臺灣博碩士論文加值系統

本研究從大型染色體蝴蝶蘭 Phalaenopsis violacea 的基因組中，選殖到兩種重複性序列的選殖株 pvr1-96 與 pvr1-57，並分析其核苷酸序列、種間分佈及在染色體上的位置。pvr1-96 之選殖片段全長為 3838 bp，其中 637~3667 核苷酸處為 7 bp (ATAAGTC) 頭尾相接排列之重複性序列。而 pvr1-57 之選殖片段長度為 4175 bp，為 Ty3/gypsy 型逆轉移子 (Ty3/gypsy-type retrotransposon) 的部份序列。進一步選殖得到包含 pvr1-57 DNA 片段之選殖株 pvr283，與 pvr1-57 的序列相連接得到全長為 7571 bp 之核苷酸序列，命名為 PVRE1。PVRE1 與 Oryza sativa 之 Ty3/gypsy 型逆轉移子最為相似，在 pol 基因區具有 52 % 的相同度。然而 PVRE1 之 pol 基因區並非完整的序列解讀區 (open reading frame, ORF)，且 3' 與 5' 兩端皆無長末端重複序列 (long terminal repeats, LTRs)。故 PVRE1 為一不完整之逆轉移子，推測可能因演化過程中基因突變或序列重組而失去功能。南方氏雜合顯示，PVRE1 廣泛存在於七種蝴蝶蘭與一種朵麗蘭的基因組中。唯染色體較小的蝴蝶蘭 P. aphrodite 及 P. equestris 之中，此種逆轉移子片段的含量較少。螢光原位雜交的結果顯示，在蝴蝶蘭所有染色體上均有雜交訊號，證明 PVRE1 為散佈型的逆轉移子。PVRE1 於小型的染色體上訊號較弱，較大的染色體上訊號較強，而在大型的染色體上多聚集於富含 AT 的異染色質區。PVRE1 為蝴蝶蘭屬植物中首次發現之逆轉移子。

Two clones containing repetitive DNA sequences, pvr1-96 and pvr1-57, were isolated from Phalaenopsis violacea and characterized. The insert of pvr1-96 (3838 bp) consists of (ATAAGTC)432 central core flanking by 636 bp and 171 bp nonrepetitive sequence at the 5' and 3' ends, respectively. The insert of pvr1-57 is 4175 bp long which shows high homology to part of the polyprotein (pol) gene of Ty3/gypsy type retrotransposon from other species. Using a 205-bp fragment in the 5' region of pvr1-57 as a probe, a clone pvr283 was isolated. Combining the insert sequences of pvr283 and pvr1-57 gives a 7571-bp sequence, named PVRE1. PVRE1 shares 52 % identity to the pol gene of the Ty3/gypsy type retrotransposon from Oryza sativa. However, several stop codons are present in the major open reading frame (ORF) of PVRE1 and there are no long terminal repeats (LTRs) flanking this sequence, suggesting that PVRE1 is inactive. Southern analysis revealed that PVRE1 is present in all eight Phalaenopsis species studied. Chromosome localization by fluorescence in situ hybridization revealed that PVRE1 is dispersed throughout the genome of P. aphrodite, P. lueddemanniana, P. venosa and P. violacea, with signals being stronger in AT-rich heterochromatic region of the large chromosomes of the later two species. PVRE1 is the first retrotransposon isolated from Phalaenopsis species.

中文摘要 ...........…………............................................................... i
英文摘要 ..........................…………................................................ ii
壹、前言 ........................................................………….................. 1
貳、材料與方法
一. 植物材料 ………………………….................…………... 6
二. 植物基因組 DNA 的抽取 ..............................…………... 6
三. 質體 DNA 的抽取 ..........................................…………... 7
四. 重複性 DNA 序列之選殖與定序 ..................................... 7
五. 缺失定序 …………….......................................................... 15
六. 基因組南方氏雜合 ….......................................................... 15
七. 螢光原位雜交 …………...................................................... 16
參、結果 .....…………..................................................................... 21
一. 重複性 DNA 序列的選殖 ................................................. 21
二. pvr1-96 的定序與分析 ….................................................... 21
三. pvr1-57 的定序與分析 ….................................................... 26
四. 逆轉移子其他部分 DNA 片段之選殖 ............................. 26
五. pvr283之定序與分析 ..…..................................................... 28
六. PVRE1 在蝴蝶蘭及朵麗蘭基因組內的存在情形 ............. 39
七. PVRE1 在蝴蝶蘭染色體上的分佈 ……………................. 41
肆、討論 ...................………..…....................................................... 44
伍、參考文獻 ......…………............................................................. 50

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