Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36181
標題: 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
Increase 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 phenotypes
作者: 朱俊穎
Zhu, Jun-Ying
關鍵字: rice
農桿菌
tiller
水稻
分蘗
出版社: 生物科技學研究所
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摘要: Part1. 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。
Part1 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.
URI: http://hdl.handle.net/11455/36181
其他識別: U0005-2208200817261500
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2208200817261500
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