臺灣博碩士論文加值系統

摘要
GA 2-oxidase擁有2-oxoglutarate-dependent dioxygenase (2-ODD) conserve domain，利用2β-hydroxylation將Gibberellins (GAs)之前驅物以及具有活性的GAs轉變成無活性的GAs。根據研究，水稻的GA 2-oxidases基因家族具有十個成員，而目前OsGA2ox4、OsGA2ox7、OsGA2ox8在水稻中之功能仍尚未有詳細的研究。為了確認GA2ox4、GA2ox7、GA2ox8是否有GA 2-oxidase之酵素功能，可將實驗分為兩部分，一為利用異位表現目標基因的方式將OsGA2ox3、OsGA2ox4、OsGA2ox7、OsGA2ox8基因構築在含 ubiquitin promoter的植物轉殖載體後，送入水稻大量表現，其中OsGA2ox3為對照組，當作GA 2-oxidase酵素強弱的基準點；另一部分則是利用原位表現目標基因的方式申請T-DNA突變株OsGA2ox4Act (M97597)、OsGA2ox7Act (M58372) 及OsGA2ox8Act (M61685)，期望目標基因受到T-DNA帶有的四套CaMV35S enhancer所活化。
當水稻植株體內GA缺乏時，最明顯之外表特徵為植株的矮化。透過轉殖株之株高與TNG67間的差異可推測OsGA2ox4、OsGA2ox7、OsGA2ox8基因是否具有GA 2-oxidase的功能。利用分子層次分析已確認在ubi:OsGA2ox3、ubi:OsGA2ox4、ubi:OsGA2ox7轉殖株，其目標基因皆大量表現。ubi:OsGA2ox3、ubi:OsGA2ox4轉殖株之平均株高分別為6.7及5.4公分，皆屬於極矮化植株；ubi:OsGA2ox7轉殖株之平均株高則為66公分，屬於半矮化的水稻植株。此外，從ubi:OsGA2ox4 T0轉殖株可發現OsGA2ox4表現量的多寡影響轉殖株株高，藉此證實OsGA2ox3、OsGA2ox4、OsGA2ox7皆具有GA 2-oxidase的功能，且隨著GA 2-oxidase基因表現量的差異而影響了植株體GA含量及株高。至於ubi:OsGA2ox8轉殖株，其目標基因OsGA2ox8並無受到ubiquitin promoter大量表現，轉殖株株高與TNG67也無明顯差異，推測可能為ubiquitin promoter之序列有所變異，因此無法確認OsGA2ox8是否具有GA 2-oxidase的功能。另一方面，原位活化之T-DNA突變株OsGA2ox4Act (M97597) 除了插入點無法確認，OsGA2ox4目標基因也沒有活化；OsGA2ox7Act (M85372) 及OsGA2ox8Act (M61685) 之突變株則利用基因型分析確認T-DNA插入位坐落於目標基因旁。然而OsGA2ox7Act (M85372) 之目標基因並無因T-DNA而活化；OsGA2ox8Act (M61685) 突變株透過RT-PCR的結果證實了插有T-DNA的M61685突變株確實活化了OsGA2ox8基因，並可透過外表性狀確認OsGA2ox8的活化會使水稻株高變矮，藉此可說明OsGA2ox8應該具有GA 2-oxidase的功能。
藉由T-DNA突變株和轉殖株兩者之間相互呼應，可推論出OsGA2ox4、OsGA2ox7、OsGA2ox8具有GA 2-oxidase的功能。同時歸納過去水稻所有GA2oxs家族成員對於植株的生長發育以及代謝過程影響之資料，比較各個GA 2-oxidase酵素對於GAs代謝能力之強弱，藉此評估水稻GA 2-oxidases家族的基因成員對於水稻育種改良上之應用價值。

