臺灣博碩士論文加值系統

已知開花對於水稻來說是決定能否獲得種子並將其性狀遺傳至子代的關鍵。在先前的文獻中指出分別大量表現OsMADS14、OsMADS15及OsMADS18基因會提早水稻的開花時間，說明此3個基因在開花路徑中可能扮演極重要的角色。根據實驗室前人搜尋T-DNA activation tagging突變株M52048與以玉米Ubiquitin啟動子大量表現OsMADS14基因之轉殖株皆具有矮株及早開花之外表性狀，且開花相關基因Hd3a、RFT1與OsMADS15表現量也有增加的現象，說明OsMADS14基因確實影響水稻開花時間。然而這3個基因對開花調控的影響並未於相同的水稻遺傳背景中進行比較，故本篇實驗利用水稻品系TNG67為背景，透過轉基因方式大量表現OsMADS14、OsMADS15及OsMADS18之方式，研究三者對開花調控的影響是否有差異。
本研究結果顯示在TNG67遺傳背景中，大量表現OsMADS14或OsMADS15轉殖株，都具有早開花及矮株的外表性狀，且其開花相關基因RFT1、Hd3a皆有活化的情形。但在Ubi:OsMADS18轉殖株中則無早開花及矮株的性狀，反而抽穗期有延遲的現象。此外，在部分的Ubi:OsMADS15轉殖株中發現若Hd3a、RFT1與OsMADS14表現無活化上升，則抽穗期會有延遲的情況。推測同時活化Hd3a、RFT1、OsMADS14及OsMADS15基因於開花調控路徑是必須的，但OsMADS14及OsMADS15調控上游開花素Hd3a及RFT1之路徑還需要進一步確認。
另一方面，本研究建立CRISPR/Cas9的轉殖株篩選平臺。透過CRISPR/Cas9技術各別破壞OsMADS14/15/18基因，觀察上述基因功能遭破壞後，對於開花時間是否有延遲的現象。從結果顯示單一破壞OsMADS14及OsMADS15基因對於水稻植株的開花時間比起野生型並無明顯的影響，然而破壞OsMADS18基因在T1植株中觀察到其開花期有提早的現象，表示OsMADS18基因對於水稻的開花路徑扮演抑制者的角色。但以上的數據皆為T0轉殖株及T1基因編輯植株的結果，因樣品數少且外表性狀還未穩定，所以還需要T2子代的種植分析，才可以確定破壞這3個基因對開花的影響。
此外，根據阿拉伯芥的開花路徑了解TEM1為開花抑制子，故推測pOsTEM1基因可能是水稻的開花抑制子。為了確認其功能，本研究利用活化pOsTEM1基因M89461突變株與Ubi:pOsTEM1轉殖株進行探討。初步觀察到活化pOsTEM1基因表現，水稻植株會有晚開花的情形，但是否確實為開花抑制子還需要後續的研究。

Flowering is an important developmental process for seed producing in plants. Previous studies had showed that respective over-expressing OsMADS14, OsMADS15 and OsMADS18 in rice would cause early flowering phenotype, implied that the three genes might play vital roles in regulating flowering process of rice. Moreover, the accumulation of OsMADS14 in both of T-DNA activation tagging mutant M52048 and Ubi:OsMADS14 transgenic rice result in dwarf and early flowering phenotype, which accompanied with activation of Hd3a, RFT1 and OsMADS15, raising the possibility that these genes might cooperate with OsMADS14 in regulating flowering. Nevertheless, the ability of the three genes to promote flowering have not investigated and compared in the same genetic background yet, to elucidate among the three genes, which gene is mainly involved in regulating flowering process, the transgenic approach and CRISPR/Cas9 knockout approach for OsMADS14, OsMADS15 and OsMADS18 were conducted in cultivar TNG67.
In the present study, most of the Ubi:OsMADS15 transgenic rice displayed dwarf and early flowering phenotypes, however, instead of promoting flowering, the accumulation of OsMADS18 in Ubi:OsMADS18 result in delayed flowering and had no obvious effect on plant height growth, indicated that OsMADS18 might not have ability to promote flowering in rice. Besides, according to the expression comparison of Hd3a, RFT1 and OsMADS14, OsMADS15 and OsMADS18 among the Ubi:OsMADS15 transgenic rice, we found that early flowering phenotype was not observed from the transgenic rice which had no activated Hd3a, RFT1 and OsMADS14, suggested that overexpression of OsMADS15 along is not enough to promote flowering. On top of that, the flowering time of Ubi:OsMADS15 might promote by coexistence of Hd3a, RFT1, OsMADS14 and OsMADS15, and how of both Hd3a and RFT1 were activated in Ubi:OsMADS14 and Ubi:OsMADS15 transgenic rice still needed to be investigated in future.
In addition, the CRIPSR/Cas9 genome editing system was applied for OsMADS14, OsMADS15 and OsMADS18 gene knockout in this study. In the flowering time comparison among these osmads14, osmads15 and osmads18 gene knockout mutants, our preliminary data showed that both of osmads14 and osmads15 had no obvious delay on flowering time. Surprisingly, the flowering time was promoted earlier in T1 progenies of osmads18, which corresponded to the result observed from Ubi:OsMADS18, implied that OsMADS18 might function as a negative regulator rather than positive regulator in regulating rice flowering. Since all of these data were observed and collected from either T0 or T1 progenies of osmads14, osmads15 and osmads18 knockout mutants, the functional inference of OsMADS14, OsMADS15 and OsMADS18 in regulating rice flowering still needed to be further confirmed in genetic stable T2 progenies.
In addition, according to the flowering pathway in Arabidopsis, which had showed that TEM1 is a flowering repressor, therefore, we speculated that pOsTEM1 might also play as flowering repressor in rice. To investigate the function of TEM1 homologous gene pOsTEM1, the pOsTEM1 activation mutant M89461 was isolated from TRIM data base, besides, the pOsTEM1 overexpression transgenic rice was also created in current study. Surprisingly, our preliminary data showed that the accumulation of pOsTEM1 in both of T-DNA mutant M89461 and Ubi:pOsTEM1 transgenic rice result in late flowering phenotype, suggested that delayed flowering phenotype might result from accumulation of pOsTEM1. However, whether the pOsTEM1 is a flowering repressor in rice still needed to be further confirmed by further experiment in future.

目錄

摘要 ⅰ
Abstract ⅱ
目錄 ⅳ
表目次 ⅵ
圖目次 ⅵ
附表 ⅶ
附圖 ⅶ
縮寫字對照表 ⅷ
前言 1
前人研究 2
一、T-DNA突變株M52048與Ubi:OsMADS14轉殖株分析 2
二、MADS-box基因家族功能 2
三、OsMADS14/15/18基因功能 3
四、參與水稻開花路徑之基因 3
五、OsMADS14及OsMADS15影響RFT1及Hd3a表現量 4
六、CRISPR系統原理 5
七、CRISPR/Cas9應用 6
材料與方法 7
一、儀器設備 7
二、實驗藥品 7
三、研究材料 7
四、大量表現基因之水稻載體構築 8
五、CRISPR基因編輯之載體構築 11
六、轉殖株基因及外表性狀分析 11
結果 14
一、活化及大量表現AP1/FUL-like基因之轉殖株 14
二、CRISPR編輯AP1/FUL-like基因之水稻植株 17
三、pOsTEM1基因功能探討 22
討論 26
一、AP1/FUL-like基因功能探討 26
二、CRISPR基因編輯植株分析平臺建立 30
三、pOsTEM1基因功能探討 31
結論 33
參考文獻 34
表 38
圖 46
附表 57
附圖 63

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