Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36825
標題: 玉米B染色體異染色質區基因組片段之特性分析
Characterization of genomic fragments from heterochromatic regions on maize B chromosome
作者: 黃珮瑜
Huang, Pei-Yu
關鍵字: B chromosome
B染色體
heterochromatic regions
CL-repeat
fluorescence in situ hybridization (FISH)
phylogenetic analysis
異染色質區
CL-repeat
螢光原位雜合
系統演化分析
出版社: 農藝學系所
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摘要: 本研究利用限制酵素剪切、南方氏雜合及螢光原位雜合對34個玉米B染色體異染色質區的基因組片段進行特性分析。利用NotI酵素剪切估計基因組片段大小介於11 kb~20 kb之間,依據XbaI片段數量與大小,剔除6個重複的基因組片段;以B染色體異染色質區專一性序列CL-repeat進行南方氏雜合分析,確認基因組片段源自B染色體異染色質區,藉由CentC的南方氏雜合分析,顯示3個基因組片段同時帶有CL-repeat與CentC序列;接著以各基因組片段作為螢光原位雜合探針,分析各片段在染色體之分布,在有絲分裂中期染色體可觀察到6種訊號分布類型;而在減數分裂粗絲期B染色體訊號,由僅在4個異染色質區至整個B染色體的分布皆有。最後,利用CL-repeat專一性引子增幅各基因組片段內的CL-repeat,可增幅出1.6-kb、0.75-kb、0.65-kb以及0.15-kb四種產物,進一步將所有產物選殖並定序,再利用已經定位的28個1.6-kb CL-repeat對本研究的21個1.6-kb CL-repeat進行系統演化分析,藉此可以將各CL-repeat及其所在的基因組片段定位在B染色體特定異染色質區。此結果使我們對玉米B染色體異染色質區有更多的了解。
In the present study, restriction enzyme digestion, Southern hybridization and fluorescence in situ hybridization (FISH) were used to characterize 34 genomic fragments from heterochromatic regions on maize B chromosome. By using NotI digestion, the size of genomic fragments were estimated to be between 11 kb to 20 kb. According to the number and size of XbaI fragments, six duplication genomic fragments were eliminated. The heterochromatic origins of each fragment were verified by Southern hybridization with the B heterochromatin specific CL-repeat. Three genomic fragments containing both CL-repeat and CentC sequences were revealed by Southern hybridization with CentC as probe. Subsequently, FISH analysis with each fragment as probe was applied to detect their distributions on chromosome. Six types of signal distributions were observed on mitotic chromosomes. The signal distributions on the pachytene B chromosome varied from signals on the four heterochromatin region to on the entire B chromosome. Finally, four CL-repeat products, 1.6-kb, 0.75-kb, 0.65-kb and 0.15-kb, amplified from those genomic fragments by a pair of CL-repeat specific primers were cloned for sequences analysis. The resulting 21 1.6-kb CL-repeat were calculated with 28 previously mapped CL-repeat by phylogenetic analysis to map each CL-repeat and their original genomic fragments onto define heterochromatic region of B chromosome. The results can help to get more understanding of the heterochromatic regions on maize B chromosome.
URI: http://hdl.handle.net/11455/36825
其他識別: U0005-2907201113182200
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2907201113182200
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