蝴蝶蘭 Phalaenopsis stuartiana 之 (GA)n 微衛星體序列的選殖與定位

Cloning and physical mapping of (GA)n microsatellites in Phalaenopsis stuartiana

劉蓉(Jung Liu)

指導教授 ： 高燕玉

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.01231

摘要

微衛星體序列是以 1-6 bp 為組成單位的簡單重複性序列，普遍存在於真核生物的基因組中。本實驗利用 (GA)11為引子進行單引子聚合酶連鎖反應，從蝴蝶蘭 Phalaenopsis stuartiana 基因組分離出 15 個富含 (GA)n 微衛星體序列的選殖體，其中 14 個為複合型，序列長度最長為 726 bp，最短為 92 bp;而 GA 重複次數最高達 28 次，TC重複次數最高達 32 次，序列間的相似度介於 35.98-78.57%，由 TIGR 資料庫比對結果，與許多植物之重複性序列相類似。挑選其中 8 個選殖體，以及朵麗蘭的 DpGA2 選殖體進行螢光原位雜交，結果訊號皆集中於 P. stuartiana 所有染色體的中節，且強度大致相同，與選殖體 DpGA2 只位於朵麗蘭一對染色體的中節不相吻合。選殖體 PstGA3 在 P. violacea 及朵麗蘭染色體上皆為散佈型，與前人研究 (GA)11 位於朵麗蘭中節的結果不符，而 PstGA16 在 P. amboinensis 為散佈型，而在 P. mannii 則聚集於中節，則與過去研究結果相符。(GA)n 微衛星體序列是否為 P. stuartiana 具功能的中節序列，將來可以免疫染色法或免疫沉澱法加以證實。

關鍵字

蝴蝶蘭Phalaenopsis stuartiana ； (GA)n 微衛星體序列

並列摘要

Microsatellites are tandemly repeated tracts of DNA composed of 1–6 base pair long units and ubiquitous in eukaryotic genomes. In this study, fifteen clones riched in (GA)n microsatellites were isolated from Phalaenopsis stuartiana by single-primer PCR using (GA)11 as a primer. All clones were compound except one imperfect repeat sequences. The insert size varied from 28 bp to 726 bp, and the maximum repeat numbers of GA and TC were 28 and 32 in these clones. The sequence identity ranged from 35.98 to 78.57% among them. According to the TIGR database, these sequences showed similarities with repetitive sequences in many plants. Nine clones, eight clones from P. stuartiana and DpGA2 from P. pulcherrima, were selected for physical mapping by fluorescence in situ hybridization (FISH). The signals were clustered at all centromeres of P. stuartiana chromosomes. It was found a quite exceptional distribution of DpGA2 which was clustered at the centromeric regions of only one pair of P. pulcherrima chromosomes. In situ hybridization to P. violacea and P. pulcherrima chromosomes with clone PstGA3 revealed dispersed signals. The result of the former but the latter was consistent with the previous study using synthetic oligonucleotides (GA)11 as a probe. The signals of the clone PstGA16 were scattered along the chromosomes of P. amboienesis, but clustered at the centromeres of P. mannii. It was well correlated with the previous study. Whether (GA)n microsatellite is the functional centromeric element of P. stuartiana remained to resolve by immunostaining or chromatin immunoprecipitation in the future.

並列關鍵字

Phalaenopsis stuartiana ； (GA)n microsatellites

參考文獻

林東杰. (2003). 蝴蝶蘭微衛星序列之選殖與特性分析. 國立成功 大學生物學研究所碩士論文.

吳凌巧. (2009). 鹿角蝴蝶蘭 Phalaenopsis cornu-cervi 含 (GA)n 微衛星體序列的選殖與定位. 國立台灣大學分子與細胞生物學研究所碩士論文.

彭欣羚. (2007). 利用數種重複性 DNA 序列的分佈建立朵麗蘭的核型. 國立台灣大學分子與細胞生物學研究所碩士論文.

Adams, S.P., Leitch, I.J., Bennett, M.D. and Leitch, A.R. (2000). Aloe L. – a second plant family without (TTTAGGG)n telomeres. Chromosoma 109, 201–205.

Avramova, Z. (2002). Heterochromatin in animals and plants. Similarities and differences. Plant Physiol. 129, 40-49.