Abstract
GA 2-oxidases (GA2oxs) is a class of 2-oxoglutarate-dependent dioxygenases (2-ODDs), could deactivate bioactive and precursor form of Gibberellins (GAs) by 2β-hydroxylation. According to previous studies, 10 putative GA 2-oxidases are identified in rice genome; however, OsGA2ox4, OsGA2ox7 and GA2ox8 have not studied yet. In order to confirm whether the OsGA2ox4, OsGA2ox7 and GA2ox8 protein possessed the enzyme function of GA 2-oxidase, two approaches were used in this study. One approach was overexpressing the OsGA2ox4, OsGA2ox7 and GA2ox8 genes ecotopically in TNG67. The second approach was utilizing the T-DNA mutant OsGA2ox4ACT (M97597), OsGA2ox7ACT (M85372) and OsGA2ox8ACT (M61685) with the expectation of the target gene actived.
The phenotype of GA deficiency in rice is dwarfism. Therefore, the plant height of transgenic plants differed to TNG67 suggest that whether OsGA2ox4, OsGA2ox7 and GA2ox8 protein possessed the enzyme function of GA 2-oxidase. The target gene were overexpressed in transgenic construct ubi:GA2ox4 and ubi:GA2ox7 but not in ubi:GA2ox8. The averge plant height of ubi:GA2ox4 was 5.4 cm, the plant height of ubi:GA2ox7 was 66 cm. The expression level of OsGA2ox4 was clearly associated to its plant height in ubi:OsGA2ox4. As for the ubi:OsGA2ox8 transgenic plant, the OsGA2ox8 gene was not overexpressed by ubiquitin promoter and the plant height was similar to TNG67. Therefore, the activity of OsGA2ox8 could not be identified in this approach.
In the second approach, among three T-DNA mutants, OsGA2ox4Act (M97597) and OsGA2ox7Act (M85372) T-DNA mutant showed the same plant height as that of TNG67, but OsGA2ox8Act (M61685) showed semi-dwarf indicated OsGA2ox8 possessed the function of GA 2-oxidase.
Taking together the result from transgenic and T-DNA mutant approach, I conclude that OsGA2ox4, OsGA2ox7 and OsGA2ox8 possessed enzyme function of GA 2-oxidase.

 

