臺灣博碩士論文加值系統

植物菌質體 (Phytoplasma)是一種細菌性病原菌，由於缺乏了生存相關的生合成與代謝之基因組及細胞壁，僅能寄生於植物的篩管細胞中，至今仍無法人工繁殖。菌質體染病的植株會有莖葉叢生、葉片畸形及變小、花器葉化及綠化、葉片黃化、髮根、不孕花及果實畸形等病徵。目前已知被解序的翠菊黃萎病菌質體 (AYWB)之基因組可編碼56種分泌性蛋白 (SAPs)。從SAP11AYWB擬南芥轉植株發現葉片皺縮及叢枝的外表型；並在本實驗室研究，已知蘋果叢枝病菌質體與花生簇葉病菌質體之分泌蛋白 SAP11CaPM及 SAP11PnWB，其擬南芥轉殖株也有叢枝外表型。在建立SAP11菸草轉殖株的過程中，發現相較於只有載體的控制組轉殖株之轉殖成功率較高，因此推測SAP11會影響轉殖之效率。首先透過針筒注射接種法配合無菌組織培養建立出三種35S::SAP11AYWB、35S::SAP11CaPM以及35S::SAP11PnWB菸草轉殖株，發現三種SAP11轉殖菸草葉培植體會形成較多處的癒傷組織且分化出較茂密的不定芽，說明三種SAP11皆會促進轉殖效率。接下來從根瘤性狀實驗中得知三種SAP11參與在農桿菌轉殖效率的過程中提升轉殖效率。最後使用AGROBEST實驗偵測GUS報導基因之表現活性，顯示三種SAP11會使GUS基因的表現活性上升，因此推測SAP11會影響其他基因之轉殖效率。

Lacking cell wall and many essential genes required for biosynthesis and metabolic functions, phytoplasmas have become unculturable and phloem-limited plant pathogens. Disease symptoms caused by phytoplasmas including proliferation, little leaves, phyllody, virescence, leaf yellowing, hairy root, flower sterility and fruit deformity. Previous studies showed that the Aster Yellows Witches’ Broom (AYWB) phytoplasma contains 56 candidated effectors. Among them, the expression of secreted AYWB protein 11 (SAP11) in plants generated leaf crinkling and branching phenotypes. Our studies further demonstrated that SAP11AYWB homologues, SAP11CaPM and SAP11PnWB, can also cause the branching phenotype. Interestingly, during the generation of SAP11AYWB-transgenic Nicotiana benthamiana lines, we observed that SAP11 transgenic plants are easier to be obtained than vector only. This phenomenon suggests that SAP11 might affect the transformation efficiency. Here, using agroinfiltration, we demonstrate that SAP11 can increase the formation of callus and differentiation of shoots in N. benthamiana. Further more, in root tumorigenesis assay, we demonstrate that SAP11 can affect transformation efficiency by increasing the formation of tumors. Finally, in AGROBEST assay, we show that SAP11 can enhance the activity GUS reporter. This result suggest that SAP11 can also affect the transformation efficiency of other genes.

中文摘要 i
ABSTRACT ii
總目錄 iii
圖目錄 iv
附錄目錄 v
壹、前言 1
一、農桿菌 1
二、影響農桿菌轉殖效率的因子 2
三、植物菌質體 7
四、植物菌質體之分泌蛋白SAP11 9
貳、研究目的與策略 12
參、材料與方法 13
※材料 13
※實驗方法 13
一、農桿菌轉殖株之建立 13
二、菸草轉殖株建立 14
三、Tricien-SDS-聚丙烯醯胺膠體電泳分析 (Tricien-sodium dodecyl sulfate-polyacrylamide gel electrophoreses; Tricien-SDS-PAGE) 15
四、擬南芥根瘤性狀分析 (Root tumorigenesis assay) 16
五、AGROBEST (Agrobacterium-mediated enhanced seedling transformation, AGROBEST) 17
肆、結果 20
一、分泌蛋白SAP11會促進轉殖效率 20
二、分泌蛋白SAP11提升農桿菌之轉殖效率 21
三、分泌蛋白SAP11影響其他基因之轉殖效率 21
伍、討論 23
陸、參考文獻 25
柒、圖 31
捌、附錄 35

呂晏婷 (2013) 菌質體之分泌蛋白在擬南芥中調節缺磷反應與防禦機制的探討。 中興大學生物化學研究所碩士論文。
吳誌堂 (2016) 細菌性分泌蛋白SAP11引起擬南芥叢枝外表行之功能分析。 中興大學生物化學研究所碩士論文。
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