水稻OsMYBS2基因的功能性分析

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李宗樺(Tsung-hua Lee) 查詢紙本館藏

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論文名稱

水稻OsMYBS2基因的功能性分析
(Functional analysis of OsMYBS2 gene in rice)

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摘要(中)

在水稻種子的萌發過程中，α-澱粉水解酶基因的表現受到糖與及吉貝素(GAs)的調控。TA box在先前研究中已被確認為糖與吉貝素調控α-澱粉水解酶基因表現的重要順式DNA序列。在我們之前的研究中已知OsMYBS1、OsMYBS2、OsMYBS3會與TA box有專一性鍵結，利用暫時性分析得知OsMYBS2會專一性的鍵結在TA box上，並且抑制TA box:: Luciferase的表現。另外，OsMYBS2在大麥中的同源基因HvMCB1根據研究也指出HvMCB1在N端的1~41個胺基酸對其抑制α-澱粉水解酶基因表現為必須的。
在本篇論文中，我們利用暫時性分析得知OsMYBS2的N端(1～53的胺基酸)對抑制轉錄活性並非為必須的，並利用轉殖植物的穩定表現系統進一步確認OsMYBS2(tr)(N端刪除的OsMYBS2)在水稻中是否對於α-澱粉水解酶基因有抑制轉錄調控的功能。與野生型比較之後發現，OsMYBS2(tr)大量表現時會讓水稻細胞的α-澱粉水解酶基因表現對糖抑制作用較敏感，而OsMYBS2基因靜默時會讓水稻細胞的α-澱粉水解酶基因表現受糖抑制的速度減緩。在缺糖的處理下，雖然水稻細胞中OsMYBS2的mRNA含量僅有少量的減少，但是OsMYBS2(tr)蛋白質含量卻有大幅的下降，表示OsMYBS2(tr)有受到轉錄後的調控。在種子萌發與生長的實驗中，OsMYBS2(tr)大量表現的種子的萌發以及生長速度會被延遲，這也表示OsMYBS2參與在水稻種子的萌發以及幼苗生長的調控中。

摘要(英)

During rice germination, α-amylase genes are controlled by sugar and gibberellins (GAs). The cis-eletment, TA box (TATCCA), has been identified and showed that it plays an important role both in sugar- and in GA-regulated α-amylase gene expression. In our previous study, TA box binding factors, OsMYBS1, OsMYBS2, and OsMYBS3, were identified. Transient expression assay showed that OsMYBS2 recognized TA box specially and functioned as a repressor. Previous studies have shown that the barley HvMCB1 is the orthologous gene of OsMYBS2 and the N-terminal (residues 1-41) of HvMCB1 is essential for negative regulation of α-amylase.
In this study, we showed that the N-terminal (residue 1-53) of OsMYBS2 is not essential for the repression of α-amylase by transient assay. Transgenic rice overexpressing OsMYBS2(tr) (N-terminal truncated version of OsMYBS2) shown that OsMYBS2(tr) is a repressor of α-amylase gene. Compared to wild-type, overexpression of OsMYBS2(tr) caused α-amylase gene hypersensitive to sugar repression, whereas RNAi of OsMYBS2 slowed down the sugar repression response. Although sugar starvation slightly decreased OsMYBS2 mRNA, protein level of OsMYBS2(tr) was much less, implying that OsMYBS2(tr) was regulated post-transcriptionally. Seed germination assay indicated that germination rate was delayed in OsMYBS2(tr) overexpression plants. Taken together, it shows that OsMYBS2 plays a role in regulating seed germination and seeding growth in rice.

