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研究生:黃麗燕
研究生(外文):Lai-In Wong
論文名稱:T-DNA插入突變株之篩選與水稻基因功能之探討---株高突變株M82268之研究
論文名稱(外文):Rice functional genomics study using T-DNA insertion mutants: characterization of plant height mutant M82268
指導教授:陳良築
指導教授(外文):Liang-jwu Chen
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:2009
畢業學年度:97
語文別:中文
論文頁數:72
中文關鍵詞:突變株株高水稻低溫逆境萌芽
外文關鍵詞:mutant lineplant heightricecold stressgermination
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  • 被引用被引用:2
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本研究從T-DNA插入突變體庫中,根據外表性狀篩選出7個與株高相關之突變株系,其中株高較野生型TNG67高約20~30%之突變株M82268經plasmid rescue方法解讀T-DNA插入點兩側序列,得知T-DNA的插入位置在水稻第5對染色體BAC clone OJ1657_H11上的第80,783鹼基對,並藉由T-DNA上CaMV35S的加強子可活化插入點上游14.0 kb OsGID1以及下游16.9 kb OsLEC14B基因的表現。觀察T2~T4子代的基因型與農藝性狀間,呈現密切共分離現象,並由南方墨點法分析顯示只有單一T-DNA插入。OsGID1為植物賀爾蒙吉貝素 (Gibberellins, GAs) 之可溶性接受器,主要在細胞核內接受具有生物活性之GAs,經細胞訊息傳遞使之逹到調控植物生長的功能;而OsLEC14B在單子葉植物中之功能尚不明瞭,因此本研究將利用M82268突變株對於OsGID1進一步瞭解及OsLEC14B功能之探討。OsGID1主要表現於快速生長分化的組織,如6到12天幼根及幼芽,而OsLEC14B則在已成熟葉片表現量較高,此兩基因都會持續在稻穗中表現,因此推測OsGID1和OsLEC14B在營養生長時期和生殖生長時期都具有不可或缺之功能。過去研究指出大量表現OsGID1會造成水稻株高較高以及稔時率偏低現象,然而在觀察M82268稔時率卻是正常,因此推測活化OsLEC14B會影響水稻稔實情形。此外,觀察M82268突變株之種子萌芽速度較野生型TNG67慢,但在進入幼芽時期其株高逐漸高於TNG67,因此推測活化之OsLEC14B 可能影響萌芽速度。在本研究中野生型水稻TNG67受低溫逆境處理時,OsGID1基因表現量增加,而OsLEC14B表現量降低,且突變株對低溫逆境較具敏感性,但回復情況較TNG67佳,可能受OsGID1和OsLEC14B共同被活化所影響,推測OsLEC14B可能參與低溫逆境反應訊息之傳遞。進一步利用ubiquitin啟動子分別大量表現OsGID1和OsLEC14B基因於野生型TNG67植株中,在ubi::GID1轉殖株中能再現突變株較高的形態,且性狀更為明顯;而ubi::LEC14轉殖株則為株高較矮,且死亡率較高,顯示此二基因之功能大不相同,且對植物之生長發育頗具重要性,值得進一步經由轉殖株外表性狀、基因與生理功能之觀察分析,以探討OsGID1和OsLEC14B在水稻中扮演之功能。
Seven rice T-DNA insertion mutant lines with altered plant height were identified in this study. The plant height of M82268 was about 20~30% higher than that of the wild type TNG67. M82268 has its T-DNA inserted at 80,783 bp of BAC clone OJ1657_H11 on the chromosome number 5. The progeny assay from T2 to T4 generations demonstrated that the taller phenotype of M82268 was co-segregated with the T-DNA insertion and there is only one T-DNA insertion event in the genome. Two flanking genes, GID gibberellin receptor and putative LEC14B, located either 14.0 kb upstream or 16.9 kb downstream of the T-DNA insertion site were activated by this insertion event. OsGID1 is a soluble receptor of gibberellin that involved in regulation of plant development while OsLEC14B has not been studied. Therefore, further characterization of M82268 will allow us to know more about OsLEC14B and its interaction with OsGID1. RT-PCR analysis revealed that both genes expressed differentially in all tissues tested, OsGID1 expressed higher in young tissues while OsLEC14B expressed much less in 6 or 12-days young leaf tissues. It has been known that over-expression of OsGID1 will increase plant height but reduce its fertility significantly. However, activation of OsGID1 did not affect the fertility in M82268. It was therefore suggested that the activation of OsLEC14B may overcome the effect of OsGID1 in the reproductive stage in M82268. We also suggested that expression of OsLEC14B may affect germination due to slowing germination in M82268 was observed. Moreover, different response of M82268 and TNG67, and