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研究生:陳佳鈴
研究生(外文):CHEN,Chia-Ling
論文名稱:台灣原生杜鵑抗病基因家族功能性演化研究
論文名稱(外文):The functional evolution of resistance gene family of rhododendron native in Taiwan
指導教授:黃士穎
指導教授(外文):Huang,Shih-Ying
學位類別:碩士
校院名稱:中國文化大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:103
中文關鍵詞:抗病基因正向選汰
外文關鍵詞:R-geneNBS-LRRpositive selection
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植物之病害抵抗基因是屬於非常大的gene family,這些基因序列結構上有一些特徵可用來判別是否屬於病害抵抗基因的一份子。植物病害抵抗基因的蛋白質結構上,主要含有核苷酸鍵結區Nucleotide-binding site domains( NBS )及C 端的Leucine rich repeats(LRR)區,再以植物病害基因蛋白質結構N端前之序列分成兩大類,分別為GroupΙ:N-ter 為TIR 區( Toll and interleukin-1 receptor genes )及GroupⅡ:N-ter 為CC 區或推測可能為LZ 區( coil-coiled or leucine-zipper)。
LRR 區主要因應相對病源之辨認而共同演化,然而NBS 及CC (TIR)兩區則分別與R 基因蛋白之構形及訊息傳導有關。因此相對於LRR 區之被動式演化而言,CC (TIR)及NBS 兩區為植物主動演化,且NBS 具有許多高度保守的功能性區域。在整個研究之架構上,藉由選殖台灣杜鵑及烏來杜鵑基因組中之R基因的CC (TIR)及NBS 區域之resistance gene analogs 以進行探討CC(TIR)及NBS 區之positively selected amino acid sites 並分析其與R 基因domain 間之結構形成之可能關係。
結果發現NBS 抗病基因有Gene duplication 的現象發生,使得序列在TIR-NBS-LRR 和nonTIR-NBS-LRR 不同類型間之序列長度差異極為明顯,且兩類型在演化的速率上亦有明顯的分歧,以最適模式free-ratio 下偵測其ω 值,大部分皆小於1,僅有少部分分支有大於1 的情形,顯示分支有存在著不同程度的選汰壓力,且這兩類型之抗病基因序列皆會在少數的胺基酸位置上,遭受到顯著之正向選汰壓力。比較TIR-NBS-LRR 和nonTIR-NBS-LRR 不同類型間之抗病基因序列,發現共同遭受正向選汰壓力的位點落在NBS 區域,但不在任何功能性domain 中,推測可能是這些功能性domain 不允許被置換以避免其抗病之功能喪失,證明了這些功能性domain 扮演著對抗病功能性之關鍵角色。
Plant disease resistance gene (R-gene) is a very big gene family, many plant disease resistance genes and related genes have been sequenced. Most of the functional sequence have some special characteristics in their structure which can be recognized whether belong to a resistance gene.
Plant disease resistance gene that encode protein sequences containing a Nucleotide-binding site domains (NBS) and C-terminal leucine-rich repeats (LRR), NBS-LRR genes are the major class of disease resistance in flowering plants, and NBS resistance gene are subdivided into two families based on the presence or absence of an N-terminal region with homology to Drosophila Toll and human interlukin-1 receptor (TIR region), namely TIR and non-TIR genes. The non-TIR type commonly has a predicted leucine-zipper (LZ)domain or putative coiled-coil (CC) domain at N-terminal region. The non-TIR family is widely distributed in both monocotyledonous and dicotyledonous species, but not seen in gymnosperm species. wherea the TIR family appears to be restricted to dicotyledonous and gymnosperm species.The research is based on cloning the resistance gene analogs within CC(TIR) and NBS regions in the R gene of Rhododenfdrom formosanum Hemsl. and Rhododendron kanehirai Wilson gene sets and analyzing their possible relationships in structure forming between these R gene domain. The results show that there’s gene duplication in both Rhododendron formosarum Hemsl. and Rhododendron kanehirai Wilson. Tajima’s relative rate test shows that there’s evolutional branching between TIR and non-TIR.
Furthermore, selective constraint analysis proves that there’s diversity between these two types of rhododendron. However, when tested with the most suitable free-ratio, most ω is less than 1, which means the end result of cloning does not allow any substitutions to form within these conserved region in order to protect it’s function.
目 錄
中文摘要.................................................................................................... 1
英文摘要.................................................................................................... 2
壹、前言.................................................................................................... 4
貳、前人研究........................................................................................... 5
(一)植物抗病基因............................................................................... 5
(二)抗病基因的作用機制................................................................. 11
(三)自然環境下之選拔力量.............................................................12
(四)基因的複製與演化上之關聯.....................................................13
(五)偵測基因序列之選汰壓力.........................................................15
參、材料與方法.....................................................................................16
一、植物樣本採集.................................................................................16
(一)台灣杜鵑.....................................................................................16
(二)烏來杜鵑.....................................................................................17
二、植物樣本處理.................................................................................18
三、DNA 之萃取....................................................................................18
四、DNA 之定量與稀釋........................................................................20
五、聚合酶連鎖反應.............................................................................20
(一)引子設計.....................................................................................20
(二)進行NBS 序列擴增....................................................................22
六、電泳..................................................................................................23
(一)電泳膠的製備.............................................................................23
(二)電泳分析充填.............................................................................23
七、T-A clning........................................................................................25
(一)接合作用.....................................................................................25
(二)轉型作用.....................................................................................25
(三)Colony PCR ..............................................................................25
(四)電泳及其資料保存.....................................................................27
八、資料分析.........................................................................................27
(一)DNA 序列分析與親緣關係分析................................................27
(二)序列之演化分析.........................................................................29
肆、結果..................................................................................................34
一、序列比對及ORF 比對分析............................................................34
二、序列之變異程度分析.....................................................................35
三、胺基酸consensus 序列親緣關係圖建立及保守區域分析.........36
四、NBS 不同類型抗病基因之遺傳距離分析....................................38
五、Tajima 相對速率測試.....................................................................38
六、選汰壓力分析.................................................................................39
七、正向選汰壓力分析.........................................................................40
伍、討論..................................................................................................44
一、植物NBS-LRR 抗病基因之Gene duplication............................44
二、植物抗病機制探討及NBS 保守功能性之組成分析...................44
三、植物NBS 抗病基因突變演化之趨勢...........................................46
四、植物NBS 抗病基因之選汰壓力探討...........................................47
五、植物NBS 抗病基因之正向選汰壓力之探討...............................48
六、台灣杜鵑與烏來杜鵑之抗病基因類型探討.................................50
陸、結論..................................................................................................52
柒、參考文獻.........................................................................................53
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