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dc.contributor.authorZhu, Jun-Yingen_US
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(2003) Agrobacterium-Mediated Root Transformation Is Inhibited by Mutation of an Arabidopsis Cellulose Synthase-Like Gene. Plant Physiology 133,1000-1010zh_TW
dc.description.abstractPart1. 農桿菌是在水稻基因轉殖技術中,最常使用的工具之一,其可幫助T-DNA插入植物基因組,目前農桿菌在水稻的轉殖效率約有30~40%以上的效率,但在其他並非皆有好的轉殖效率,利用大量表現參與分子是否能促進轉殖效率提升。T-DNA的傳送過程需要許多參與因子,包括分別來自農桿菌與水稻的蛋白質,例如BTI、NiR、VIP1和KU80。其中BTI為一個VirB2-interaction蛋白質;KU80功能為在細胞核內幫助T-DNA插入體染色體;VIP1是引導T複合物進入植物細胞和接入染色體;而NiR是一種亞硝酸還原酵素,可將較毒的亞硝酸鹽轉化成銨,幫助水稻的再生。其中有些已被證實假如大量表現,可提升擬南芥的轉殖效率,我們測試這些基因在水稻的效果,故選殖出BTI、NiR、VIP1和KU80的水稻同源基因,構築於載體,並進行對水稻轉殖。在T0世代的試驗,帶有 BTI的產生再生芽的效率相較控制組35ST來的高,約1.6到1.9倍,而在GUS報導基因實驗中也提升了1.9倍,但在擬南芥中則無明顯效果,可能與物種差異有關。 Part2 本實驗室先前利用SA1613.1和TNG67所產生的F2族群進行基因組分析鑑定,已知1號和11號染色體上各有一個調控穗數性狀的數量遺傳基因座qPN1與qPN11,分別可解釋22.6%和22.9%的性狀變化。在此研究我們僅針對qPN11的基因座定位,來自SA1613.1的qPN11是一個使分蘗數減少的對偶基因,為了進行qPN11精準定位,首先須建立帶有qPN11(S)的TNG67近同源品系,使之帶有SA1613.1的qPN11對偶基因,本實驗即利用SSR分子標誌,針對BC1F1世代植株進行全基因組檢測,目的為挑選較佳的回交世代。因植株生長勢極差與開花期嚴重延遲,亦發現水稻不定根的缺失情形比分蘗的情形更為嚴重,故進行根的結構分析。zh_TW
dc.description.abstractPart1 Agrobacterium-mediated transformation of higher plants is a well-known and powerful tool for transgene delivery to plant cells. The long journey for T-DNA transferring requires not only bacteria proteins but also is aided by various plant factors. Among the identified plant factors, several proteins were demonstrated to enhance plant transformation efficiency if overexpressed. We have isolated the orthologous genes for BTI, NiR, VIP1, and KU80 from rice and subcloned in an overexpression vector under controlled by the 35S promoter. By examination of the transformation efficiency in the T0 generation, BTI was found to generate more regenerating shoot 1.5~1.9 fold compared to transformation of vector only. BTI also enhance the transferring and expression of GUS reporter gene 1.9 fold to that of vector control. However, BTI is not effective in Arabidopsis, indicting its species-specificity in T-DNA delivery. Part2 Linkage map constructed from a F2 population of SA1613.1/TNG67 identified two quantitative trait loci (QTLs) responsible for tillering control. Located on chromosomes 1 and 11, qPN1 and qPN11 explained 22.6% and 22.9% of phenotypic variance, respectively. qPN11 loci was chosen for further analysis and molecular cloning. For fine-mapping and eventually cloning of qPN11 gene, a TNG67 near-isogenic line containing qPN11(S) needs to be constructed. In this study, a whole genome scanning on BC1F1(T) populations was performed and the target lines were identified. However, those identified BC1F1 grew very weak and flowering extremely late. Examination of root tissue revealed a structure difference between normal plant and plant with very low panicle numbers.en_US
dc.description.tableofcontents中文摘要 4 Abstract 5 前言 6 前人研究 8 一、農桿菌感染植物細胞的機制 8 二、影響植物轉殖效率的蛋白 8 三、與水稻再生的研究 9 二、與水稻根生長的相關基因 10 三、賀爾蒙對植物根生長的調控 11 四、數量性狀遺傳 12 五、分子標誌與同源品系建立 13 六、NIL品系的用途與建立之方法 13 材料方法 15 一、試驗材料 15 (一)台農67號 (Oryza sativa L. cv. Tainung 67, TNG67) 15 (二)SA1613.1 15 (三)SA1613.1×TNG67之BC1F1與BC1F2子代 15 二、試驗方法 15 (一)水稻轉殖與轉殖株的篩選 15 (二)萃取植物蛋白 19 (三)蛋白質定量 20 (四)GUS活性定量分析 20 (五)擬南芥的種植 20 (六)農桿菌感染擬南芥根 20 (七)水稻基因組DNA之小量萃取 21 (八)SSR分子標誌的聚合酶鏈反應(Polymerase chain reaction)條件及步驟 21 (九)分子標誌的分析 21 (十)石蠟包埋切片法 22 結果 23 一、受測基因是否提升自身基因轉殖效率 23 二、受測基因是否能幫助GUS報告基因轉殖進入植物(水稻癒傷組織暫時性轉殖分析) 23 三、受測基因是否能幫助GUS報告基因轉殖進入異源植物(擬南芥根暫時性轉殖分析) 24 一、分析qPN8的RSq值以判別此基因座對性狀的影響力 25 二、建立帶有qPN11基因座之TNG67近同源品系 25 三、SA1613.1×TNG67雜交子代根部的觀察分析 26 討論 28 參考文獻 31 表 36 圖 43 附錄一、農桿菌轉殖之質體構築 53 附錄二、兩個控制穗數性狀之QTL關係圖 57 附錄三、pBISN1與pCAMBIA1305.1質體 58 附錄四、蛋白質萃取液和GUS試驗相關試劑 59 附錄五、SSR分子標誌多型性分析之相關試劑 60 附錄六、石蠟包埋切片法相關試劑 61 附錄六、水稻組織培養所需之培養基成分 62 附錄七、水稻組織培養所需之培養基成分 63zh_TW
dc.title以表現輔助蛋白增進植物農桿菌轉殖法的效率 與 建立帶有qPN11(S)基因座之TNG67近同源品系與其性狀分析zh_TW
dc.titleIncrease Agrobacterium-mediated plant transformation efficiency by expression of accessory protein and Establishment of TNG67 near-isogenic lines containing qPN11(S) allele and characterization of its phenotypesen_US
dc.typeThesis and Dissertationzh_TW
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item.openairetypeThesis and Dissertation-
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