關鍵字(中)

★ OsMYBS2

關鍵字(英)

★ OsMYBS2

論文目次

中文摘要 I
Abstract II
縮寫表 III
本文目錄 IV
圖目錄 XII
表目錄 XIII
本文目錄
壹、緒論 1
ㄧ、 α-澱粉水解酶 (α-amylase) 1
二、水稻種子的萌發與α-澱粉水解酶基因表現的調控 1
三、 GA對α-澱粉水解酶基因的調控機制 2
四、糖類對α-澱粉水解酶基因的調控機制 3
五、 MYB蛋白家族介紹 4
六、 OsMYBS2對水稻α-澱粉水解酶基因的調控 6
貳、研究動機與研究問題 8
參、實驗材料與方法 9
一、載體構築 9
1. cDNA製備 9
1.1. DEPC-treated ddH2O (Rnase-free)製備 9
1.2. RNA的萃取與純化 9
1.3. 去除RNA樣品中的DNA 10
1.4. cDNA合成 10
2. 以PCR合成cDNA片段 11
2.1. primer設計 11
2.2. 以PCR合成DNA 11
3. 瓊脂膠體回收限制酵素切過的DNA 12
4. 接合反應 13
4.1. 黏頭端載體DNA與黏頭端DNA片段的接合 13
4.2. 齊頭端DNA片段的接合 13
5. 細菌的轉殖作用 14
5.1. 製備E. coli competent cell 14
5.2. 細菌轉殖 15
5.3. 細菌質體DNA小量抽取法 15
快速少量製備法 15
高速少量製備法 16
5.4. 細菌質體DNA大量製備法 17
二、水稻基因轉殖 19
6. 水稻胚利用微粒子投射法進行短暫活性分析 19
6.1. 水稻胚的準備 19
6.2. 金粒子的製備 19
6.3. DNA附著 19
6.4. 基因槍型號及設定 20
6.5. 轉殖水稻胚樣品處理及分析 20
6.6. Firefly Luciferase酵素活性分析 20
6.7. GUS酵素活性分析 21
7. 水稻細胞利用農桿菌(Agrobacterium)轉殖的方法 21
7.1. 製作Agrobacterium competent cell(EHA105) 21
7.2. 農桿菌的轉殖方法 22
7.3. 農桿菌的生化檢測 22
7.4. 水稻癒傷組織的誘導 22
7.5. 水稻癒傷組織的轉殖 23
三、轉殖水稻分析 25
8. GUS 染色 25
9. 基因組DNA PCR 25
9.1. 基因組DNA 的抽取與純化 25
9.2. PCR 26
10. 原生質體的GFP螢光觀察 26
10.1. 水稻懸浮細胞的建立 26
10.2. 水稻原生質體的分離 26
10.3. GFP螢光觀察 26
11. 西方點墨法 27
11.1. 蛋白質萃取 27
11.2. 蛋白質定量 27
11.3. 蛋白質電泳(SDS-PAGE) 27
11.4. PVDF膜轉印以及抗體雜合 28
肆、實驗結果 29
1. 選殖全長的OsMYBS2基因 29
1.1. OsMYBS2蛋白質功能性區域序列分析 29
1.2. OsMYBS2與HvMCB1胺基酸序列比較分析 30
2. OsMYBS2(tr)與OsMYBS2對SRC的轉錄活性分析 30
3. OsMYBS2(tr)大量表現與OsMYBS2基因靜默轉殖水稻的分析 31
3.1. OsMYBS2(tr)大量表現與基因靜默轉殖T2種子篩選 31
3.2. OsMYBS2(tr)大量表現與基因靜默轉殖T2細胞株中OsMYBS2(tr)表現量的分析 32
3.3. OsMYBS2(tr)大量表現與基因靜默轉殖T2細胞株中α-澱粉水解酶基因表現調控分析 33
4. OsMYBS2的調控分析 34
4.1. 分析OsMYBS2在含糖與缺糖細胞內的位置 35
4.1.1. 建立GFP::OsMYBS2(tr)與GFP::OsMYBS2大量表現的轉殖水
稻 35
4.1.2. UGS2(tr)與UGS2轉殖水稻的轉殖基因表現分析 36
4.1.3. 分析GFP::OsMYBS2(tr)與GFP::OsMYBS2在含糖與缺糖細胞內
的位置 36
4.2. GFP::OsMYBS2(tr)累積量的分析 36
5. OsMYBS2(tr)基因對於水稻種子的生理影響 37
