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研究生:王孜勻
研究生(外文):Tzu-Yun Wang
論文名稱:AtJ3藉蛋白質法尼酯化修飾以改變植物耐受高溫逆境能力之研究
論文名稱(外文):The study of the mechanism of AtJ3 on protein farnesylation mediated heat stress responses in plants
指導教授:吳少傑
指導教授(外文):Shaw‑Jye Wu
學位類別:碩士
校院名稱:國立中央大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:77
中文關鍵詞:阿拉伯芥耐熱機轉熱休克蛋白蛋白質法尼酯化
外文關鍵詞:ArabidopsisHeat-tolerance mechanismHIT5Protein Farnesylation
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植物無法自由移動,是以演化出許多抵禦機制來面對不利的生長環境。高溫對植物而言是主要的非生物逆境之一。為了了解植物如何抵禦高溫逆境,我們利用前向式遺傳學法,篩選對熱敏感的突變植株,藉此找尋植物耐熱相關的基因,瞭解植物的耐熱機制。我們所篩選到的其中一棵熱敏感突變株,命名為hit-intolerant 5(hit5) 。hit5突變株具有對持續性溫和高溫敏感及對短期熱休克高溫耐受的熱反應性狀。經實驗證實,造成hit5突變株熱反應性狀的突變點,位於編號At5g40280的基因(HIT5)上。此基因所編碼表現出的蛋白質為蛋白質法尼酯轉移酶(PFT)之β次單元。PFT是由α與β兩個次單元所構成的異二具體,其功能為將含有15個碳原子的法尼脂基團接到C端帶有CaaX保守序列的蛋白質上進行轉譯後修飾。阿拉伯芥HSP40家族成員J2(J2)與AtJ3(J3)被認為是HIT5的受質蛋白。雖然J2與J3的胺基酸序列有90%相同,但只有j3突變株具有與hit5突變株相同的熱反應性狀。本研究進一步測試j3表達J3C417S之轉植株的耐熱能力。J3C417S是將J3 C端的CaaX序列改為SaaX,以阻斷J3被法尼酯化。結果顯示,J3C417S/j3植株對熱逆境的反應與hit5相同,證明J3為法尼酯化調控植物熱逆境反應之媒介分子。此外,hit5與j3突變株在室溫下比野生型植株具有較高的HSP101基礎表現量,hit5/hsp101與j3/hsp101耐受熱休克的能力雖不如hit5與j3,但比野生型佳,顯示hit5耐受熱休克逆境能力部分因素來自於J3無法被法尼酯化,進而影響下游HSP101的表現所造成。此結果也指出,尚有其他未知的PFT受質參與hit5所顯現的熱逆境反應。本研究也證實了,去法尼酯化會阻礙J3蛋白質與其他HSPs的交互作用,進而降低植株對持續性高溫的耐受力。
Plants are sessile organisms they must evolve unique protecting mechanisms enabling them to react to unfavorable growth environment. High temperature is one of the major abiotic stresses for plants. To understand how plants cope with high-temperature stress, we have used a forward genetic approach to screen Arabidopsis mutants and isolated the hit-intolerant 5 (hit5) mutant. Incubation at 37°C for 4 days was lethal for hit5 but not for wild-type plants. However, hit5 is better able to tolerate heat-shock stress than the wild type. Map‐based cloning shows that HIT5 encodes the β‐subunit of the protein farnesyltransferase (PFT), which adds a farnesyl group on protein’s CaaX domain. Two of the Arabidopsis HSP40 homologs, AtJ2 (J2) and AtJ3 (J3), are considered to be HIT5 substrates. Although J2 and AtJ3 sharing 90% amino acid sequences identity, only j3 but not j2 have the same heat stress phenotypes as hit5. These phenotypes are confirmed to be related to the lack of the farnesylation of J3 protein. The basal transcript levels of HEAT-SHOCK PROTEIN 101 (HSP101) in hit5 and j3 were higher than those in the wild type. hit5/ hsp101 and j3/hsp101 are not as tolerant to heat shock as hit5 and j3, but still more tolerant than wild-type. These results show that the heat shock phenotypes of hit5 are partly caused by the modulation of HSP101 activity, and also indicates that (a) mediator(s) other than J3 is (are) involved in the PFT-regulated heat-stress response. Furthermore, we confirmed that J3 protein interacts with other heat shock proteins, and these interactions are promoted by farnesylation of J3, and are likely to play an important role in plant survival to sustained heat stress.
中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
圖表目錄 vi
一、 緒論 1
二、 實驗材料與方法 7
1. 實驗材料 7
1-1 植物材料 7
1-2 培養基 7
2.高溫處理之耐熱性測試 8
2-1 持續性高溫處理 8
2-2 熱休克高溫處理 8
3.及時定量聚合酶連鎖反應(quantitative real time polymerase chain reaction, qRT PCR) 8
4. AtDjA3與HSP70-4 BiFC assay(bimolecular fluorescence complementation assay)質體建構 9
4-1 構築pDONRTM221- J3(J3C417S)及pDONRTM221- HSP70-4質體 9
4-2 純化Plasmid DNA 11
4-3 構築pE3130-J3(J3C417S)及pE3136-HSP70-4質體 12
5. AtDjA3與HSP70-4之雙分子螢光互補分析(BiFC assay) 13
5-1 萃取阿拉伯芥原生質體 13
5-2 萃取大量pE3130-J3(J3C417S)及pE3136-HSP70-4質體 13
5-3 阿拉伯芥原生質體基因轉殖 13
6.螢光蛋白表現實驗之載體構築 15
6-1 插入DNA之製備 15
6-2 載體製備 15
6-3 接合作用 (ligation) 16
6-4 大腸桿菌(DH5α)轉型作用(transformation) 16
7. 雙分子螢光互補分析之平均明度與平均綠色色階計算 17
三、 實驗結果與方法 18
四、 討論 25
五、 參考文獻 31
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