臺灣博碩士論文加值系統

Regulation of gene expression is an essential issue for plant growth, development and environmental responses. In this study, we focus on the gene expression study of a white back associated gene, OsWB, in rice. The accumulation of OsWB protein had been shown to be negatively correlated with chalkiness during rice grain formation under high temperature. However, the regulation of OsWB gene expression and its corresponding physiological function remains to be addressed. To reach this goal, we first characterized the alternative splicing gene expression pattern of OsWB1 at post-transcriptional level. Also, we carried out the promoter activity analysis of OsWB1 either with transient gene activation analysis or transgenic rice plant. By RT-PCR analysis, we identified putative 11 alternative splicing transcript variants (ASTVs) of OsWB1 in TNG 67 rice. The length of these ASTVs can be distributed from 210 bp to 1.5 kb. Comparison of various OsWB1 ASTVs sequence revealed that the process of alternative splicing is dependent on short direct repeat sequence. We are also interested in understanding whether the alternative splicing gene expression of OsWB1 can be affected under different tissues, developmental stages and different abiotic stresses. The results showed that among various OsWB1 transcript variants, the expression of OsWB1-c transcript is highest in booting tissues of rice. OsWB1-b transcript expression was decreased under high temperature but the amount of OsWB1-c transcript remained constant. In addition, the gene expression pattern of OsWB1 is unique in mature rice grains. To further understand how OsWB1 gene expression is regulated at transcriptional level, the promoter sequence of OsWB1 is analyzed and searched for various responsive cis-acting DNA elements. Several elements can be found, including ABA、Me-JA、LTRECOREATCOR15 within OsWB1 promoter. We then used transient gene expression assay to study the relationship between OsWB1 gene expression and plant hormone ABA. Preliminary result indicated that the expression of OsWB1 (1.4 Kb)::GUS can be induced at least ten folds by ABA. To fully understand the subcellular-level gene expression and regulation of OsWB1, the transgenic rice plant of OsWB1 (1.4 Kb)::GUS was also produced. It may due to the weak activity of OsWB1 promoter, the GUS staining did not provide any positive result in various tissues of transgenic rice. Taken together, this study proved that OsWB1 expression is not only controlled by its own promoter but also regulated by alternative splicing.

致謝 i
圖表目錄 ii-v
縮寫字對表vi
中文摘要 vii
英文摘要 viii
第一章 前言 1
第二章 前人研究 2
1. 水稻穀粒心腹白相關基因OsWB之發現 2
2. 基因選擇性剪接目前研究 3
2.1 選擇性剪接研究概況及基本分類 4
2.2 SR蛋白之選擇性剪接 5
2.3 植物中選擇性剪接方式 6
2.4 選擇性剪接發生位置 7
3. 植物抗病機制與基因選擇性剪接關係 8
4. 植物非生物逆境訊息傳遞與基因選擇性剪接關係 9
5. 基因啟動子之研究 11
6. 研究目標與研究策略 12
第三章 材料方法 13
1. 試驗材料 13
2. 不同非生物逆境與植物荷爾蒙處理條件 13
3. OsWB基因啟動子分析、胺基酸比對以及
選擇性剪接分析 14
4. OsWB基因表現分析 15
5. OsWB1啟動子選殖及構築 17
6. 暫時性表現分析OsWB11.4 Kb啟動子活性 21
7. 利用水稻基因轉殖分析OsWB11.4 Kb啟動子組織表現差異 23
第四章 實驗結果 26
1. OsWB基因結構、啟動子序列與
胺基酸序列之比對分析 26
1.1 OsWB結構分析 26
1.2 OsWB1啟動子順式調控DNA序列分析 27
1.3 OsWB蛋白質結構及胺基酸比對分析結果 28
2. OsWB1基因選擇性剪接分析 29
2.1 OsWB1不同選擇性剪接產物確認 29
2.2 OsWB1基因選擇性剪接模式 30
2.3不同生長發育及水稻組織中OsWB表現分析 30
2.4 OsWB在不同逆境與荷爾蒙處理下基因表現分析 31
2.5 OsWB於不同發育天數水稻未成熟穀粒中
基因表現分析 31
3. OsWB1啟動子暫時性表現及轉基因水稻分析 32
3.1 OsWB1 ATG上游1.4 Kb啟動子片段暫時性表現分析 33
3.2 OsWB1 ATG上游1.4 Kb啟動子在轉基因水稻
不同組織中GUS表現結果 33
第五章 討論 34
1. OsWB1基因intron 及 exon 序列與選擇性剪接關係 34
2. 短片段重複序列 (Short direct repeat sequence)
可能是單子葉植物中控制選擇性剪接之特殊模式 34
3.OsWB1基因3’端不轉譯區域 (un-translated region)結構
以及選擇性剪接產物之探討 35
4.非生物逆境與OsWB1基因選擇性剪接之關係 36
5. ABA 與OsWB1基因表現之關係 37
6.啟動子水稻轉殖無法呈現GUS染色結果可能原因之探討 37
第六章 未來工作 39
參考文獻 41

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