臺灣博碩士論文加值系統

植物的選擇性自噬作用(selective autophagy)在逆境反應、延遲老化以及營養短缺中扮演重要角色。其主要功能為回收再利用特定物質如胞器、蛋白質聚合物或是特定蛋白質。先前研究指出，靜默phytoene desaturase (pds)基因表現的菸草，會導致葉綠素以及類胡蘿蔔素表現量下降，同時呈現葉片白化的外表型。為了解pds靜默與葉片白化的關聯性，我們首先藉由植物切片觀察到白化現象與葉綠體的數目有密切關連性。依據文獻得知，葉綠體的降解是Rubisco-Containing Bodies (RCBs)為主的自噬作用，會選擇性降解含有葉綠體基質的內含物，可是目前為止，對於何種因子決定RCBs的選擇性降解仍然不清楚。本實驗發現pds靜默產生的白化葉片會使選擇性自噬作用接合分子Joka2蛋白的表現量上升。同時，藉由Amitrole抑制PDS蛋白活性產生的白化葉片，在Joka2靜默的情況下可以觀察到細胞中會有明顯的胞狀物質堆積。由此推測Joka2可能參與葉綠體胞器的降解回收。另外一方面，已知Xanthomonas會藉由第三型分泌系統分泌致病性蛋白並影響植物生理。當植物大量表現單一致病性蛋白XopQ或XopN會引發抵抗病原菌感染之過敏性反應(hypersensitive response)導致細胞壞死。本實驗發現到此過敏性反應也會提升Joka2蛋白的表現量，因此推測Joka2也可能參與植物免疫機制中的過敏性反應。

Plant selective autophagy plays a significant role in stress responses, delay aging and nutrient shortage. The main function of selective autophagy is to recycle the specific elements like organelles, aggregated proteins or specific proteins. Previous studies indicated that phytoene desaturase (pds) silencing not only lead to the decrease of carotenoid and chlorophyll contents, but also cause an albino phenotype. Here, we characterized the relationship between pds silencing and leaf albino were characterized. We observed the number of chloroplasts was reduced in the albino leaves via tissue slices. Chloroplast degradation has been reported via Rubisco-Containing Bodies (RCBs), which was regarded as autophagy. However, it is still unknown how specific chloroplast substrates are recognized and obtained in RCBs. In this study, we found that the expression level of cargo receptor Joka2 was highly increased in albino leaves. Moreover, in Joka2 silencing plant, vesicle-like structures were observed and accumulated after Amitrole, a PDS inhibitor, treatment. Taking together, these results suggest that Joka2 is involved in chloroplast degradation.
It has been known that Xanthomonas can interfere to plant immunity by virulence effectors, which are secreted via type III secretion system. The overexpression of XopN(Xanthomonas outer protein N) and XopQ effectors in leaves could trigger severe hypersensitive response (HR) and cause cell death in tobacco leaves. In this study, we found that HR can also induce the expression of Joka2. Thus, these results suggest that Joka2 is also involved in HR triggered by Xanthominas type III effectors.

摘要 i
Abstract ii
總目錄 iii
圖目錄 vi
表目錄 vii
附錄 viii
第一章、緒論 1
一、植物體中葉綠體的降解 1
二、類胡蘿蔔素 2
三、自噬作用 2
四、選擇性自噬作用與接合分子 3
五、Xanthomonas簡介 4
六、Xanthomonas的第三型致病性蛋白 4
七、研究目的與策略 5
第二章、材料與方法 7
一、實驗材料 7
二、載體之構築 7
聚合酶連鎖反應(Polymerase Chain Reaction, PCR) 7
DNA膠體電泳 7
PCR產物之純化 7
PCR產物和質體之限制酶作用 7
PCR產物和質體之接合反應 8
大腸桿菌勝任細胞之製備 8
大腸桿菌之轉殖 8
重組DNA之鑑定 8
三、利用穿透式電子顯微鏡觀察細胞構造變化 9
樣品塑膠包埋 9
塑膠切片 9
超薄切片之染色 9
穿透式電子顯微鏡之使用 10
四、十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (sodium dodecyl sulfate polyacrylamide gel electrophoresis，SDS-PAGE) 10
五、西方墨點法(western blotting) 10
轉漬作用 10
抗體雜合 11
呈色反應 11
六、農桿菌轉殖菌株之建立 11
農桿菌勝任細胞(argobacterial competent cell)製備 11
農桿菌轉形 11
七、菸草短暫性表現系統之農桿菌表現系統 12
第三型致病性蛋白之菸草短暫性表現針筒注射法 12
植物組織研磨 12
八、圓葉菸草VIGS (virus-induced gene silencing )建立 12
菸草針筒注射法 12
九、靜默Joka2轉殖圓葉菸草之建立 13
十、Amitrle溶液配置 13
第三章、實驗結果 14
一、 觀察藉由VIGS產生的白化葉片與葉綠體數量的關係 14
二、 VIGS產生的白化葉片與選擇性自噬作用接合分子Joka2蛋白表現量偵測 14
三、 Amitrole引發菸草葉片白化外表型與接合分子Joka2蛋白表現量偵測 15
四、 天然白化園藝植物與接合分子Joka2蛋白表現量偵測 15
五、 探討接合分子Joka2與白化葉片之間的關聯性 16
六、 大量表現不同Xanthomonas第三型致病蛋白與細胞死亡之過敏性反應篩選 16
七、 探討Joka2與Xanthomonas第三型致病性蛋白形成細胞死亡之過敏性反應之間的關聯性 17
第四章、討論 18
一、 葉綠體降解與選擇性自噬作用的關係探討 18
二、 Joka2蛋白自我調控機制的推測 18
第五章、參考文獻 20
第六章、圖、表及附錄 22

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