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研究生：	王正鈺 Wang, Cheng-Yu
論文名稱：	葉綠體第一型DnaJ同源蛋白質調控之種子健康 Seed fitness modulated by the type I chloroplast DnaJ homologues
指導教授：	邱啟洲 Chiu, Chi-Chou
共同指導教授:	蘇百祥 Su, Pai-Hsiang
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 熱帶植物科學研究所 Institute of Tropical Plant Sciences
論文出版年：	2017
畢業學年度：	105
語文別：	中文
論文頁數：	66
中文關鍵詞：	阿拉伯芥、葉綠體、J蛋白、伴護子、種子充實
外文關鍵詞：	Arabidopsis, chloroplast, J protein, molecular chaperone, seed fitness
相關次數：	點閱：91 下載：6
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熱休克蛋白是一群保守性的伴護蛋白，廣泛存在於原核和真核生物中，根據其分子量，可被細分為不同的家族。葉綠體擁有自己的熱休克蛋白Hsp70伴護子系統，其可能參與多個生物過程，並且透過指定含有J結構域的共同伴護子 (J蛋白)，將之招募到特定過程。阿拉伯芥 (Arabidopsis thaliana) 中，存在19個葉綠體的J蛋白，其中有4個是屬於第一型的J蛋白，被命名為DJA4、DJA5、DJA6和DJA7，至今，關於它們的生物功能了解甚少。我們的初步研究，顯示葉綠體DJA蛋白對種子健康是重要的。我們觀察到dja四重突變株 (dja quadruple knockout, dja-qko) 所產生的種子，表現出不規則形狀的皺縮外觀。生化分析顯示，皺縮種子含有較低含量的儲存性脂肪酸 (三酸甘油酯)。生理特徵顯示，dja-qko突變株種子無法在無糖MS培養基上生長，這可能是由於在突變株種子中用於早期建立幼苗的儲存物儲備不足所致。此外，dja-qko突變株幼苗，在含高濃度糖的MS培養基上，也表現出敏感性外表型，表示葉綠體DJA蛋白可能參與糖代謝或訊息傳遞。總而言之，這些數據表示DJA蛋白是種子發育所必需的，並且對於早期萌芽階段之幼苗是十分重要的。

Heat-shock proteins (Hsps) are conserved chaperone proteins that widely exist in the prokaryotic and eukaryotic organisms. According to their molecular weight, these proteins could be classified into different subfamilies. Chloroplast has its own Hsp70 chaperone system, which may be involved in multiple biological processes and is recruited to specific processes by designated J domain-containing co-chaperones (J proteins). In Arabidopsis, there are 19 chloroplast-targeted J proteins; of them 4 are belonged to type-I J proteins naming DJA4, DJA5, DJA6 and DJA7. To date, little is known about the biological function of DJAs. Our study showed that chloroplast DJA proteins are important for the seed fertility and fitness. Viable seeds produced by the dja quadruple knockout (dja-qko) mutant exhibited a wrinkled appearance with irregular shape. Biochemical analyses revealed that the wrinkled seeds contain a reducing level of storage fatty acid (triacylglycerol). Physiological characterization showed that the dja-qko seedlings could not grow up on sugar-free MS medium, which might due to an insufficient storage reserve in mutant seeds for early establishment of young seedlings. Furthermore, the dja-qko seedlings also exhibited a sensitive phenotype to a higher level of sugar in MS medium, implying that chloroplast DJA proteins may be involved in sugar metabolism or signaling. To sum up, our data suggested that chloroplast DJA proteins are required for seed development and important for seedling establishment at the early germinating stage.

