| 摘要: | 全球暖化威脅未來糧食供給。找出並研究植物耐熱基因，作為培育耐熱作物品種之學理基礎，有其必要性與迫切性。據此，吾人以阿拉伯芥為材料，篩選出一突變株heat-intolerant 5 (hit5)。相較於野生型，hit5對37°C 4 day 熱處理敏感，但對44°C 40 min熱處理更具耐受性。HIT5編碼產物為蛋白質法尼脂轉移酶 (PFT) 之β次單元。PFT催化之反應，是將法尼脂共價鍵結到C端含有CaaX motif的蛋白質上。是以由hit5的性狀可以知，必然有特定PFT受質，其法尼脂化與否，影響了植物耐受不同形式高溫逆境的能力。本研究計畫之目的，即是同時以前向式及反向式遺傳學的策略，尋找PFT控制熱反應路逕的下游分子(們)。前向式遺傳學的部份，吾人已從hit5群體中，篩選出兩株能將hit5性狀回復成野生型的突變株(roh1與roh2)。反向式遺傳學的部份，吾人已根據文獻資料及初期實驗，得出阿拉伯AtDjA3應是PFT調控植物熱逆境反應的媒介者。本研究計畫將以roh1, roh2 及AtDjA3為出發點，設計相關實驗。可預期執行之結果，能對此一全新植物耐熱調控機轉，有更完整與深入的闡釋。 ;Recent global warming can be traced back to the 18th century as industrial revolution released immense amount of greenhouse gases into the atmosphere. High temperature can inhibit plant growth and development which has been indicated to threaten crop production. Thus, understanding how plant sense and response to heat stress which can aid in devising strategies to enhance heat stress tolerance in crops is a necessary and imperative task. To do so, Arabidopsis heat intolerant 5 (hit5) mutant was isolated by its heat stress dependent phenotypes. Prolong heat incubation at 37°C for 4 d is lethal to hit5 but not to wild-type seedlings. On the contrary, sudden heat shock treatment at 44°C for 40 min is lethal to wild-type but not to hit5 seedlings. Positional cloning determined that the HIT5 gene encodes the β subunit of the heterodimeric protein farnesyl transferase (PFT). In eukaryotes, PFT catalyzes the reaction of addition of a 15-carbon polyunsaturated farnesyl lipid to the proteins whose C terminus ends with a CaaX motif, where “C” is cysteine for thioester linkage of farnesyl group, “a” is aliphatic amino acid, and “X” can be any amino acid. This lipidation reaction is one of the essential post translational modifications (PTMs). The isolation and characterization of hit5 thus not only reveals for the first time that plant heat stress response involves protein farnesylation at the PTM level, and indicates that there must be molecules involve in downstream of HIT5-mediated plant heat stress response. The aim of this proposed study is therefore to identify these molecules. Both forward genetics and reverse genetics approaches will be employed to reach this goal. In forward genetics, we have isolated two mutants from hit5 plants, named revertant of hit5 1 (roh1) and 2 (roh2), respectively, that can revert the heat dependent phenotypes of hit5 to those of wild type. Gene mapping of roh1 and roh2 will be performed to determine the responsible genetic determents. In reverse genetics, according to available sequence databases and literatures, we identified that one of the Arabidopsis DnaJ proteins, AtDjA3, contains the conserved CaaX motif and demonstrated that atdja3 knockout mutant is sensitive to 37°C 4 d heat treatment like hit5. These results suggest that AtDjA3 is potentially a mediator in the HIT5-contolled plant heat stress response. Experiments related to AtDjA3 are therefore designed and will be performed to deduce its specific functions and roles in this regard. Together, results from this proposed study will provide valuable insight into the novel protein farnesylation mediated plant heat stress response for future agriculture application. |
