臺灣博碩士論文加值系統

植物菌質體(Phytoplasma)是一種無細胞壁的植物病原菌，寄生於植物的篩管細胞內，至今仍然無法以人工方式培養，所以致病機制目前仍無法被研究清楚。植物菌質體在宿主植物上會引起許多病徵產生，如發育遲緩、葉片黃化、花器綠化、莖葉簇生、葉片捲曲等外表型。藉由基因體的解序可知翠菊黃萎簇葉病菌質體基因組可以編碼出許多分泌性致病蛋白SAPs (serected AY-WB proteins)，其中SAP11可以改變植物外表性狀例如造成叢枝，且可以降解CIN-TCPs轉錄因子。近年來，隨著植物菌質體基因體被大量解序，可以發現在不同菌株的SAP11胺基酸序列具有保留性，而這些保留性序列可能為造成叢枝共通性的主要區域。因此，為了瞭解更多植物菌質體間分泌蛋白的差異度與功能性，我們分析五種不同SAP11，分別是SAP11CaPS、SAP11ICPP、SAP11WBDP、SAP11VWBP與SAP11CaPPr。其中SAP11ICPP、SAP11WBDP與SAP11CaPS在序列上較為相近。進一步，藉由轉殖植物的建構與分析，發現只有SAP11VWBP與SAP11CaPS會造成植物葉片外表型的改變。雖然並非所有轉殖植物都有葉片外表型的改變，但皆有叢枝外表型的出現。接著，利用菸草短暫性表現系統以及西方墨點法分析，發現不同之SAP11皆會使調控植物叢枝的TCP12與TCP18轉錄蛋白不穩定而降解。透過生物資訊學分析，發現SAP11可能具有結合自噬作用之重要因子Atg8蛋白的AIM/LIR (Atg8-interacting motif/LC3-interacting region)區塊。將AIM/LIR區塊中重要的苯丙胺酸(Phenylalanine, F)突變成丙胺酸(Alanine, A)，可以發現TCP12與TCP18轉錄因子無法被降解。此結果表示AIM/LIR區塊在SAP11促進TCP轉錄因子的降解作用中扮演重要的角色。

Phytoplasmas are bacterial pathogens without cell wall and live in phloem sieve elements. They are unculturable and cause several disease symptoms including dwarf, witches’ broom, leaf yellowing, virescence and phyllody. The whole genome of Aster Yellows phytoplasma strain witches’ broom has been sequenced, and 56 candidate effectors were identified and named as SAPs (secreted AYWB proteins). Among them, SAP11AYWB can change plants phenotype and destabilize the plant Class II TCP transcription factors. Recently, SAP11 proteins were identified from different phytoplasma strains via whole genome sequencing. To understand the function and difference of individual SAP11 proteins, SAP11s, SAP11CaPS、SAP11ICPP、SAP11WBDP、SAP11VWBP and SAP11CaPPr were characterized. Among them, only SAP11CaPS、SAP11ICPP and SAP11WBDP have similar sequences. However, all of them can cause branching phenotype when they were expressed in planta. According to bioinformatics analyses, we found that SAP11 might contain a conserve motif called AIM/LIR (Atg8-interacting motifs/LC3-interacting regions) ,which is involved in autophagy. By point mutation analyses, we identified critical amino acid which is responsible for the degradation of TCP12 and TCP18. Results suggest that the AIM/LIR motif plays an important role in SAP11-mediated degradation of TCP transcription factors.

總目綠
中文摘要 i
Abstract ii
總目綠 iii
圖目綠 v
附錄 vi
第一章　前言 1
一、植物菌質體之介紹 1
二、植物菌質體致病性蛋白與分泌系統 2
三、不同植物特有轉錄因子TCP 家族功能及介紹 4
四、自噬作用 5
五、植物菌質體之免疫膜蛋白 7
第二章 研究目的與策略 8
第三章 材料與方法 9
一、實驗材料 9
二、載體構築 9
聚合酶連鎖反應(Polymerase Chain Reation, PCR) 9
DNA瓊脂膠體電泳 9
PCR產物之純化 10
PCR產物和質體之限制酶作用 10
PCR產物和質體之接合反應 10
大腸桿菌勝任細胞之製備 10
大腸桿菌之轉殖 11
重組DNA之鑑定 11
三、擬南芥轉殖株之建立 11
農桿菌勝任細胞之製備 11
農桿菌之轉殖及鑑定 12
農桿菌轉殖基因至阿拉伯芥(花序沾黏法) 12
擬南芥轉殖株之篩選 12
四、重組蛋白質之表現 13
大腸桿菌重組蛋白質之小量表現 13
SDS-PAGE蛋白質電泳 13
大腸桿菌重組蛋白質之大量表現 14
蛋白質之純化 14
五、抗體製備 15
抗體製備 15
西方墨點法 15
六、菸草短暫性表現系統之農桿菌注射法 (Agroinfiltration) 15
農桿菌勝任細胞製備 15
菸草短暫性表現菸草注射法 16
七、定點突變(Point mutation) 16
點突變引子設計 16
聚合酶連鎖反應(Polymerase Chain Reation, PCR) 16
重組DNA之鑑定 17
第四章 結果 18
一、 植物菌質體分泌蛋白SAP11質體構築與轉基因植物構築 18
二、 植物菌質體分泌蛋白SAP11之轉基因植物擬南芥外表型分析 18
三、 不同SAP11轉殖植株蛋白表現量檢測 19
四、 植物菌質體分泌蛋白SAP11與TB1/CYC-TCP轉錄因子降解作用探討 20
五、 植物菌質體分泌蛋白SAP11CaPPr特殊區域點突變之功能探討 20
六、 Anti-ImpEPWB之抗體製備 21
第五章 討論 22
一、 植物菌質體分泌蛋白SAP11與轉基因植物外表型關聯性 22
二、 植物菌質體分泌蛋白SAP11關鍵區域點突變與TCPs降解關聯性 22
第六章 附圖與表 23
第七章 參考文獻 31
第八章 附錄 34
 

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