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研究生:蔡靜琪
研究生(外文):Ching-Chi Tsai
論文名稱:自水稻突變體分析鑑定出一個受體激活酵素為控制葉色性狀的重要顯性基因
論文名稱(外文):From Study a Rice Mutant Reveal a Receptor-like Kinase Gene Dominantly Involved in Control of Leaf Color
指導教授:呂維茗
指導教授(外文):Wei-Ming Leu
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
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:2004
畢業學年度:92
語文別:中文
論文頁數:91
中文關鍵詞:香米受體激活酵素葉尖黃化葉色
外文關鍵詞:SA0420receptor-like kinaseleaf tip yellowingleaf color
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香米(SA0420)乃由台農67號誘變後自交十代以上純化育成,與台農67號性狀具有許多顯著且獨立分離的差異,包括:香米的成熟葉與榖粒具有濃厚芋香、植株矮小、葉尖黃化、早開花及稔實率低等。為了選殖與此些性狀相關而表現升高或降低的基因,本實驗以雜合扣除技術構築cDNA庫,並製成colony array以探針篩選可能的標的基因,經由RT-PCR實驗確認SA1與SA2基因表現量於二種水稻品系間具有顯著差異,故選定作為深入研究的對象。SA1及SA2均屬於抗水稻白葉枯病基因Xa21家族成員之一,與Xa21蛋白具有約65%序列相似度,南方點墨分析顯示兩者皆為單套基因,然而SA1為SA0420水稻所專有,相反的, SA2基因則僅存在於台農67號水稻。由於現有的水稻基因組資料庫中並無SA1或SA2的對應序列,故進行分子選殖獲得全長的基因,預測可轉譯出一包含千餘個胺基酸的蛋白,N端有白胺酸高度重複區域,中間為穿膜區域,C端則為典型的激活酵素區域。為探討SA1與SA2基因之功能,本實驗檢查F子代 (SA0420 × TNG67 ) 個體中SA1與SA2之基因型,配合性狀紀錄進行變方統計分析,發現葉尖黃化性狀與SA2基因缺失顯著相關,此結果亦與分析其他SA突變株或是F6、F7子代基因型的結果相符。為了進一步確認SA2基因本身可調控葉色性狀,而非鄰近的基因所導致,於是構築SA2基因靜默的轉殖水稻,發現SA2基因靜默會導致轉殖植物出現葉色較淡的性狀,且存活率降低,而存活者葉綠素與胡蘿蔔素含量均下降,與SA0420性狀類似,因此認為SA2基因是調控葉色的重要顯性基因。
SA0420, a rice mutant derived from cultivar TNG67, exhibits multiple distinctive traits including a taro fragrance in the mature leaf and grain, a dwarf appearance of the whole plant, leaf-tip yellowing, early maturity and low fertility, etc. To characterize genes involving the above phenotypes, we aimed to isolate both up- and down-regulated genes in SA0420. Subtracted cDNA libraries were constructed and colony arrays were screened by hybridizations. The differential expressions of two clones, SA1 and SA2, were confirmed by gene-specific RT-PCR. Both sequences predicted proteins sharing ~65% sequence similarity to Xa21, a protein that confer resistance to rice bacterial blight. Although belonged to the same multigene family, SA1 and SA2 exhibit an opposite expression pattern while SA1 is SA0420-specific, SA2 is TNG67-specific. Genomic Southern analysis indicated that both genes are single copy in rice but SA1 is present only in SA0420 whereas SA2 is only in TNG67. Since searching of current database did not reveal their corresponding sequences, PCR on adaptor-ligated genomic DNA were used to pursue the full-length gene information. The predicted SA1 and SA2 proteins contain ~1000 amino acids with a leucine-rich repeat followed by a trans-membrane region and a kinase domain characteristic for Xa21 gene family. To address their gene functions in rice, variance analyses of SA genotypes and phenotypes were performed on F2 plants (SA0420´TNG67). A significant correlation between the leaf-tip yellowing and the absence of SA2 gene was revealed. Data from analysis of F6 plants, F7 plants, and other SA mutants lead to similar conclusions. To demonstrate if the SA2 gene itself, but not its neighboring gene, is critical for greening, transgenic rice putatively silencing SA2 expression by RNA interference (pSA2-9) was generated. In contrast to vector-transformed regenerates, most shoots regenerated from the pSA2-9-transformed callus showed leaf yellowing and ~25% of them died after transplanting to soil. Moreover, the amount of chlorophyll a/b and carotenoid in the survival plants were reduced, similar to the observations on SA0420. We conclude that SA2 gene dominantly plays a key role in regulating leaf greening in rice.
