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研究生： 蕭昱
Hsiao, Yu
論文名稱： 水稻大米基因OsBG1突變品系之研究及組織表現分析
Characterization and Tissue Expression Profile of OsBG1 (Oryza sativa Big Grain Mutant 1) in Rice
指導教授： 余淑美
Yu, Su-May
學位類別： 碩士
Master
系所名稱： 生物科學與科技學院 - 熱帶植物科學研究所
Institute of Tropical Plant Sciences
論文出版年： 2013
畢業學年度： 101
語文別： 中文
論文頁數： 63
中文關鍵詞： 水稻 、產量 、穀粒大小 、OsBG1
外文關鍵詞： Rice, yield, grain size, OsBG1.
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    • 截自2012年，世界人口總數已達到70億，聯合國估計在2050年，全球人口數將超過90億。世界人口數增加所面臨的第一個重要問題，便是糧食供給不足，原因包括可耕地面積減少、氣候變遷、原物料價格上漲等。因此研究如何提高糧食作物產量可減輕未來糧食不足的危機。控制稻米產量的主要因素包含了米粒的大小、單穗的穀粒數及單株的總穗數，本研究利用正向遺傳學的策略，從Taiwan Rice Insertional Mutagenesis（TRIM）Database篩選出具有比野生型米粒變大的突變株，與本突變株相關的基因已被確認並命名為OsBG1 (Oryza sativa Big Grain Mutant 1) ，這個新基因是首次在水稻及其他物種中被研究。因此本研究的目標是找出OsBG1的功能及表現位置，利用OsBG1的啟動子結合GUS報導基因，結果顯示OsBG1表現在根尖、葉的維管束、年輕的葉頸、花藥、外穎頂端，並配合RT-PCR的結果，發現OsBG1在開花時期大量表現。同時利用生物資訊分析胺基酸序列，發現在水稻內有另一個Os01g69290基因與OsBG1有30%的相似性，同時也是水稻內唯一一個被比對到的基因，但還需要做進一步的確認，以利了解其生物作用機制。

    In 2012, the world’s population has reached 7 billion. The United Nations estimated that the world population may be over 9 billion in 2050. When the population keeps growing, the major problem human will face is global food crisis, which could be worsened by decreasing arable lands, climate change and rising prices of raw materials. Therefore, the breeding of high-yield crops may alleviate food crisis in the future. The grain size, grain numbers of each spike and the spike number of each plant have contributed to the traits of yield. By screening the Taiwan Rice Insertional Mutagenesis (TRIM) Database, we identified a mutant with grains longer and larger than the wild type. The responsible gene was identified and designated as OsBG1 (Oryza sativa Big Grain Mutant 1). OsBG1 is a novel gene and has not been studied in any organism other than rice. The biological functions of OsBG1 are studied by fusing OsBG1 promoter with the GUS reporter gene. OsBG1 is expressed at tip of root, vascular of leaf, young collar, anther, and tip of lemma. By using the RT-PCR analysis, OsBG1 is found to be expressed at inflorescence stage. Blast of OsBG1 with the NCBI database indicates that Os01g69290 shares a 30% identity of amino acid with OsBG1, and it is the only homologous gene identified in the rice genome. Further study of OsBG1 is in progress.

