臺灣博碩士論文加值系統

若植物本身抗旱能力佳，則木材形成效率可能隨之提高，因此我們想找出與抗旱能力相關的基因。本篇研究利用實驗室所提供的乾旱處理後的毛果楊(Populus trichocarpa)以及其控制組的RNA-seq資料，進行基因表達量分析，得到差異表達的轉錄因子 (Transcription factor, TF)，為後續建立調控網路的一環。
為了檢驗TF與下游基因啟動子 (Promoter)之間是否有交互作用，以建立轉錄調控網路，我們研發出一套新的酵母單雜交系統 (Yeast one-hybrid, Y1H)，Meiosis-directed Y1H (簡稱Meiosis)，此系統結合了傳統Haploid transformation (簡稱Haploid)以及Diploid mating (簡稱Diploid) Y1H系統的優點。以colony counter來計算上述三種Y1H系統所得到的每個酵母菌落的面積，用自行設計的程式來分析這些面積，結果顯示，Meiosis陽性結果的發現率為三者中最高。因此得知，實驗室成功開發出了一套結果好，且耗時短的高效率Y1H系統。
次級細胞壁合成在木材形成途中扮演著重要角色。毛果楊中屬於NAC (NAM, ATAF1/2, and CUC2)蛋白家族的PtrSND1-A2 (Secondary Wall-Associated NAC Domain 1s)與PtrVND6-C1 (Vascular-Related NAC Domain 6s)會因為的intron 2 retention差異剪切的發生，抑制PtrSND1-A2與PtrVND6-C1活化下游基因的功能，進而影響次級細胞壁的合成。本篇研究中，我們嘗試探討在NAC家族中，這種intron retention現象最早出現在植物演化過程中的哪一時間點，以及該現象是否廣泛分布在植物界中。
分析了10個物種，包含2個蘚苔類、1個蕨類、以及7個被子植物，其中僅有1個蘚苔類植物未篩選出任何一個intron 2或intron 3 retention現象。目前發現，物種中出現前述intron retention的NAC基因數量比例 (該物種中有所求intron retention的NAC基因數量/該物種中所有NAC基因的比例)在蘚苔蕨類組與被子植物組之間並沒有看到顯著差異。

Wood formation efficiency can increase as plant drought resistance ability increases, so we tried to find out drought resistance related genes. Our laboratory conducted RNA-seq data of Populus trichocarpa drought treatment groups and their control group to obtain a set of differentially expressed genes (DEGs) including transcription factors (TFs). These drought response TFs were used to build a transcriptional regulatory network for wood formation.
In order to identify interactions between TFs and promoters to construct a regulatory network, our laboratory developed a novel yeast one-hybrid (Y1H) system, Meiosis-directed Y1H (hereinafter called Meiosis), which integrated the advantages of the two traditional Y1H systems, Haploid transformation (hereinafter called Haploid) and Diploid mating (hereinafter called Diploid). With colony counter, areas of all yeast colonies obtained by the three mentioned Y1H systems would be calculated. Results showed that the discovery rate of Meiosis is the highest among these three systems. Our laboratory successfully developed a highly efficient Y1H system, which can generate high quality results and consume short time.
Biosynthesis of secondary cell wall plays an important role in wood formation. SND1 (Secondary Wall-Associated NAC Domain 1s) and VND6 (Vascular-Related NAC Domain 6s) family proteins are significant regulators of secondary cell wall differentiation. In previous studies, PtrSND1-A2 and PtrVND6-C1, which belong to SND and VND families of Populus trichocarpa, can interfere in the activation of downstream genes due to intron 2 retention. In this research, we try to explore the evolution of this kind of intron retention in plant species.
Analyzing 10 species, including 2 bryophytes, 1 fern, and 7 angiosperms, we found that neither intron 2 nor 3 retention can’t be observed in only 1 bryophyte among these 11 species. Statistics showed that there was no statistically significant difference in the rate of NAC genes which the specific conditions of intron retention occur between bryophyte-fern group and angiosperm group.