目錄
中文摘要 1
Abstract 2
前言 3
前人研究 5
1. Xa21水稻抗白葉枯病基因 (rice blight resistance gene) 5
2. 植物黃化因素探討 6
材料與方法 8
一、 實驗材料 8
(一) 構築雜合扣除基因庫與檢測SA1及SA2基因表現 8
(二) 分析SA2基因在水稻各時期組織的基因表現 8
(三) 檢測SA0420 × TNG67 F2子代基因型 9
(四) 檢測SA0420 × TNG67 F6、F7子代基因型 9
(五) 檢測SA突變株基因型 9
二、 萃取水稻基因組DNA 9
(一) phenol/chloroform method (Muttler, I. J., 1987) 9
(二) Monocot DNA isolation method (from Tanksley’s lab) 9
三、 檢測基因表現 10
四、 建立香米品系 (SA0420) 與台農67號 (TNG67) cDNA雜合扣除基因庫 (subtracted cDNA library) 11
(一) cDNA的萃取 11
(二) 建立香米 (SA0420) 與台農67號 (TNG67) cDNA雜合扣除基因庫 (subtracted cDNA library) 11
五、 雜合探針之製備 11
六、 南方點墨法 (Southern blotting) 12
七、 自動放射顯影及定量分析 12
八、 Inverse PCR 13
九、 GenomeWalker 13
十、 水稻白葉枯病感染試驗 13
十一、MAPMAKER分析 14
十二、Real-time PCR 14
十三、色素分析 14
十四、農桿菌轉型作用-電穿孔法:見附錄五 15
十五、水稻轉殖:見附錄三、四 15
十六、阿拉伯芥轉殖:見附錄六 15
結果 16
一、 選殖香米 (SA0420) 與台農67號 (TNG67) 中具差異性表現的基因SA1與SA2 16
(一) 建立SA0420與TNG67 cDNA雜合扣除基因庫 (subtracted cDNA library) 菌落陣列分析膜 16
(二) 篩選表現量差異大的基因 16
(三) 反轉錄增幅反應和基因組南方點墨法 (RT-PCR and Genomic Southern blot) 檢查SA1與SA2基因 17
(四) 分子選殖SA1與SA2全長cDNA與基因組DNA 18
(五) SA1與SA2蛋白結構特性與序列比對分析 18
二、 水稻白葉枯病感染試驗 19
三、 SA1的功能與特性探討 19
(一) F2子代分析 19
(二) 轉殖植物分析 21
四、 SA2的功能與特性探討 21
(一) F2子代分析 21
(二) 檢測其它實驗材料之SA2基因型及其是否與葉尖黃化性狀相關……………………………………………………………………….22
(三) 分析SA2基因在水稻各時期組織的基因表現 24
(四) 以基因轉殖植物分析SA2基因的功能 24
討論 30
參考文獻 34
附錄一、實驗中所使用的引子 66
附錄二、於南方點墨法所用的探針 67
附錄三、水稻轉殖與轉殖株的篩選 68
附錄四、水稻組織培養所使用之培養基成份 73
附錄五、以電穿孔法進行農桿菌轉形作用 74
附錄六、阿拉伯芥轉殖方法 75
附錄七、質體構築………………………………………………………………..76
附錄八、SA0420 × TNG67 F2子代紀錄的十種性狀 89
附錄九、Real time PCR引子配對濃度 90
附錄十、水稻白葉枯病培養基配方……………………………………………...91
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