目錄
摘要 i
Abstract ii
縮寫字對照表 x
前言 1
前人研究 3
水稻基因功能之探討 3
植物賀爾蒙吉貝素Gibberellin (GA) 之生合成及代謝路徑 4
Gibberellin (GA) 訊息傳遞 4
GA與其他植物荷爾蒙之相關研究 5
GA2-oxidases (GA2oxs) 之相關研究 6
材料與方法 9
一、儀器與設備 9
二、藥品 9
三、水稻轉殖株之構築與分析 9
(一) 植株表現載體之構築 9
(二) 勝任細胞之製備 9
(三) 質體DNA之萃取 10
(四) 黏合作用 10
(五) 質體電穿孔轉型作用 10
(六) 菌落聚合酶連鎖反應 11
(七) 轉殖載體之酶切確認 11
(八) 植物表現載體之水稻轉殖與再生 11
(九) 水稻基因組序列DNA之萃取 12
(十) 轉殖株之聚合酶鏈反應 12
四、轉殖株基因偵測表現 13
(一) 水稻轉殖株RNA之抽取 13
(二) 逆轉錄聚合酶鏈反應 13
(三) 基因表現量偵測之聚合酶鏈反應 13
五、T-DNA插入突變株處理 14
六、T-DNA插入位之確認及突變株基因型之分析 14
(一) 利用生物資訊進行插入點兩側序列之定位及插入點確認 14
(二) T-DNA突變株之DNA萃取 15
(三) T-DNA突變株之聚合酶鏈反應 15
(四) T-DNA突變株之基因偵測表現：RNA萃取 15
(五) T-DNA突變株之基因偵測表現：RT-PCR 15
七、蛋白抗體製備 15
(一) 蛋白表現載體之構築 15
(二) OsGA2ox4、OsGA2ox7 及OsGA2ox8重組蛋白之小量蛋白誘導 15
(三) OsGA2ox4、OsGA2ox7 及OsGA2ox8重組蛋白之大量蛋白誘導 16
(四) OsGA2ox7可溶性蛋白之回收 16
(五) OsGA2ox7不可溶性蛋白之回收 16
八、轉殖基因蛋白表現分析 17
(一) 萃取水稻葉片之目標蛋白 17
(二) 蛋白質定量 17
(三) 蛋白質膠體電泳分析 17
(四) 西方轉漬法 18
九、水稻植株體內的Gibberellin恆定試驗 18
結果 20
一、OsGA2ox3之功能探討分析 20
(一) OsGA2ox3之基因序列分析 20
(二) OsGA2ox3之選殖、載體構築與轉殖再生 20
(三) Ubi:OsGA2ox3 T0轉殖株之確認分析 21
(四) Ubi:OsGA2ox3 T0轉殖株之農藝性狀觀察 21
二、OsGA2ox4之功能探討分析 22
(一) OsGA2ox4之基因序列分析及選殖 22
(二) OsGA2ox4之選殖、載體構築與轉殖再生 22
(三) Ubi:OsGA2ox4 T0轉殖株之確認及分析 22
(四) GA2ox4ACT (M97597) T-DNA突變株插入點之確認及分析 23
(五) Ubi:OsGA2ox4 T0轉殖株與GA2ox4ACT (M97597) 水稻T-DNA突變株 農藝性狀觀察 23
三、OsGA2ox7之功能探討分析 24
(一) OsGA2ox7之基因序列分析及選殖 24
(二) OsGA2ox7之選殖、載體構築與轉殖再生 24
(三) Ubi:OsGA2ox7 T0轉殖株之確認及分析 24
(四) GA2ox7ACT (M85372) 水稻T-DNA突變株插入點之確認及分析 25
(五) Ubi:OsGA2ox7 T0水稻轉殖株與M85372水稻T-DNA突變株農藝性狀觀察 25
四、OsGA2ox8之功能探討分析 26
(一) OsGA2ox8之基因序列分析及選殖 26
(二) 水稻OsGA2ox8之選殖載體構築與轉殖再生 26
(三) Ubi:OsGA2ox8 T0水稻轉殖株之確認及分析 26
(四) GA2ox8ACT (M61685) 水稻T-DNA突變株插入點之確認及分析 27
(五) Ubi:OsGA2ox8 T0水稻轉殖株與GA2ox8ACT (M61685) 水稻T-DNA突變株農藝性狀觀察 27
五、Ubi:OsGA2ox3、Ubi:OsGA2ox4、Ubi:OsGA2ox7及Ubi:OsGA2ox8轉殖株之分析 28
六、OsGA2ox4、OsGA2ox7及OsGA2ox8蛋白表現載體之分析 28
討論 30
一、OsGA2ox3、OsGA2ox4、OsGA2ox7、OsGA2ox8基因功能之探討 30
OsGA2ox3基因功能之探討 30
OsGA2ox4基因功能之探討 31
OsGA2ox7基因功能之探討 32
OsGA2ox8基因功能之探討 33
二、水稻GA 2-oxidase家族：OsGA2ox1-OsGA2ox9之差異與特異性 34
三、OsGA2ox4、OsGA2ox7及OsGA2ox8蛋白分析之探討 36
結論 37
參考文獻 38


表目次
表一、OsGA2ox3、OsGA2ox4、OsGA2ox7和OsGA2ox8基因之詳細資訊……..45
表二、Ubi:GA2ox3、Ubi:GA2ox4、Ubi:GA2ox7、Ubi:GA2ox8之T0 轉殖株獨立株系數目及植株總數量 46
表三、轉殖株及T-DNA突變株之農藝性狀分析 47
(A) Ubi:GA2ox3、 Ubi:GA2ox4 、Ubi:GA2ox7、 Ubi:GA2ox8轉殖株之株高及分蘗數 47
(B) GA2ox4ACT (M97597)、GA2ox7ACT (M85372)、GA2ox8ACT (M61685) T-DNA突變株之株高及分蘗數 47

