臺灣博碩士論文加值系統

轉位子可在基因組中任意移動且複製，在玉米的基因組中，約有85%由轉位子所構成。當轉位子插入基因的隱子中時，轉位子有機率透過其所帶有的剪接供位，而造成該基因透過選擇性剪接產生帶有轉位子部分片段的轉錄物。許多研究亦指出，當遭受逆境時，植物會大量啟動選擇性剪接以因應外在環境的變化。為了解玉米DNA轉位子－Ds在非生物逆境下對於玉米選擇性剪接的影響，本研究將53個與非生物逆境相關的玉米RNA-Seq資料進行組裝與分析，所包含之非生物逆境有滲透勢、乾旱、鹽分、熱、冷、淹水及紫外線逆境。分析結果顯示，所組裝出的轉錄物中，約有68%為已知的轉錄物、25%為已知基因的未知轉錄物、7%是屬於基因間區段的轉錄物。在大部分的逆境處理中，其轉錄物總數高於其所相對應的控制組處理，且已知基因的未知轉錄物及基因間區段的轉錄物在逆境處理中的比例在大部分的逆境下也高了1~3%。而在逆境下，差異表現已知轉錄物的基因與轉錄出未知轉錄物的基因在功能性註解上有很大的差異。綜合以上結果，顯示在逆境下選擇性剪接在非生物逆境下可能扮演重要的角色。再透過對玉米基因組中的轉位子Ds進行分析，我們發現在9個內含Ds序列的基因中，Ds序列會藉由4個不同的剪接機制進行選擇性剪接，並將部分的Ds訊息帶入轉錄物中。這四種機制分別為：內含子保留、顯子化、選擇性轉錄起始點及選擇性轉錄終止點，且其中有些包含Ds序列的轉錄物會在特定的非生物逆境下被誘導。我們的結果提供了相關證據以證實透過Ds轉位所造成的轉錄物可能影響玉米在非生物逆境下的後轉錄調控。

Transposable elements (TE) are the mobile and replicable sequences within the genome. Particularly, 85% of maize genome is composed of TEs. It has been demonstrated that a TE insertion can possibly introduce alternative splicing (AS) junction sites in the intron of a gene and result in new isoforms. AS events in plants have also been reported to be responsive to environmental stresses. To understand the roles of alternative splicing caused by insertions of the Ds elements, a family of well-studied DNA TE, during abiotic stresses, we assembled and analyzed 53 stress-related RNA-Seq libraries of maize under drought, osmotic, salt, heat, cold, water flooding, and UV stresses.
Among all assembled transcripts, 68% are known transcripts, 25% are novel isoforms of known genes, and 7% are intergenic transcripts. In most stressed samples, the total transcripts would be greater than those in the corresponding controlled samples. The functional annotations of the genes differentially expressing known transcripts were distinct from those of novel isoforms, together, highlighting the importance of AS in enhancing transcriptomic diversity during abiotic stresses. The identification of partial Ds sequences in the isoforms of 8 annotated and 1 putative intergenic genes suggested that the Ds elements can affect the splicing of inserted genes through 4 mechanisms including intron retention, exonization, alt-transcription start site, and alt-polyadenylation site. Several Ds-related isoforms appeared to be induced by a specific abiotic stress. In conclusion, our results implied that the AS isoforms resulted from TE insertion may contribute to the transcriptomic responses to diverse abiotic stresses in maize.

口試委員審定書 i
致謝 ii
摘要 iii
Abstract iv
Table of Contents vi
Table index viii
Figure index ix
Introduction 1
Materials and Methods 6
2.1 Data processing and reads mapping 6
2.2 Transcript assembly using Cufflinks 6
2.3 Combination with reference annotation using Cuffmerge 7
2.4 Differential expression (DE) analysis 7
2.5 GO enrichment analysis 8
2.6 Ds elements discovery 8
Results 10
3.1 RNA-Seq Transcriptome Analysis reveals novel transcripts 10
3.2 Numbers of isoform increased during abiotic stresses 13
3.3 Transcriptome differential expression between control and stresses and GO enrichment of the genes expessing DE isoforms 14
3.4 Ds transposons involved in alternative splicing 17
3.5 Ds-related isoforms may be induced by abiotic stresses, or expressed in specific tissues and growth stages 21
Discussion 25
4.1 The role of alternative splicing in the response to abiotic stresses 25
4.2 Ds transposons involved in alternative splicing during abiotic stresses 28
Conclusion 32
Reference 33
Supplementary Sequences 59
Supplementary Tables 63
Supplementary Figures 64
Appendix 73

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