目錄

中文摘要......................................................................................................................i
英文延伸摘要...............................................................................................................ii
誌謝...........................................................................................................................xi
目錄...........................................................................................................................xii
表目錄.......................................................................................................................xv
圖目錄......................................................................................................................xvi
附圖目錄...................................................................................................................xvi
符號及縮寫索引表.....................................................................................................xviii

主文
壹、	背景介紹與前人結果..............................................................................................1
一、	葉綠體J蛋白的分類...............................................................................................1
二、	葉綠體J蛋白的生理功能.........................................................................................2
三、	阿拉伯芥葉綠體第一型J蛋白命名與dja突變株特性....................................................2
四、	種子發育過程........................................................................................................3
五、	種子儲存物與經濟意義...........................................................................................3
六、	種子充實與熱逆境.................................................................................................4
七、	基因調控種子儲油之例子.......................................................................................4
八、	高溫逆境影響種子充實之例子.................................................................................5
九、	葉綠體第一型J蛋白抵抗高溫逆境影響.....................................................................5
十、	實驗研究目標.......................................................................................................5

貳、	材料與方法...........................................................................................................6
一、	植物相關材料製備.................................................................................................6
1.	植物生長條件...........................................................................................................6
2.	阿拉伯芥突變株來源…..............................................................................................6
二、	含DJA基因片段之載體構築....................................................................................7
1.	本篇文章內所使用的 E. coli 勝任細胞品系與用途.........................................................7
2.	培養液成分...............................................................................................................7
3.	對應DJA之引子設計..................................................................................................7
4.	聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) ...................................................9
5.	瓊脂膠體電泳 (Agrose gel electrophoresis) 和 DNA 純化 (DNA purification)....................9
6.	限制酶剪切和基因黏合與轉殖 (Restriction enzyme digestion, ligation and transformation) ..................................................................................................................................10
7.	菌落聚合酶連鎖反應檢測 (Colony PCR) 和質體抽取 (Plasmid mini-preparation) ............11
8.	pCambia1390載體構築............................................................................................11
三、	DJA轉基因互補試驗 (Complementation test) .........................................................12
1.	本篇文章內所使用的農桿菌品系與轉殖 (transformation) .............................................12
2.	農桿菌 (Agrobacterium tumefaciens GV3101) 勝任細胞之製備 (化學法) ......................12
3.	花序浸漬法-阿拉伯芥轉殖 (Floral dip： Agrobacterium-mediated transformation of Arabidopsis thaliana) ..................................................................................................12
4.	篩選轉基因植株......................................................................................................13
5.	植物DNA萃取 (Genomic DNA extraction) .................................................................13
6.	基因分型 (genotyping) 檢驗......................................................................................13
四、	種子儲存物分析...................................................................................................14
1.	總蛋白質粗萃取與定量 (Total protein extraction and quantification) .............................14
2.	SDS-PAGE............................................................................................................14
3.	脂質萃取 (Lipid extraction) 與薄層層析法 (TLC, Thin Layer Chromatography) ...............15
4.	脂肪酸組成 (Fatty acid composition) 與氣相層析質譜儀檢測 (GC-MS, Gas Chromatography-Mass spectrometry) ..........................................................................16
5.	碳水化合物分析 (Carbohydrate analysis) ..................................................................17
6.	澱粉定量 (Starch quantification) ..............................................................................17
五、	植物生理測試......................................................................................................18
1.	種子萌芽試驗與子葉變綠時間點觀察.........................................................................18
2.	葉綠素含量測定......................................................................................................19
3.	碳源控制條件下之生長觀察......................................................................................19
4.	種子直播土壤之生長觀察.........................................................................................20