differential regulation of OsGID1 and OsLEC14B genes to cold stress suggested that the interactions of these two genes were involved in cold stess response and recovery. In order to understand the function of individual gene, transgenic rice over-expression either OsGID1 or OsLEC14B genes by ubiquitin promoter were obtained. The ubi::GID1 transgenic rice showed much taller phenotype than M82268 but with low fertility which revealed the same phenotype as previous results. The ubi::LEC14B transgenic rice, however, was more difficult to obtain and the survived transgenic plants showed sereval distinct phenotype. Therefore, to understand the possible function of OsLEC14B, further study on more stable transgenic lines are required.
中文摘要............................................. i
英文摘要............................................ ii
目錄................................................ iii
圖表目錄............................................. v
縮字對照表.......................................... vii

前言................................................... 1
前人研究
一、水稻基因功能之研究..................................................... 2
二、植物荷爾蒙吉貝素 (Gibberellins, GAs) 之相關研究
1.生合成及功能......................................... 3
2.其他植物荷爾蒙和環境因素對GAs之調控.................. 4
3.參與種子萌芽之調控機制............................... 5
4.訊號傳遞路徑之探討................................... 5
三、GID1 gibberellin receptor基因之功能分析........... 6
四、植物抵抗低溫逆境的防禦反應........................ 7
五、 LEC14B基因可能之功能分析......................... 8
材料與方法
一、儀器及設備........................................ 9二、藥品.............................................. 9
三、T-DNA插入突變株處理............................... 9
四、T-DNA插入點鄰近序列之分析........................ 9
五、南方墨點法....................................... 12
六、基因表現偵測..................................... 13
七、ubi::GID1和ubi::LEC14B水稻轉殖株之構築與分析..... 14
八、蛋白抗體製備..................................... 16
九、蛋白質表現之分析................................. 18
十、萌芽試驗......................................... 20
十一、低溫逆境處理................................... 20
結果
一、T-DNA插入水稻突變株之篩選........................ 21
二、水稻突變株T-DNA 插入數目及插入點之分析
1. 水稻突變株T-DNA 插入數目之分析.................... 21
2. 水稻突變株T-DNA 插入位置之分析.................... 21
3. 水稻M82268突變株T-DNA 插入位置之確認.............. 22
三、M82268水稻T-DNA插入突變株田間性狀之觀察.......... 23
四、M82268突變株T-DNA插入點附近基因表現之分析........ 24
五、OsGID1和OsLEC14B 在不同發育時期與不同組織部位之表現分 析................................................... 25
六、OsGID1和OsLEC14B於TNG67中大量表現之構築
1. 植物表現載體之構築與水稻轉殖及再生............... 25
2. ubi::GID1 (T0)水稻轉殖株之確認及分析............. 26
3. ubi::LEC14B (T0)水稻轉殖株之確認及分析........... 26
七、M82268水稻T-DNA插入突變株之萌芽觀察............. 27
八、OsGID1與OsLEC14B在低溫逆境下之表現分析.......... 28
九、OsGID1和OsLEC14B的蛋白表現之分析................ 28
討論................................................ 30
結論................................................ 33
參考文獻............................................ 34
表.................................................. 39
圖.................................................. 43
附表................................................ 66
附圖................................................ 67
附錄................................................ 70
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