5.1.OsMYBS2(tr)大量表現、野生型與OsMYBS2 RNAi T3代種子胚萌發
分析 38
5.2.OsMYBS2(tr)大量表現、野生型、OsMYBS2 RNAi T3代種子糊
粉層中α-澱粉水解酶表現調控分析 38
5.3. OsMYBS2(tr)大量表現、野生型、OsMYBS2 RNAi T3代種子萌
發分析 39
6. OsMYBS2啟動子在水稻植株中表現的組織特異性分析 40
6.1. OsMYBS2 promoter::GUS轉殖水稻的建立 40
6.2. OsMYBS2在水稻植株中表現的組織特異性 40
伍、討論 42
1. OsMYBS2(tr)在水稻植物中對α-澱粉水解酶基因調控的功能 42
2. OsMYBS2在有糖與缺糖處理下的細胞內位置 43
3. OsMYBS2蛋白在有糖與缺糖的處理下的蛋白質含量調控 44
4. OsMYBS2對水稻種子萌發及生長的影響 44
5. OsMYBS2在水稻中表現的組織特異性 45
陸、參考文獻 75
附錄I 79
附錄II 95
附圖目錄
附圖一、水稻種子萌芽過程中，糖、GA以及α-澱粉水解酶之間的作用關係圖 2
附圖二、新生成蛋白對α-澱粉水解酶基因轉錄速率的影響 6
圖目錄
圖一、 OsMYBS2與OsMYBS3胺基酸序列與大麥中同源基因的胺基酸序列比較 49
圖二、 OsMYBS2的N端53個胺基酸對抑制轉錄活性並非為必須的 50
圖三、 OsMYBS2(tr)大量表現與OsMYBS2基因靜默T2轉殖細胞株分析 51
圖四、 OsMYBS2在水稻細胞作為轉錄抑制子調控α-澱粉水解酶基因 52
圖五、在OsMYBS2(tr)大量表現與基因靜默不同的T2轉殖細胞株中分析OsMYBS2對α-澱粉水解酶基因表現的調控 53
圖六、 GFP::OsMYBS2(tr)與GFP::OsMYBS2大量表現T0轉殖細胞株鑑定 54
圖七、 UGS2(tr)與UGS2 T0轉殖細胞株轉殖基因表現分析 55
圖八、 OsMYBS2(tr)在細胞中的位置不受到糖的調控 56
圖九、 UGS2以及UGS2(tr) T0轉殖細胞株重組蛋白質表現含量分析 57
圖十、 OsMYBS2(tr)累積量的調控主要在轉錄後的調控階段 58
圖十一、 OsMYBS2基因表現受抑制可使分離胚在無糖的澱粉培養基有
較良好的生長 59
圖十二、 OsMYBS2在水稻胚乳中GA對α-澱粉水解酶的調控作為一轉
錄抑制子 61
圖十三、 OsMYBS2(tr)大量表現會延緩水稻幼苗的生長 62
圖十四、 S2P::GUS轉殖癒傷組織以及轉殖植株分鑑定 64
圖十五、 OsMYBS2在水稻植株中的葉、葉鞘、根的維管束中以及保衛
細胞、花粉、柱頭均有表現均有表現 66
表目錄
表一、 OsMYBS2(tr)大量表現T2代種子以50 mg/L Hygromycin篩選之發芽率統計表 68
表二、 OsMYBS2基因靜默T2代種子以50 mg/L Hygromycin篩選之發芽率統計表 69
表三、 OsMYBS2(tr)大量表現、野生型、OsMYBS2基因靜默T3代種子列表 69
表四、 OsMYBS2(tr)大量表現T3代種子以50 mg/L Hygromycin篩選之發芽率統計表 70
表五、野生型8714 T3代種子以50 mg/L Hygromycin篩選之發芽率統計表 71
表六、 OsMYBS2基因靜默T3代種子以50 mg/L Hygromycin篩選之發芽率統計表 72
表七、篩選後可能的T3同型合子轉殖水稻列表 72
表八、 OsMYBS2(tr)大量表現、野生型、OsMYBS2基因靜默T3轉殖種子分離胚的萌發率統計表 73
表九、 OsMYBS2(tr)大量表現、野生型、OsMYBS2基因靜默T3轉殖種子萌發幼苗莖上部長度統計表 74

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指導教授

陸重安(Chung-an Lu)

審核日期

2008-8-25

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