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研究生:鄭雅銘
研究生(外文):Ya-Ming Cheng
論文名稱:玉米B染色體的分子結構
論文名稱(外文):Molecular organization of maize B chromosome
指導教授:林伯耀
指導教授(外文):Bor-Yaw Lin
學位類別:博士
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:2004
畢業學年度:92
語文別:英文
論文頁數:104
中文關鍵詞:B染色體螢光原位雜交B-10L易位染色體玉米B染色體專一序列粗絲期染色體
外文關鍵詞:B chromosomefluorescence in situ hybridization (FISH)B-10L translocationZea maysB-specific sequencepachytene chromosomemicrodissectionCL-repeat
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玉米B染色體與正常染色體(A染色體)之間的高度同源性,是選殖該染色體DNA的最大難題。該困難已在本論文中利用microdissection技術克服,以此技術直接取得粗絲期B染色體,再以PCR增幅並選殖B染色體DNA片段。所得到的DNA片段可藉由B染色體劑量反應分析(B-dosage response)及螢光原位雜交技術證實其來源是B染色體。以此方式總共獲得19個B染色體DNA序列,它們都是重複性序列,而且都與A染色體具有同源性(只有一個例外)。DNA序列比對顯示,其中12個序列與玉米染色體節重複序列(knob repeat)及一些基因的附近區域具有相似度;而其他6個則與目前已發表的序列都不具有相似度。最後一個(pBPC51)是B染色體專一的序列,利用七個B-10L易位染色體及螢光原位雜交分析,將此序列定位在B染色體長臂的遠端異染色質區。
爲了能進一步了解B染色體的分子結構,利用pBPC51從帶16個B染色體的染色體組基因庫中,篩選大片段的B染色體DNA序列。總共獲得5個菌株,每一個菌株都帶有大於10 kb的插入DNA,根據南方氏雜交分析和螢光原位雜交(FISH)分析,這些插入DNA都是與A染色體具有同源性的重複性序列。接著,對其中2個菌株做進一步的分析及定序,將它們分成數個限制酵素切割片段後,發現每一片段具有程度不一的重複性;有些片段是B染色體專一,而其他片段則與A染色體具有不同程度的同源性。DNA序列比對顯示,這2個序列(約26 kb)除了與轉位子(transposable element)、B染色體及A染色體中節序列具有相似度外,與目前已發表的基因都沒有相似。當中也發現一種B染色體專一的重複性序列(重複單位為1.6 kb),稱之為CL-repeat,此序列大都是以銜接排列(tandem arrange)的方式存在於B染色體上。此外,DNA序列分析的結果顯示轉位子在B染色體的演化過程中,扮演著相當重要的角色。

Isolation of sequences from the maize B chromosome is always hampered by its high homology with the normal complements (A chromosomes). In this study, this hindrance was overcome by cloning sequences through microdissection of the pachytene B chromosome on a microscopic slide. Nineteen sequences were obtained and found to hybridize with genomic DNA in a B-dosage-dependent manner and with the pachytene B chromosome by fluorescence in situ hybridization (FISH), corroborating with their B origin. All but one sequences are repetitive and homologous to the A chromosome(s). Sequence comparison revealed that 12 sequences have homology with the knob repeat and flanking regions of several maize genes. Six have no homology to any documented sequence. The remaining one, pBPC51, is B specific and was mapped to the central portion of distal heterochromatic region in the B long arm by seven B-10L translocations and FISH analysis.
To gain insights into the molecular structure of the B chromosome, pBPC51 was used to isolate large B fragments from a genomic library carrying 16 B’s. Five clones (> 10 kb each) were isolated, and all were repetitive, sharing homology with A chromosomes in Southern and FISH analyses. Two of them were further characterized and sequenced. Each is composed of a number of restriction fragments with different levels of repetitiveness. Some of these are B specific and others share various degrees of homology with the A chromosomes. Their sequences (about 26 kb) show no homology with any published gene other than sequences of transposable elements (retrotransposons and MITEs) and the B as well as A centromeres. A 1.6 kb B-specific repeat (CL-repeat) that mostly arrange in tandem array on the B chromosome was identified in the two clones. Moreover, results of sequence analysis suggested that transposable elements play important roles in the B chromosome evolution.

中文摘要………………………………………………………………………1
Abstract………………………………………………………………………2
Chapter One Literature review…………………………………………3
Chapter Two Cloning and characterization of maize B chromosome sequences derived from microdissection…………………………… 26
Chapter Three Molecular organization of large fragments in maize B chromosome: indication of a novel repeat…………………62
Chapter Four Conclusion………………………………………………103

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