紅豆杉科與傘松科植物色質體基因組分析:洞悉其核質 體DNA 與重組基因群

Study on the Plastomic Organizations of Taxaceae and Sciadopityaceae: Insights into Their Nuclear Plastid DNAs and Chimeric Gene Clusters

許智堯(Chih-Yao Hsu)

指導教授 ： 趙淑妙

國立臺灣大學/生命科學院/基因體與系統生物學學位學程/博士(2016年)

https://doi.org/10.6342/NTU201601257

摘要

針葉樹植物 (conifers)分為松科 (Pinaceae) 與柏門 (Cupressophyta) 兩大群，其中以柏門植物最具多樣性及高經濟價值。現生的柏門植物又分為五個科，共約有400多種。種子植物 (seed plants) 色質體基因組 (plastomes)結構相當保守，但柏門植物的色質體基因組結構卻有高度的變異性。為了更深入的探討色質體基因組重組在演化上的意義與影響，本論文利用比較色質體基因體學的方法研究以下兩個題目。
第一，提出新的方法篩選及研究紅豆杉科植物核質體DNA (nuclear plastid DNA or nupt)。核質體DNA是一群由色質體基因組轉移到核基因組中的DNA片段。核質體DNA的移轉為核基因組提供了豐富的遺傳資源，也提高了核基因組的遺傳多樣性。但目前為止，沒有針葉樹植物核質體DNA的相關研究，因此本研究定序了台灣穗花杉 (Amentotaxus formosana) 及台灣紅豆杉 (Taxus mairei) 的完整色質體基因組，並利用比較基因體學的方法分析穗花杉、紅豆杉及粗榧屬的色質體基因組排列方式，進而推測出紅豆杉科植物祖先色質體基因組的組成與排列方式。由此，我們設計專一性的引子來增幅祖先型及現生紅豆杉科植物色質體基因組的非共線型區域 (non-syntenic region)。利用這方法我們一共篩選出12.6 kb的核質體DNA，這些核質體DNA明顯地累積較多GC變成AT的突變。此外，藉由比較祖先型核質體DNA與現生核質體DNA的rps8基因，我們發現現生核質體DNA的rps8基因轉譯起始碼子包含了一個C變成U的RNA修飾。我們的研究進一步的指出紅豆杉科植物的色質體基因組大約在白堊紀就已經轉移到核基因組中，這些核質體DNA不僅保留了祖先型色質體基因組排列方式與核酸組成，也提供了線索，讓我們可以瞭解與研究針葉樹色質體基因組的演化過程。
第二，利用實驗的方法驗證與探討色質體基因組序列重組對於傘松科植物演化上的意義與影響。種子植物的色質體基因轉錄時大多是以操縱組 (operon) 的模式進行，也就是多個基因同時轉錄 (polycistronic transcription); 種子植物的色質體基因組操縱組相當保守，即使像針葉樹的色質體基因組排列方式經過多次的重組，但它們的操縱組也很少遭到破壞。本研究定序了完整的傘松(Sciadopitys verticillata)色質體基因組。傘松科植物目前僅存一種，基因體比較分析後發現傘松的色質體基因組有三項特點: 色質體基因組排列經過多次的重組、包含了三組連續並退化的tRNA基因 (trnV-GAC, trnQ-UUG及trnP-GGG )、有一個特殊的反向重複序列 (inverted repeat)，而這個反向重複序列可形成不同構型 (isomeric) 的色質體基因組。此外，傘松的色質體基因組因重組打斷了多個原本的操縱組，而這些斷裂的操縱組卻重新組合而形成四個重組基因群 (chimeric gene clusters)。我們的數據顯示這些新的重組基因群保留有原本的啟動子(promoter) ，且可以順利地轉錄出其相對應的RNA序列。本研究結果使我們能更深入的了解為何針葉樹植物色質體基因組具有多樣性及高複雜度的特性。

關鍵字

色質體基因組 ； 基因組重組 ； 演化 ； 核質體DNA ； 基因群 ； 紅豆杉科 ； 傘松科 ； 針葉樹

並列摘要

Cupressophytes (Cupressophyta or conifers II) is the largest of the two conifer groups, the other being Pinaceae. Cupressophytes comprise about 400 species in five families. They are the most diversified and economically valuable group in conifers. Our previous studies showed that gene organizations of their plastid genomes (plastomes) are highly variable among the few elucidated plastomes of cupressophytes. To further decipher the evolution of plastomic re-organization, I carried out comparative plastome studies of two cupressophytes families; Taxaceae and Sciadopityaceae.
Here we reported two major findings. First, I proposed a new strategy for identification and evolutionary studies of nuclear plastid DNAs (nupts) in Taxaceae. Plastid-to-nucleus DNA transfer provides a rich genetic resource to the complexity of plant nuclear genome architecture. To date, the evolutionary fates of nupt remain unknown in conifers. We have sequenced the complete plastomes of two yews, Amentotaxus formosana and Taxus mairei. Comparative plastomic analyses revealed possible evolutionary scenarios for plastomic reorganization from ancestral to extant plastomes in the three sampled Taxaceae genera, Amentotaxus, Cephalotaxus, and Taxus. Specific primers were designed to amplify non-syntenic regions between ancestral and extant plastomes, and 12.6 kb of nupts were identified based on phylogenetic analyses. These nupts have significantly accumulated GC-to-AT mutations, suggesting a nuclear mutational environment shaped by spontaneous deamination of 5-methylcytosin. The ancestral initial codon of rps8 is retained in the Taxus nupts, but its corresponding extant codon is mutated and requires C-to-U RNA-editing. These findings suggest that nupts can help recover scenarios of the nucleotide mutation process. We also demonstrated that the Taxaceae nupts we retrieved may have been retained since the Cretaceous period of Mesozoic Era and they carry the information of both ancestral genomic organization and nucleotide composition, which offer clues for understanding the plastome evolution in conifers.
Second, we used experimental data to show the evolutionary impact of plastomic rearrangements in Sciadopityaceae. Many genes in the plastid genomes of seed plants are organized in polycistronic transcription units known as operons. These plastid operons are highly conserved, even among conifers whose plastomes are highly rearranged. We sequenced the complete plastome sequence of Sciadopitys verticillata (Japanese umbrella pine), the sole member of Sciadopityaceae. The Sciadopitys plastome is characterized by extensive inversions, pseudogenization of tRNA genes after tandem duplications, and a unique pair of inverted repeats involved in the formation of isomeric plastomes. We showed that plastomic inversions in Sciadopitys have led to the shuffling of remote operons, resulting in the birth of four chimeric gene clusters. Our data also suggested that these chimeric gene clusters have adopted pre-existing promoters for the transcription of genes. This newly deciphered plastome of Sciadopitys advances our current understanding of how the conifer plastomes have evolved toward increased diversity and complexity.

並列關鍵字

Plastome ； Genomic reorganization ； Evolution ； Nupt ； Gene cluster ； Taxaceae ； Sciadopityaceae ； Conifer

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