臺灣博碩士論文加值系統

粒線體基因體（mitochondrial genome）在過去十年間已被廣泛的研究。大部分動物的粒線體基因體具有13個蛋白質轉譯基因、2個rRNA及22個tRNA的環形結構。而刺絲胞動門中的花蟲綱（Anthozoa）的粒線體基因體為環形結構，被認為與後生動物的粒線體具有共同祖先的特徵。但是花蟲動物的粒線體基因體也具有獨特的特徵，包括失去大部分的tRNA，第ㄧ型介入子（group I intron）的插入，與不同的基因排列等。即使在花蟲綱的幾個主要類群中，部分基因第一型介入子的插入則會因分類群而有所不同。因此，藉由進一步解析更多類群的花蟲動物粒線體基因體，可以幫助我們了解花蟲粒線體基因的演化。本研究以古老的石珊瑚屬絲珊瑚（Siderastrea spp.）做材料，分析其粒線體基因體結構，並比較與其他已發表的石珊瑚粒線體基因的異同與第ㄧ型介入子的分子演化。
絲珊瑚的粒線體基因排列順序與其他目前已發表的石珊瑚粒線體基因體(16138-18338 bp)相似，但不同的地方為（ㄧ）絲珊瑚具有較長（> 19 kb）且較為鬆散的基因體構；（二） 在細胞色素氧化酶次單位1（cytochrome oxidase subunit,cox1）基因中有第ㄧ型介入子插入；和（三）控制區可能是位在細胞色素b (cytochrome b)基因與NADH去氫酶2 (NADH dehydrogenase 2) 基因之間。這些不同的特性顯示絲珊瑚在石珊瑚演化上的特殊性。
除了海葵外，絲珊瑚的細胞色素氧化酶次單位1基因介入子為目前石珊瑚中已知的首例。由於細胞色素氧化酶次單位1基因介入子中具有一LAGLI-DGEG內切酶(homing endonuclease)，可使細胞色素氧化酶次單位1基因介入子借該內切酶轉置（translocation）到其他生物的基因體中。分析其與海葵細胞色素氧化酶次單位1基因介入子，發現與絲珊瑚細胞色素氧化酶次單位1基因介入子有相同的插入點及較高的相似度，顯示這兩組介入子有較高的同源性，介入子插入的事件可能是在石珊瑚與海葵共同祖先就已發生，而在後來石珊瑚的演化過程中發生個別支序的介入子掉落事件。
絲珊瑚屬雖然分布於三大洋但是分布範圍相當侷限，而細胞色素氧化酶次單位1基因介入子在三大洋的絲珊瑚種均有被發現，顯示細胞色素氧化酶次單位1基因介入子在絲珊瑚屬分歧之前就已存在，支持絲珊瑚屬是ㄧ支相當古老的石珊瑚。而在介入子與其他粒線體片段所發現的高歧異度可能是由於早期孓遺（relict）在各個地理區後長期累積變異所導致。這項結果也說明由傳統分類的謬誤在robust group珊瑚造成的屬內深度分歧在絲珊瑚屬內跨洋性的三物種內並未發生。因此，古老而又可靠的分類群絲珊瑚，可能可以在未來的研究中為石珊瑚的演化路徑提供新的證據。

Animal mitochondrial genome was well studied in past decades with a closed circular structure containing 13 protein coding regions, 2 rRNAs and 22 tRNAs. Nevertheless, mitochondrial genomes of the Anthozoa are different from the typical animal mt genome in several characteristics including, losing most of the tRNAs, possessing group I introns, incompact gene arrangement, and slow evolution. Even within the major lineages of the Anthozoa, the gene containing group I intron and gene arrangement could be very different. Thus, more information should be obtained in order to address the molecular evolution of mitochondrial genome in the Anthozoa. In this study, I determined 3 mt genomes of Siderastrea corals and compared to the published mt genomes of the scleractinian corals.
Mt genome gene arrangement of Siderastrea is nearly identical to the published scleractinian mt genomes except the existence of cox1 intron. Mt genomes are longer (19387-19619 bp) than those of other scleractinian corals (16138-18338 bp). Comparing the phylogenetic relationship of these corals, the loosen mt genome of Siderastrea is probably an ancestral character of the mitochondrial evolution in the Scleractinia.
Cox1 group I introns were detected in Siderastrea species with a LAGLI-DGEG homing endonuclease to perform intron translocation function. Cox1 group I introns were also found in Actiniaria (Metridium senile) with the same insertion site and higher similarity than that of fungi’s cox1 intron, suggesting that the insertion event was ancient probably prior to the split of the common ancestor of Scleractinia and Actiniaria. The group I intron was probably lost several times during the course of evolution in most of the scleractinian lineages.
The finding of coxI group I introns in the three Siderastrea species from the Caribbean, Indian Ocean, and Pacific Ocean suggested that this intron existed before the divergence of the common ancestor of Siderastrea. The genetic divergence was accumulated in both intron as well as the other mitochondrial regions after these species become relict and endemic in these Oceans. The result also demonstrated that the deep divergence within genus might not be caused by obscuring of conventional taxonomy. Siderastrea represents an old and reliable taxonomic group and may provide a new perspective view for evolution of the Scleractinia.

CONTENTS

摘要 --------------------------------------------------------------------------------I
Abstract ------------------------------------------------------------------------ III
Introduction ----------------------------------------------------------------------1
Molecular evolution of Scleractinia --------------------------------------1
Mitochondrial DNA among metazoan -----------------------------------2
Mitochondrial genome in Cnidaria ----------------------------------------3
Siderastrea --------------------------------------------------------------------5
Materials and Methods ---------------------------------------------------------8
Sample collection and species identification -----------------------------8
DNA extraction, PCR amplification and sequencing --------------------8
Mitochondrial genome analyses --------------------------------------------9
Survey of cox1 intron in the complex group and analysis --------------10
Sequence alignment, nucleotide statistics and phylogeny analysis ----11
Results ----------------------------------------------------------------------------12
Gene content and organization ---------------------------------------------12
Codon usages among Siderastrea, Acroporidae, & Montastreaea -----13
Nucleotide composition in Siderastrea ------------------------------------14
Intergenic regions and putative control region ---------------------------15
Genetic distance and phylogeny of Siderastrea --------------------------17
Cox1 group I intron and their origin ---------------------------------------17
Discussion -----------------------------------------------------------------------20
Different mt genomic characteristics with Acroporidae ----------------20
Deep and reliable monophyletic taxonomic group ----------------------22
Cox1 intron insertion among scleractinian corals -----------------------23
Cox1 intron origin -----------------------------------------------------------25
Conclusions ---------------------------------------------------------------------28
References -----------------------------------------------------------------------30
Tables -----------------------------------------------------------------------------38
Figures ---------------------------------------------------------------------------45
Appendix ------------------------------------------------------------------------58

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