綠體到細胞核的逆向信號(retrograde signaling)及交叉抗性機制中。後續實驗將探討該基因是否參與在HIT4所調控的耐熱機制中，及其回復hit4突變株熱敏感性狀之機轉。

摘要(英)

As sessile organisms, plants have developed sophisticated mechanisms to cope with both biotic and abiotic stresses from their living environment. In order to understand how plants survive under high temperature conditions, we used a forward genetic approach to isolate the Arabidopsis mutant heat tolerant 4 (hit4). The hit4 mutant is tested to be sensitive to either prolonged heat stress or sudden heat stress treatment. HIT4 was reported to mediate heat induced decondensation of chromocenters, which facilitates release of transcriptional gene silence(TGS). To better understand the underly mechanisms of and to identify more genetic determinants involved in the heat-triggered HIT4-dependent responses, I employed a forward genetic approach again to isolate two independent revertants of hit4. They were named revertant 13 (r13) and revertant 34 (r34), respectively. r13 is tolerant to prolonged heat stress but not sudden heat stress. DAPI-staining analysis showed that the chromocenters of r13 decondensed after heat treatment at 37°C for 30 hr. The map-based cloning indicated that r13 mutated locus is located at a region between AGI map 19,623 kb~19,698 kb of the chromosome II. r34 can tolerate both prolonged heat stress and sudden heat stress. The r34 mutated locus is mapped to At2g03050 which is known as SINGLET OXYGEN-LINKED DEATH ACTIVATOR 10(SOLDAT10). r34 is a missense mutation resulting in a change of the 442nd nucleotide from guanine to adenine which leads to a change of the 148th amino ac-id of the SOLDAT10 protein from glycine to arginine. SOLDAT10-GFP fusion protein was observed to locate in chloroplasts. Meanwhile, in r34, the chromocenters remained con-densed, and the heat-induced TGS release remaind attenuated under heat stress condition as observed in hit4. These results suggest that the better heat tolerant ability of r34 is not likely to involve HIT4 mediated mechanisms but derived from the enhanced thermotolerance of mutation at SOLDAT10.

關鍵字(中)

★ 高溫逆境
★ 前向式遺傳學
★ 阿拉伯芥
★ 染色質重塑
★ 逆向信號
★ 交叉抗性

關鍵字(英)

★ heat stress
★ forward genetic approach
★ Arabidopsis
★ chromatin remodeling
★ retrograde signaling
★ cross tolerance

論文目次

中文摘要 i
Abstract ii
誌謝 iii
目錄 iv
圖表目錄 vii
緒論 1
材料與方法 5
1. 突變株位址定位 5
1-1 植物材料 5
1-2挑選定位之突變株 5
1-3以分子標記定位出突變基因所在位置 6
1-4 阿拉伯芥基因體萃取 8
2. 突變基因顯隱性之鑑定 9
3. 突變種之核苷酸定序 9
4. SOLDAT10基因之載體構築 10
4-1 插入DNA(SOLDAT10)之製備 10
4-2 載體製備 11
4-3 接合作用 (ligation) 12
4-4 大腸桿菌(DH5α)轉型作用(transformation) 12
4-5質體DNA萃取 13
5. 短時間致死性熱處理(Sudden Heat Stress, SHS)、長時間後天耐熱性獲得(Long term Acquired Thermotolerance, LAT)與短時間後天耐熱性獲得(Short term Acquired Thermotolerance, SAT)對r34生長表現型之測試 14
6. 及時定量聚合酶連鎖反應(quantitative real time polymerase chain reaction, qRT PCR) 15
7. SOLDAT10-GFP 載體在原生質體中表現 16
7-1 阿拉伯芥原生質體(protoplast)抽取 16
7-2 原生質體轉型(transformation) 16
8. DAPI(4′,6-diamidino-2-phenylindole)染色 18
9. 報導基因GUS (β-glucuronidase)染色實驗 18
實驗結果 19
1. r13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 19
2. r13突變株之顯、隱性鑑定 19
3. r13突變株之突變點位址定位 19
4. r13突變株熱誘導染色質中心結構變化 20
5. r34突變株之表現型 20
5-1 r34突變株具有回復hit4突變株對PHS敏感之表現型 20
5-2 r34突變株對短時間致死性高溫(Sudden Heat Stress, SHS)之表現型 21
5-3 r34突變株對短期後天耐熱性(Short term Acquired Thermotolerance, SAT)之表現型 21
5-4 r34突變株對長期後天耐熱性(Long term Acquired Thermotolerance, LAT)之表現型 21
6. r34突變株之顯、隱性鑑定 22
7. r34突變株之突變點位址定位 22
8. r34突變株之核甘酸定序 23
9. r34突變株之胺基酸序列分析 23
9-1胺基酸結構及突變點所在位置 23
9-2胺基酸序列對比 23
10. SOLDAT10基因之構築 23
10-1互補試驗(complementary test)所用質體 23
10-2 SOLDAT10-GFP融合蛋白載體 24
11. SOLDAT10在細胞中表現位置 24
12. r34突變株熱誘導染色質中心結構變化 24
13. 以qPCR分析加熱前後內生性TGS loci genes之表現 25
14. 熱處理前後外源性TGS loci gene之表現 25
15. r34/HIT4之耐熱能力比較 26
討論 27
1. r13突變株分析及突變基因定位結果 27
2. r34突變基因定位結果與胺基酸序列探討 27
3. SOLDAT10蛋白質所在位置及功能之探討 28
4. SOLDAT10與非生物逆境之探討 29
4-1SOLDAT10與ROS和交叉抗性(cross-tolerance)之探討 29
4-2SOLDAT10與逆向信號之探討 31
5. r34回復hit4不耐熱性狀之探討 33
參考文獻 34

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指導教授

吳少傑(Shaw-Jye Wu)

審核日期

2019-7-18

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