臺灣博碩士論文加值系統

在本研究中，確定了魚類中鯔魚科(Mugilidae)，Liza屬和金線魚(Nemipterus virgatus)的特異性 DNA片段。自15個不同魚科之魚中取血液，萃取出基因組DNA 樣本，藉由80個隨機的引子，隨機擴增多態性 DNA指紋。試驗結果發現，以OPAV04當做引子擴增基因組DNA時，可以在Mugilidae科魚的PCR產物中發現此科魚之特異性DNA片段，其片段長度為856 bp；以OPAV10隨機引子擴增基因組DNA時，可以在Liza屬擴增出本屬魚之特異性DNA片段，其片段長度為418 bp。若以OPAA11當做引子擴增基因組DNA時，僅能在金線魚的PCR產物中發現金線魚的特異性DNA片段，此片段之長度為535 bp。將這些特異DNA片段之序列與基因庫(GenBank) 中前人發表之序列進行比對，並未發現相似的序列。根據特異DNA序列設計科或屬之特異引子，用這些特異引子進行PCR增殖魚之基因組DNA，可成功地增殖出專屬於Mugilidae科和Liza屬特異性的DNA片段，其長度分別為569 bp和186 bp；在金線魚方面，則可增殖出其特有之372 bp長的的片段。試驗結果也顯示，利用Mugilidae科的特異引子進行PCR增殖，可區分陸生和水生動物。將金線魚之特異DNA序列與GenBank中前人發表之序列進行比對，亦未發現相似的序列。根據此特異序列來設計金線魚的特異引子，此引子僅能在金線魚增殖出明顯的372 bp的特異片段。綜合上述結果，顯示利用逢機增殖多態性DNA指紋之應用，可以建立物種的特異性分子標記，提供未來做為快速且能準確鑑別常見食用魚類物種之工具。

In the present study, Both of novel family- and genus-specific DNA markers in Mugilidae fish and golden threadfin bream (Nemipterus virgatus). Genomic DNA was isolated from the blood of fish of 15 families and eighty random primers were used for random amplified polymorphic DNA (RAPD) fingerprinting. When the primer OPAV04 was employed, a novel specific PCR product was observed in the Mugilidae family. In addition, another novel specific PCR product was also observed in the Liza genus while using primer OPAV10. Sequencing analysis revealed that the novel family- and genus-specific DNA fragments were 856 bp and 418 bp, respectively, and no similar sequence was found in GenBank. Two primer sets were designed based on the family- and genus-specific sequences to confirm the RAPD results and the 569 bp and 186 bp predicted bands were successfully amplified by PCR. Intriguingly, Mugilidae family specific DNA markers were also effectively used for terrestrial and aquatic animal discrimination. A species-specific band amplified from Nemipterus virgatus using the OPAA11 random primer was selected for sequencing analysis, and a 535 bp sequence which had no similarity with any others in the nucleotide database was obtained. A primer set was designed based on the Nemipterus virgatus specific sequence to validate the RAPD-PCR results. A predicted 372 bp amplicon was significantly observed by PCR. Therefore, the novel family- and genus-specific DNA markers identified in this study can be used as an effective tool for rapid and accurate determination of which fish, and even for cross-species identification.

content
中文摘要.................................................i
Abstract ...............................................iii
Part 1：Novel family- and genus-specific DNA markers in Mugilidae.............................................. 1
Summary...............................................1
Introduction..........................................3
Materials and Methods.................................5
Results...............................................10
Discussion............................................21
Part 2：A unique molecular marker for golden threadfin bream (Nemipterus virgatus) species identification.............25
Summary................................................25
Introduction...........................................26
Materials and Methods..................................28
Results................................................34
Discussion.............................................39
Conclusion...............................................43
References...............................................44
Appendix.................................................51


List of Figures
Figure 1-1. RAPD fingerprints of 15 popular food fish families in Taiwan.......................................12
Figure 1-2. A novel family-specific DNA sequence (856 bp) of Mugilidae cloned from the RAPD fingerprints.............................................13
Figure 1-3. RAPD fingerprints of 10 popular food fish families in Taiwan…………...14
Figure 1-4. A novel genus-specific DNA sequence (418 bp) of Liza cloned from the RAPD fingerprints.............................................15
Figure 1-5. PCR analysis for family and genus identification in 10 popular food fish families in Taiwan...................................................18
Figure 1-6. Discrimination of terrestrial and aquatic animals by PCR...........................................20
Figure 2-1.The RAPD fingerprints of 10 species of popular food fishes in Taiwan....................................34
Figure 2-2. A unique species-specific sequence ( 535 bp) of Nemipterus virgatus isolated from the RAPD fingerprints.............................................35
Figure 2-3. The Nemipterus virgatus species identification by PCR analysis using SpeAA11NemiF1/R1 specific primer set......................................................37
Figure 2-4. The species identification in 10 species of common food fishes by PCR analysis ......................38

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