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研究生:邱仕華
研究生(外文):Chiu Shis Hua
論文名稱:利用逢機擴增多型性DNA(RAPD)標記偵測甘藷扦插繁殖世代基因組DNA之變異性
論文名稱(外文):Genomic variations in sucesssive cutting-propagated clones of sweet potato (Ipomoea batatas (L.)Lam.) revealed by RAPD markers.
指導教授:陳榮芳陳榮芳引用關係
指導教授(外文):Long-Fang Oliver Chen
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:1999
畢業學年度:87
語文別:中文
論文頁數:115
中文關鍵詞:逢機擴增多型性DNA甘藷扦插南方式雜交DNA甲基化
外文關鍵詞:RAPDSweet potatocuttingSouthern hybridization analysisDNA methylation
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甘藷在臺灣可週年栽培,田間的栽作上一般以採取扦插苗繁殖,育苗上主要是以種藷苗和本田苗兩種方式進行。本田苗由本田中採苗直接扦插繁殖,其所需的勞力與土地成本較低,為一般農民喜愛採用的栽作方式。但此種栽作方式,當連續採用同系的甘藷扦插苗扦插繁殖數代以後,會產生收量減少與藷型縮小的情形,須以種藷重新育苗,更新甘藷扦插苗。本研究探討甘藷無性扦插繁殖苗與基因組之變異性有無關係,以為進一步防治扦插苗品系衰敗研究之基礎。
試驗利用本省甘藷栽培品系臺農57(TN57)與臺農66(TN66),選其經由試管苗繁殖而來的健康植株,探討甘藷不同扦插世代其基因組的變異性,並輔以不同氮肥量的試驗,了解氮肥對該變異性有無影響。所利用之方法係分子標示因子中,最簡單便捷的逢機擴增多型性DNA(Random Amplification Polymorphic DNA: RAPD),分析甘藷扦插苗在不同氮肥量處理與不同扦插代數材料其基因組DNA是否有變異產生。
計逢機選用了35個引子,利用AP-PCR(Arbitrarily primed polymerase chain reaction)方法篩選兩甘藷品系不同組織部位(芽、幼葉與成葉)間之變異性,共得到7個具多型性的引子。利用這些具多型性的引子在甘藷不同扦插世代與不同氮肥量處理材料,進行遺傳變異性的檢定;每個引子在不同代數與不同氮肥量處理的材料,均可以檢定出具差異性的片段。另外,選擇了四條在不同代數與不同氮肥處理具多型性的DNA擴增片段給予選殖定序,並與基因資料庫進行比對。資料顯示不同代數與不同氮肥量處理差異產生基因組變異之片段,其中一選殖系明顯與粒線體基因有相似性,一與細胞質Cu/Zn超氧物岐化(Cu/Zn-SOD),一與逆轉位子有相似性,而另一與阿拉伯芥第五染色體之片段有相似處。在南方氏雜交分析,也顯示不同代數與不同氮肥量處理植株間,其基因組間存有差異性。
本試驗之結果顯示繼代扦插繁殖苗確有基因組變異存在,使用上須加以小心。至於此變異之產生與塊根產量之減少與藷型之縮小有無直接關聯有待進一步研究。
Abstract
In Taiwan, sweet potato can be grown in all seasons. Cutting propagation is the most popular method for most farms. However, it was found that successive cutting for several generations from the same varietal clone tended to lower the yield of root tuber and generally the root tuber became to get smaller. In this study, we are interesting to know whether the above phenomenon is associated with genomic variations in different cutting generations.
Two cultivars, Tainung 57 (TN 57) and Tainung 66 (TN 66) were used for this study. Original shoots of each cultivar were derived from a micropropagated shoot in vitro. Random amplification polymorphic DNA (RADP) derived from arbitrarily primed polymerase chain reaction (AP-PCR) was used as a tool for detecting genetic variations in plants from successive cutting generations and cuttings grown under different nitric treatment.
A total of 35 random primers were examined on TN57 and TN66 to see if there was any polymorphism among sprout, primary leaf and mature leaf. Seven primers, which had the capacity of revealing the differences among different tissue DNAs, were further served for this study. It was found that all these seven primers were also able to detect the differences in clones derived from different cutting generations as well as cutting clones grown under different nitric managements. Four polymorphic bands were cloned for sequencing. In comparison with sequences in Gene bank (GenBank and EMBL) it was noted that one might associated with mitochondria gene, another had the similarity with Cytosolic Cu/Zn-superoxide dismutase (Cu/Zn-SOD), the third showed the similarity with retrotransposable like-element and the other was similar to Arabidopsis genomic sequence.
Southern hybridization also indicated that degree of methylation might also involved for the genomic variation in differrnt cutting generations and nitric treatments.
Results from this study indicate that Genomic variations tend to occur among different cutting generations. Therefore, cutting propagation should be used cautions.
摘 要................................................................................1
Abstract.............................................................................3
壹. 緒 論...........................................................................5
貳. 前 人 研 究.................................................................7
一.植物組織繁殖之變異性.........................................
二.遺傳變異性之種類.................................................
三.遺傳變異性之檢定技術.........................................
參. 試 驗 設 計.................................................................21
肆. 材 料 與 方 法............................................................22
材 料 處 理.....................................................................22
試 驗 方 法.....................................................................23
1.甘藷葉片組織DNA的抽取.......................................
2.逢機擴增多型性核酸PCR (RAPD-PCR)...................
3.特定基因PCR分析.....................................................
4.RAPD-PCR 分析得到差異DNA片段之定序.............
5.南方式雜交................................................................
伍. 結 果...........................................................................38
陸. 討 論...........................................................................49
參 考 文 獻.......................................................................57
表......................................................................................67
圖......................................................................................85
附 註.................................................................................98
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