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研究生:周琪英
研究生(外文):Chi-Ying Chou
論文名稱:利用差異表現法自水稻突變體篩選抗褐飛蝨相關基因
論文名稱(外文):Screening of Brown Planthopper Resistance-related Genes from Rice (Oryza sativa L.) Mutants by Differential Diaplay
指導教授:古新梅王強生
指導教授(外文):Hsin-Mei KuChang-Sheng Wang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農藝學系
學門:農業科學學門
學類:一般農業學類
論文種類:學術論文
論文出版年:2002
畢業學年度:91
語文別:中文
論文頁數:111
中文關鍵詞:差異表現法褐飛蝨
外文關鍵詞:differential displaybrown planthopper
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褐飛蝨(Nilaparvata lugens Stal., brown planthopper, BPH)常造成水稻生產上極嚴重的損失,育成抗性品種為根本防治之道。以疊氮化鈉(sodium azide, NaN3)誘變不抗褐飛蝨的台農67號(TNG67)品種,所得之同源突變品系TM6與TM268具有高度的抗蟲性,且均為一對顯性基因所控制。以抗(TM6, TM268, Mudgo)、感(TNG67)褐飛蝨品種(系)為材料,利用差異性表現法(differential display, DDRT-PCR)進行抗褐飛蝨相關基因片段之篩選。經篩選78個引子組合,共獲得95個差異片段。利用dT載體選殖差異片段,並進行DNA定序分析及序列比對(BLAST)分析,進而以此片段為探針進行南方雜交分析,確認選殖的片段是否為抗、感蟲品系之差異片段。
南方雜交分析確認OSDD51、OSDD54、OSDD81、OSDD166及OSDD220等5個殖系為抗、感差異殖系;OSDD166殖系為水稻phenylalanine ammonia-lyase(PAL)之基因片段;OSDD51及OSDD81兩殖系可能為CBS domain protein;OSDD54與OSDD220兩殖系可能為新的抗蟲相關基因。褐飛蝨危害過程中,OSDD51殖系於抗、感品種(系)間具有表現量上的差異;OSDD166則於抗、感品種(系)均有表現,表現量隨著褐飛蝨危害程度而增加。
利用同切位酵素Hpa II及Msp I剪切進行南方雜交分析結果,發現OSDD51、OSDD81、OSDD166及OSDD220等4個差異殖系之表現與DNA甲基化有關。由序列比對分析發現15個殖系與逆轉位子及其活性相關蛋白質有關。推測DNA甲基化可能與褐飛蝨抗性相關基因的表現及誘變早期逆轉位子的活性有關。
上述5個與褐飛蝨抗性相關的差異殖系均將進一步篩選基因全長及基因功能性分析等相關研究。未來研究必須選殖到直接參與誘變反應的逆轉位子,以進一步探討逆轉位子的活化是否與抗性的產生有關,期能早日解開誘變及突變體抗蟲作用機制。
Brown planthopper (Nilaparvata lugens Stal., BPH) usually cause serious damage in most rice production areas, breeding for resistant varieties is the best solution for the pest control. Two mutants, TM6 and TM268, derived from the BPH susceptible variety TNG67 by sodium azide (NaN3) mutagenesis showed BPH resistance and controlled by a single dominant gene. To clone the genes responsible for the BPH resistance in mutants, differential display (DDRT-PCR) strategy was applied to screen the differential expressed genes by comparing the BPH resistant mutants TM6 and TM268 with the susceptible variety, TNG67. A total of 95 differential displayed fragments were obtained by screening 78 sets of primers. These fragments were amplified and cloned into the dT vector, and characterized by sequencing analysis and BLAST characterization. They were further used as probes in the Southern analysis to investigate the genomic difference between the resistant and susceptible lines.
Five clones, OSDD30, OSDD51, OSDD54, OSDD81, OSDD166, and OSDD220 related with the BPH resistance were found with polymorphisms by Southern analysis. The OSDD166 clone was identified as a phenylalanine ammonia-lyase (PAL) gene in rice. Clones OSDD51 and OSDD81 showed 42~46% identity to the CBS (cystathionine beta-synthase) domain protein. No similarity in either nucleotide or amino acid sequences of OSDD54 and OSDD220 were found in the database therefore, they maybe the new genes in rice related to the BPH resistance. During BPH attacked, the expression of the CBS clone, OSDD51, was slightly higher in the BPH resistant lines by northern hybridization.
The difference in DNA methylation patterns in rice mutants were detected by four clones OSDD51, OSDD81, OSDD166 and OSDD220 in Southern analysis using restriction isoschizomers Hpa II and Msp I to digest the genomic DNAs. Fifteen of the differentially expressed clones were correlated with retrotransposon or its activity related proteins by nucleotide BLAST. It is hypothesized that the expression of gene in response to BPH challenge in the resistant mutants may associate with the activation of retrotransposon in the early mutagenesis stage and the changes of DNA methylation pattern.
Further experiments will be conducted to clone the five BPH resistance-related genes as well as the retrotransposon related genes from mutants and to investigate their functions in order to unravel the mechanisms of mutagenesis and BPH resistance in rice mutants.
中文摘要...................................................VIII
英文摘要.......................................................X
壹、前言------------------------------------------------------ 1
貳、前人研究-------------------------------------------------- 3
一、褐飛蝨與水稻之關係........................................ 3
二、褐飛蝨之綜合防治............................................................ 4
三、水稻褐飛蝨之遺傳育種及抗性基因之研究...................... 8
四、利用疊氮化鈉(sodium azide, NaN3)誘變擴大水稻
遺傳變異 .................................................12
五、逆轉位子(retrotransposon).............................. 14
六、基因修飾作用-甲基化(methylation)作用................... 17
七、利用生物技術選殖抗褐飛蝨相關基因......................... 19
叄、材料與方法----------------------------------------------- 23
一、台農67號誘變品系褐飛蝨抗性檢定........................... 23
二、TNG67及其誘變品系樣品之準備.............................. 23
三、試驗方法................................................. 25
1. 總量RNA之萃取............................................. 25
2. 差異表現法................................................ 27
3. 差異片段之選殖............................................ 29
4. 簡易水稻基因組DNA之萃取................................... 32
5. 氯化銫(CsCl)梯度離心純化水稻基因組DNA................... 33
6. 水稻抗褐飛蝨相關殖系之核酸自動定序分析.................... 34
7. 褐飛蝨抗性相關殖系之序列分析.............................. 36
8. 褐飛蝨抗性相關殖系之基因表現分析.......................... 36
肆、結果----------------------------------------------------- 40
一、水稻抗褐飛蝨相關基因片段之差異性篩選..................... 40
二、水稻抗褐飛蝨相關基因之選殖及序列比對分析................. 41
三、水稻抗褐飛蝨相關基因之表現及特性分析..................... 44
伍、討論----------------------------------------------------- 53
一、水稻抗褐飛蝨相關基因片段之差異性篩選..................... 54
二、基因序列之比對分析........................................................... 56
三、選殖基因與抗蟲性之可能關係...............................58
四、DNA甲基化與抗褐飛蝨相關基因表現之關係................... 61
陸、結語----------------------------------------------------- 66
柒、參考文獻------------------------------------------------- 67
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