圖目次
圖一、OsGA2ox3/UMP/pCAMBIA-1301之構築 49
圖二、Ubi:GA2ox3 T0 水稻轉殖株之確認 50
圖三、Ubi:GA2ox3 T0 水稻轉殖株之GA2ox3基因表現分析 51
圖四、OsGA2ox4/UMP/pCAMBIA-1301之構築 53
圖五、Ubi:GA2ox4 T0 水稻轉殖株之確認 54
圖六、Ubi:GA2ox4 T0 水稻轉殖株之GA2ox4基因表現分析 55
圖七、T-DNA 突變株GA2ox4ACT (M97597) 之插入點訊息 56
圖八、OsGA2ox4基因在T-DNA突變株GA2ox4ACT (M97597) 之基因分型分析 57
圖九、OsGA2ox4基因在T-DNA突變株GA2ox4ACT (M97597) 之表現分析 58
圖十、OsGA2ox7/UMP/pCAMBIA-1301之構築 60
圖十一、Ubi:GA2ox7 T0 水稻轉殖株之確認 61
圖十二、Ubi:GA2ox3 T0 水稻轉殖株之GA2ox3基因表現分析 62
圖十三、T-DNA 突變株GA2ox7ACT (M85372) 之插入點訊息 63
圖十四、OsGA2ox7基因在T-DNA突變株GA2ox7ACT (M85372) 之基因型分析 64
圖十五、OsGA2ox7基因在T-DNA突變株M85372之表現分析 65
圖十六、OsGA2ox8/UMP/pCAMBIA-1301之構築 67
圖十七、Ubi:GA2ox8 T0 水稻轉殖株之確認 68
圖十八、Ubi:GA2ox8 T0 水稻轉殖株之GA2ox3基因表現分析 69
圖十九、T-DNA 突變株M61685之插入點訊息 70
圖二十、OsGA2ox8基因在GA2ox8ACT (M61685) T-DNA突變株之基因分型之分析 71
圖二十一、OsGA2ox8基因在GA2ox8ACT (M61685) T-DNA突變株之表現分析 72
圖二十二、Ubi:GA2ox3、Ubi:GA2ox4、Ubi:GA2ox7及GA2ox8轉殖株其GA2-oxidase基因表現分析 73
圖二十三、蛋白表現載體OsGA2ox4/pET-30a(+)、OsGA2ox7/pET-30a(+)、
OsGA2ox8/pET-30a(+)之構築 74
圖二十四、蛋白表現載體OsGA2ox4/pET-30a(+)/Rosetta、OsGA2ox7/pET-30a(+)/ Rosetta及OsGA2ox8/pET-30a(+)/Rosetta之小量蛋白誘導 75
圖二十五、OsGA2ox7/pET-30a(+)/Rosetta之大量蛋白誘導及純化 76
圖二十六、蛋白表現載體OsGA2ox7/pET-30a(+)/Rosetta 之抗體偵測 77
圖二十七、Ubi:GA2ox7之T0轉殖株與TNG67之OsGA2ox7蛋白含量之比較 78
圖二十八、10 DAI之TNG67水稻分別生長於10 μM GA及1 μM PAC，其合成或代謝活性GA之基因分析 79
圖二十九、GA2ox8ACT (M61685) T-DNA突變株之葉片捲曲示意圖 80
圖三十、Ubi:GA2ox3、Ubi:GA2ox4、Ubi:GA2ox7、Ubi:GA2ox8轉殖株及GA2ox4ACT
(M97597)、GA2ox7ACT (M85372)、GA2ox8ACT (M61685) 之株高比較圖 81

 
附錄目次
附表 82
附錄一、Gibberellin (GA) 生合成及代謝之路徑 85
附錄二、水稻不同時期之GA代謝酵素GA2-oxidase的基因表現分析 86
附錄三、T-DNA突變株外表性狀圖及T-DNA插入點示意圖。 87
附錄四、不同GA 2-oxidase之水稻轉殖株 88
附錄五、蛋白表現載體OsGA2ox2/pET-30a(+)/Rosetta、OsGA2ox5/pGEX-5X/BL21與OsGA2ox6/pGEX-5X/BL21 89

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