點帶石斑魚微衛星基因座之選殖與特性分析

Isolation and characterization of microsatellite loci from orange-spotted grouper, Epinephelus coioides

林渤昀(Bou-Yun Lin)

指導教授 ： 張繼堯

國立臺灣大學/生命科學院/漁業科學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.00538

摘要

近年來微衛星序列 (microsatellites) 被應用在許多領域的研究而成為相當重要的分子標記工具。微衛星基因座有下列特性：普遍存在於基因體(Genome)中，故能夠很方便的經由聚合酵素連鎖反應(PCR)的方式偵測其多型性。微衛星基因座優於其他分子標記的主要原因為其能夠顯現來自父親及母親的遺傳訊息，這就是所謂的共顯性(Co-dominance)。目前，這種標誌在水產上被應用於親源鑑定及純化品系的工具，以增加養殖魚隻的競爭力造就更大的市場價值。
本實驗目的在於從點帶石斑魚(Epinephelus coioieds)中分離出微衛星基因座並分析其特性。以了解各基因座之總性狀數及性狀出現頻度，以便計算各基因座在種內的鑑別力(Polymorphic Information Content, PIC)。本實驗分離微衛星基因座的策略是依照其本身的一些特性所設計。微衛星基因座之片段長度約為 300 bp 到 500bp；以及含有 2 個鹼基重複數次或以上的重覆序列。首先將基因體核酸從點帶石斑魚中萃取出來，利用特定酵素將長且完整得片段作用成更小的片段，接下來利用特定含有 2 個鹼基重複序列的探針(probe) 去和這些小片段進行雜合反應(hybridization)，最後篩選出微衛星基因座。
有 28 個微衛星基因座經過篩選後被分離出來，針對其游離區域(flankingregions)設計引子(primer)，在分析樣本前，嘗試找出最佳的黏合溫度(annealingtemperature)以降低非專一性的產物干擾分析結果。經過數據分析結果發現 Ec-21之 PIC 值為 0.85 是這些基因座中最高的，同時代表 Ec-21 為很有潛力的分子標誌，將來可用來分析種內個體之間的差異。而在跨物種的分析中也發現 Ec-19 這個基因座能夠分辨同科魚種和點帶石斑魚之間的差異。此外本實驗五個高度多型性的基因座被篩選出來，未來可發展多基因座增幅(multiloci amplification)，如此一來就能同時分析其中的兩個基因座，將能夠更有效率的進行更深入的分析。

關鍵字

點帶石斑魚 ； 微衛星基因座 ； 跨物種分析 ； 多基因座增幅

並列摘要

In recent years, microsatellites have become one of the most popular molecular markers used with applications in many fields. The objective of this study is to isolate the microsatellite loci from orange-spotted grouper, Epinephelus coioides and characterize those microsatellite loci.
The approach to achieve the target is based on the characteristics of the microsatellite loci. First, the genomic DNA extracted from the orange-spotted grouper had to be cut into smaller fragments by RsaI. Then, 6 sets of specific di-nucleotide probe (CG15, GA15, AC15, AT15, TG15 and CT15) were used to select the microsatellite-enriched fragments by the selective hybridization with two different hybridization buffers. Select totally 470 colonies from the microsatellite-enrich library. There were 201 positive colonies confirmed by the digestion check. Among all these positive colonies, 28 microsatellite loci have been successfully isolated. Before the samples were analyzed, finding the optimized annealing temperature was essential.
Total 54 samples E. coioides were collected from Cha-Dong, then the allelic distribution and the allele frequency of those microsatellite loci were analyzed. The number of the allele range from 0 to 8 alleles was found. Locus Ec-21 has the most PIC value of 0.85 and consider as the highest polymorphic locus. In the result from the analysis of cross-species amplification, locus Ec-19 can distinguish different species of Serranidae. Base on the distribution of separating alleles, the approach of mutiloci PCR amplification can be used in the further research.

並列關鍵字

Epinephelus coioides ； Serranidae ； microsatellite DNA ； polymorphic information content (PIC)

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