目錄.............................................................................................................I
致謝..........................................................................................................III
中文摘要..................................................................................................IV
Abstract....................................................................................................VI
圖目錄...................................................................................................VIII
表目錄.......................................................................................................X
1. 緒論.......................................................................................................1
1.1 毛果楊 (Populus trichocarpa)在乾旱條件下的轉錄組分析...................1
1.2 酵母單雜交 (Yeast one-hybrid, Y1H).................................................2
1.3 PtrSND1-A2IR與PtrVND6-C1IR的交互調控現象...............................3
2. 實驗材料與方法...................................................................................6
2.1 網路資料庫 (Database)提供的材料....................................................6
2.1.1 Phytozome裡物種的基因組資料.................................................6
2.1.2 毛果楊乾旱處理之下的RNA-seq................................................6
2.1.3 PlantTFDB裡的TF家族基因ID..................................................7
2.1.4 本研究需要的參考序列資料......................................................8
2.1.5 NCBI裡的RNA-seq資料...........................................................9
2.2 序列比對.........................................................................................9
2.2.1 序列比對程式 – BWA...............................................................9
2.2.2 Sam檔內容與格式..................................................................10
2.2.3 Sam檔處理程式 – Samtools.....................................................11
2.3 差異表達分析................................................................................12
2.4 Intron retention判斷........................................................................14
2.4.1 程式分析................................................................................14
2.4.2 篩選出有intron retention的基因...............................................18
2.5 Y1H結果分析................................................................................18
2.5.1 實驗材料來源.........................................................................19
2.5.2 分析掃描結果.........................................................................21
2.5.3 建立調控網路.........................................................................24
2.6 實驗材料與方法總結......................................................................24
2.6.1 在乾旱脅迫下的轉錄組分析 (Transcriptome analysis during drought stress)...................................................................................................24
2.6.2 TFs與promoters的交互作用 (Interaction between TFs and promoters)............................................................................................25
2.6.3 NAC家族的分子演化 (Molecular evolution of NAC family)........26
3. 結果.....................................................................................................28
3.1 在乾旱脅迫下的轉錄組分析...........................................................28
3.1.1 Day0, Day5, Day7之間的差異性...............................................28
3.1.2 Day0與Day5的差異表達分析..................................................29
3.1.3 Day0與Day7的差異表達分析..................................................30
3.2 TFs與promoters的交互作用...........................................................32
3.2.1 有交互作用的TFs與promoters.................................................32
3.2.2 基因調控網路.........................................................................33
3.3 NAC家族的分子演化.....................................................................34
4. 討論.....................................................................................................35
4.1 在乾旱脅迫下的轉錄組分析...........................................................35
4.2 TFs與promoters的交互作用...........................................................36
4.3 NAC家族的分子演化.....................................................................37
5. 參考文獻.............................................................................................38

圖1.1 傳統Y1H方法的優缺點...................................................................3
圖1.2 PtrSND1-A2IR與PtrVND6-C1IR的交互調控現象.................................5
圖2.1 毛果楊乾旱處理示意圖....................................................................7
圖2.2 gff檔案形式....................................................................................9
圖2.3 sam檔案形式.................................................................................11
圖2.4 merged_rawreads檔案形式..............................................................13
圖2.5 EdgeR輸出Excel檔案形式.............................................................14
圖2.6 Python程式流程圖.........................................................................15
圖2.7 Statistics.csv檔案形式....................................................................16
圖2.8 機械手臂塗酵母菌在生長盤 (SC、AbA1和AbA2)上.........................19
圖2.9 PhenoBooth Colony Counter儀器.....................................................21
圖2.10 酵母菌落矩陣示意圖......................................................................21
圖2.11 矩陣分析程式輸入提示與輸出結果.................................................24
圖2.12 差異表達基因篩選流程圖...............................................................25
圖3.1 Day0-Day5-Day7相似度結構分析圖................................................28
圖3.2 Day0-Day5 smear plot.....................................................................29
圖3.3 Day0-Day5差異表達的TFs.............................................................30
圖3.4 Day0-Day7 smear plot.....................................................................31
圖3.5 Day0-Day7差異表達的TFs.............................................................31
圖3.6 Haploid, Diploid, Meiosis篩選結果文氏圖........................................32
圖3.7 TF-promoter調控網路.....................................................................33
圖4.1 Phytozome上毛果楊的基因功能註解...............................................35
圖4.2 調控網路擴建示意圖......................................................................36

表3.1 節點名稱與基因ID對照表..............................................................33
表3.2 候選基因列表................................................................................34

Li Q., et al. (2012). Splice variant of the SND1 transcription factor is a dominant negative of SND1 members and their regulation in Populus trichocarpa. Proc Natl cad Sci USA. 109:14699–14704.
Li H., Durbin R. (2009). Fast and accurate short read alignment with Burrows-Wheeler Transform. Bioinformatics, 25:1754-60.
Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R., 1000 Genome Project Data Processing Subgroup. (2009). The Sequence alignment/map (SAM) format and SAMtools. Bioinformatics, 25:2078-9.
Lin, Y. J., Chen, H., Li, Q., Li, W., Wang, J. P., Shi, R., Tunlaya-Anukit, S., Shuai, P., Wang, Z., Ma, H., Li, H., Sun, Y. H., Sederoff, R. R., Chiang, V. L (2017). Reciprocal cross-regulation of VND and SND multigene TF families for wood formation in Populus trichocarpa. PNAS, 114:E9722-E9729.
Robinson M. D., McCarthy D. J., Smyth G. K. (2010). edgeR：a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics, 26(1):139–140.
Shi, R. et al. (2017). Tissue and cell-type co-expression networks of transcription factors and wood component genes in Populus trichocarpa. Planta 245:927-938.
Stuart, J. M., Segal, E., Koller, D., Kim, S. K. (2003). A gene-coexpression network for global discovery of conserved genetic modules. Science. 302:249–55.
Tuskan G. A., et al. (2006). The genome of black cottonwood, Populus trichocarpa
(Torr. & Gray). Science. 313:1596-604.
Wang, J.P. et al. (2014). Complete proteomic-based enzyme reaction and inhibition kinetics reveal how monolignol biosynthetic enzyme families affect metabolic flux and lignin in Populus trichocarpa. Plant Cell. 26:894-914.
Zhong, R., Demura, T., Ye, Z. H. (2006). SND1, a NAC Domain Transcription Factor,
Is a Key Regulator of Secondary Wall Synthesis in Fibers of Arabidopsis. Plant Cell. 18:3158-3170.