參、	結果...................................................................................................................21
一、	dja-qko突變株之主要外表型..................................................................................21
二、	含DJA基因之pCambia1390質體構築.....................................................................21
1.	構築pCambia1390-DJA6gDNA質體...........................................................................22
2.	構築pCambia1390-DJA7gDNA質體...........................................................................22
3.	構築pCambia1390/35S-DJA5gDNA質體....................................................................22
4.	構築pCambia1390/35S-DJA4gDNA質體....................................................................23
5.	構築pCambia1390/DJA7pro-DJA5gDNA質體.............................................................23
三、	DJA轉基因互補測試 (Complementation test) ........................................................24
四、	種子儲存物分析...................................................................................................25
1.	總蛋白分析.............................................................................................................25
2.	脂質分析................................................................................................................26
3.	脂肪酸組成分析......................................................................................................26
4.	碳水化合物分析......................................................................................................27
五、	外源性碳源影響種子萌芽率與子葉變綠時間點........................................................27
六、	外源性碳源影響植株生長......................................................................................28
七、	dja-qko突變株種子播種於土壤之植株生長..............................................................29
八、	結論...................................................................................................................30
(一)	DJA轉基因互補測試.............................................................................................30
(二)	種子儲存物分析...................................................................................................30
(三)	植物生理分析.......................................................................................................30

肆.	討論 ....................................................................................................................31
一、	探討葉綠體第一型J蛋白對植物生理之影響.............................................................31
二、	葉綠體DJA蛋白參與種子發育與充實.....................................................................32
三、	葉綠體DJA蛋白可能影響脂質生合成途徑..............................................................32
四、	葉綠體DJA蛋白可能參與糖代謝或訊息傳遞...........................................................34
五、	葉綠體DJA蛋白可能參與光合作用........................................................................34

伍.	參考文獻...............................................................................................................36

陸.	表圖.....................................................................................................................40
表.............................................................................................................................40
圖.............................................................................................................................42
附圖.............................................................................................................................57

表目錄

表  1、 本研究使用之專一性引子列表............................................................................40

圖目錄

圖  1、 野生株與dja-qko突變株成熟果莢裡的種子形態分析..............................................42
圖  2、 以專一性引子克隆DJA4、DJA5、DJA6、DJA7基因............................................43
圖  3、 pCambia1390-DJA6gDNA之質體構築.................................................................44
圖  4、 pCambia1390-DJA7gDNA之質體構築.................................................................45
圖  5、 pCambia1390/35S-DJA5gDNA之質體構築..........................................................46
圖  6、 pCambia1390/35S-DJA4gDNA之質體構築...........................................................47
圖  7、 pCambia1390/DJA7pro-DJA5gDNA之質體構築....................................................48
圖  8、 DJA互補系種子之外表型...................................................................................49
圖  9、 成熟種子之儲存性蛋白質含量分析......................................................................50
圖  10、 成熟種子之TAG含量分析.................................................................................51
圖  11、 TAG之FA組成分析..........................................................................................52
圖  12、 培養基含糖有無對種子萌芽之影響....................................................................53
圖  13、 培養基含糖有無對子葉變綠時間點之影響..........................................................54
圖  14、 培養基含糖有無對幼苗生長之影響....................................................................55
圖  15、 播種於土壤之幼苗生長觀察..............................................................................56

附圖目錄

附圖  1、野生株與dja-qko突變株種子之綜合比較............................................................57
附圖  2、阿拉伯芥種子發育過程之葉綠體第一型DJA基因表現量.......................................58
附圖  3、 T1子代 pCambia1390-DJA6gDNA轉殖株之基因分型檢驗..................................59
附圖  4、 T1子代 pCambia1390-DJA7gDNA轉殖株之基因分型檢驗..................................60
附圖  5、 T1子代 pCambia1390/35S-DJA5gDNA轉殖株之基因分型檢驗............................61
附圖  6、 T1子代 pCambia1390/35S-DJA4gDNA轉殖株之基因分型檢驗............................62
附圖  7、 T1子代 pCambia1390/DJA7pro-DJA5gDNA轉殖株之基因分型檢驗.....................63
附圖  8、 T1子代 pCambia1390/35S-DJA5gDNA轉殖株之白化致死外表型 (Albino lethal phenotype) ................................................................................................................64
附圖  9、 植株葉綠素含量測定......................................................................................65
附圖  10、 黑暗條件下培養基含糖有無對幼苗生長之影響….............................................66
 

                                    

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校內：2022-01-18公開
校外：2022-01-18公開

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