    目錄 中文摘要 I ABSTRACT II 誌謝 III 目錄 IV 表目錄 VII 圖目錄 VIII 第一章 研究背景 1 1-1 水稻與全球糧食危機 1 1-2 利用T-DNA插入水稻突變株研究基因之功能 1 1-3 影響產量的因素 3 1-4 水稻基因與產量的關係 3 1-4-1. 穀粒數目 3 1-4-2. 器官大小 4 1-4-3. 穀粒充填 5 1-4-4. 植株高度 5 1-4-5. 分蘗 6 1-4-6. OsBG1前人研究 7 第二章 研究動機及目的 8 第三章 材料與方法 9 3-1 研究策略 9 3-2 水稻種植、樣本採集及收種 9 3-3 突變株性狀與基因的關係 9 3-3-1. 相鄰突變株輔助大米基因的篩選 9 3-3-2. DNA萃取 9 3-3-3. 質體救援（Plasmid rescue） 9 3-3-4. T-DNA 側翼序列分析 10 3-3-5. 植株基因型分析 10 3-3-6. Co-segregation analysis 10 3-4 OSBG1的表現量 10 3-4-1. Total RNA萃取 10 3-4-2. DNase處理 11 3-4-3. 反轉錄作用（Reverse transcription） 11 3-4-4. RT-PCR 11 3-5 T-DNA插入數目 12 3-5-1. Southern blot分析 12 3-6 OSBG1組織特異性 12 3-6-1. 基因構築 12 3-6-2. 農桿菌轉殖 13 3-6-3. GUS染色 13 3-6-4. 切片 13 3-7 找出和OSBG1有交互作用的蛋白 14 3-7-1. pGBKT7-OsBG1的構築 14 3-7-2. 將質體轉型至酵母菌 14 3-7-3. 西方點墨法 14 3-7-4. Bait自體活性測試 14 3-7-5. 利用酵母菌接合作two-hybrid library screening 15 3-7-6. 利用X-gal作培養基內 In vivo plate assay 15 3-7-7. 菌落轉移呈色分析（Colony-lift filter） 15 3-7-8. 萃取酵母菌蛋白 16 3-7-9. 萃取酵母菌質體 16 3-7-10. 將酵母菌質體轉型到E.coli 17 3-7-11. 利用PCR分析bait plasmid 17 3-7-12. 定序及分析 17 第四章 結果 18 4-1 植株性狀及考種分析 18 4-2 OSBG1影響植株性狀總結 18 4-3 OSBG1胺基酸序列分析 19 4-4 與OSBG1共同參與調控的基因 20 4-5 OSBG1基因的表現位置 20 第五章 討論 23 5-1 T-DNA種原庫於水稻基因研究之貢獻 23 5-2 細胞週期的調控 23 5-3 植物的分生組織 24 5-4 葉片折角與植物的發育 24 5-5 大米基因之應用性 25 第六章 參考文獻 26 第七章 圖表 30 第八章 附錄 61 表目錄 Table 1. 2009第一期作考種資料 30 Table 2. 2010第二期作考種資料 31 Table 3. 轉殖株T1代性狀 32 Table 4. Os01g69290 T-DNA突變株2012第二期作考種資料 33 圖目錄 Figure 1. 大米植株OsBG1ACT（#147 He）性狀及相對位置。 34 Figure 2. 基因與突變株位置相對圖及性狀。 35 Figure 3. 野生型，OsBG1ACT（#147 Ho），Ubi:OsBG1植株性狀比較圖。 36 Figure 4. 野生型，OsBG1ACT（#147），Ubi:OsBG1種子長度比較圖。 37 Figure 5. OsBG1與其他物種間相似基因比對。 40 Figure 6. Os01g69290 T-DNA突變株相對位置及RT-PCR表現。 41 Figure 7. Os01g69290 T-DNA突變株性狀。 42 Figure 8. Os04g39020相關資料圖表 43 Figure 9. OsBG1、Os01g69290、Os04g39020在野生型各時期RT-PCR結果。 44 Figure 10. OsBG1:eGFP-GUS構築。 45 Figure 11. 幼穗原始體（1~2公分）即有GUS表現。 46 Figure 12. 3~5公分幼穗在花梗有大量表現。 47 Figure 13. 6~10公分幼穗表現區域與3~5公分相似。 48 Figure 14. 11~15公分未成熟穗表現在花梗之連接區。 49 Figure 15. 16~20公分未成熟穗主要表現於花藥。 50 Figure 16. 大於20公分的穗GUS表現在花梗切口、花藥、小花頂部。 51 Figure 17. 授粉前後小花之GUS表現。 52 Figure 18. T1種子發芽後12～48小時外表GUS表現。 53 Figure 19. T1種子浸種24小時，縱切之後做GUS染色。 54 Figure 20. T1種子浸種5天後莖和根GUS表現。 55 Figure 21. 根作連續切片染色的型態。 56 Figure 22. GUS表現於葉子切口的葉肉及維管束。 57 Figure 23. GUS在葉頸（collar）的表現量相較於葉片及葉鞘要高。 58 Figure 24. 植株20天大T1植株的葉頸染色後連續切片。 59 Figure 25. T1植株66天大的葉子頂芽分生組織（SAM）和側芽皆有GUS表現。 60 Supplemental Figure 1.OsBG1ACT T-DNA插入數目及RT-PCR 61 Supplemental Figure 2. OsBG1在不同的處理下表現量的改變 62 Supplemental Figure 3. ARALYDRAFT_914401在Genevestigator作表現位置預測。 63

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