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研究生:沈盟倪
研究生(外文):Meng-Ni Shen
論文名稱:利用T-DNA插入突變體探討水稻基因之功能-抗病能力異常突變體M0023454基因功能之研究
論文名稱(外文):Rice functional genomics study with T-DNA insertion mutants-Functional analysis of a disease susceptible mutant M0023454
指導教授:陳良築
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
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:2008
畢業學年度:96
語文別:中文
論文頁數:68
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M0023454為T-DNA插入的水稻(Oryza sativa L. cv. Tainung 67)突變株。在本實驗室過去研究中指出,M0023454在水稻生長第六十到七十天期間開始,會出現嚴重的稻熱病病斑,並且在榖粒黃熟期間,整株出現枯萎的現象。稻熱病在台灣,為受到真菌類病原菌Pyricularia oryzae Cavara寄生所引起,在溼度高的情況下,有利病原菌胞子發芽,入侵植物組織,嚴重影響水稻收成。進一步分析M0023454突變株後代的基因型與農藝性狀間的遺傳模式,發現有共分離的現象。經由RT-PCR分析,顯示454-10基因(Os04g32620)的表現量會受到T-DNA構築上之8個CaMV35S增強子而活化。推測454-10的大量表現可能造成植株抵抗病菌能力產生變異。利用植物基因轉殖技術,生產以玉米Ubiquitin啟動子大量表現 454-10基因之水稻轉植株(Ubi::454-10),以進一步確定M0023454水稻突變株所觀察到之抗病能力異常為454-10基因的活化大量表現所導致,然而Ubi::454-10水稻轉植株並未觀察到易感病徵的性狀。經胺基酸比對及系統演化樹的分析,454-10蛋白內含有一個AP2區域,歸類在AP2/EREBP (APETALA2/ethylene response factor)家族ERF subgroup中的B4 subcluster,主要參與調控防禦病菌入侵的生理反應。與抗病機相關的植物荷爾蒙中,Ethylene、Jasmonic acid和Salicylic acid會誘導454-10的表現;然而Abscisic acid卻會抑制454-10的表現,顯示454-10可能與這些植物荷爾蒙的訊號傳遞有關。分析可能受454-10基因調控的下游Pathogenesis-related (PR)之相關基因表現情形,在沒有病徵之M0023454和Ubi::454-10水稻轉植株葉片,OsPR10a/PBZ1和OsPR1a的表現會受到抑制,然而OsChia1c與TNG67並無明顯的差別,推測454-10基因可能參與OsPR10a/PBZ1和OsPR1a基因的調控。在M0023454突變株中,推測除454-10基因外,尚有其它未知因子共同參與植物病原防禦機制,目前在M0023454偵測到插入點上游12,442 bp處之454-9 (Os04g32610)基因表現亦受T-DNA插入而活化。因此,也進一步將454-9基因大量表現於TNG67中,探討其功能是否與水稻疾病防禦機制有關。
Rice (Oryza sativa L. cv. Tainung 67) mutant line, M0023454, is susceptible to rice blast that may resulted by the activation of a T-DNA insertion. Eight CaMV35S enhancers were constructed in T-DNA that can activate the expression of flanking genes next to its insertion. AP2/ERF (APETALA2/ethylene response factor) domain-containing gene 454-10 (Os04g32620), a flanking gene next to the T-DNA insertion in M0023454, is classified into B4 subcluster of ERF subfamily in AP2/ERF superfamily. Phylogenic analysis suggested that 454-10 is an AP2/ERF-like transcription factor. In the present study, we demonstrated that 454-10 can be activated by pathogen defense-related plant hormones, such as ethylene (ET), jasmonic acid (JA), and salicylic acid (SA) but was suppressed by abscisic acid (ABA). The constitutive expression of 454-10 in the Ubi::454-10 transgenic line can be detected, however no distinct phenotype was observed. It was suggested that AP2 domain can bind the GCC box to regulate gene expression. Several pathogenesis-related (PR) genes, such as OsPR1a, OsChi1c and OsPR10a/PBZ1 that contain putative GCC box were identified and their expression in M23454 and Ubi::454-10 were examined. We demonstrated that OsPR10a/PBZ1 and OsPR1a were partially suppressed, but OsChi1c was normally expressed in M0023454 and Ubi::454-10. Interestingly, the expression of 454-10 in Ubi::454-10 is stronger than that in M0023454, while the suppression of OsPR1a in Ubi::454-10 is weaker than that in M0023454. This observation suggests that at least one more factor is involved in the regulation of OsPR1a and this may be involved in defense response of M0023454. In addition, a hypothetic gene, 454-9 (Os04g32610) was recently identified to be activated in M0023454, and the study of its functional role in mutant M0023454 is underway.
中文摘要…………………………………i
英文摘要…………………………………………ii
目錄………………………………………………iii
圖表目錄…………………………………………v
縮字對照表…………………………… vi
前言…………………………………………1
前人研究
水稻基因功能的研究……………………………1
植物抵抗病原菌的過程…………………………………3
Pathogenesis-related (PR)基因的介紹…………………4
植物荷爾蒙與植物防禦反應……………………………5
轉錄因子與病原菌關係的研究………………………5
AP2/ERF superfamily…………………………………6
材料……………………………………………9
方法
一、Ubi::454-10水稻轉殖株之分析…………9
二、南方墨點法……………………………………………………12
三、基因表現偵測…………………………………14
四、蛋白抗體製備…………………………………15
五、蛋白質表現之分析…………………………16
六、Ubi::454-10水稻轉殖株感病試驗………………18
七、植物荷爾蒙與淹水試驗………………………19
結果
一、M0023454突變株T-DNA插入點兩側基因表現情形之探討…………20
二、454-10蛋白抗體的製備………………………………21
1.蛋白表現載體454-10/pET30a之構築與His6x-454-10之生產……21
2.誘導His6x-454-10融合蛋白的產生及純化…………………………21
3.His6x-454-10蛋白抗體血清的製備………………………21
三、Ubi::454-10轉殖株之分析…………………………21
1.植物表現載體之構築與水稻轉殖及再生……………………………22
2.Ubi::454-10 (T0)水稻轉殖株之確認及分析…………………………22
3.Ubi::454-10 (T1)水稻轉殖株同質株系篩選及分析……22
4.Ubi::454-10 (T2)水稻轉殖株和M0023454突變株之性狀比較分析……23
四、Ubi::454-10水稻轉殖株感病試驗……………………24
五、454-10和AP2/EREBP家族基因親緣關係建立………………………25
1.阿拉伯芥AP2/EREBP家族基因的建立……………………………25
2.水稻AP2/EREBP家族基因的建立…………………………………25
六、454-10與疾病防禦系統相關之植物荷爾蒙關係…………………26
七、M0023454和Ubi::454-10水稻轉殖株可能影響下游基因之分析……26
八、454-9基因在M0023454 T-DNA插入突變株之表現分析………28
討論
M0023454 T-DNA插入突變株之觀察與分析…………………31
454-10基因功能之探討…………………………………31
454-9基因結構及功能分析………………………35
結論……………………………………………………35
圖………………………………………………………36
參考文獻…………………………………………………59
附表………………………………………………65
附錄……………………………………………67
李彥璇(2006)利用T-DNA插入突變體探討水稻基因之功能-抗病能力異常突變體M0023454及穀粒發育異常突變體M0039314之特性及基因表現

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