水稻小分子熱休克蛋白質-OsHSP16.9A累積對稻米品質影響之研究

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梁佑德(You-De Liang) 查詢紙本館藏

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生命科學系

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水稻小分子熱休克蛋白質-OsHSP16.9A累積對稻米品質影響之研究
(Study on the effect of accumulation of OsHSP16.9A, a rice small heat shock protein, on the rice quality)

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摘要(中)

實驗室先前研究發現，在DNA微陣列中，過量表現OsHSP16.9A轉殖株中ADP-glucose pyrophosphorylase large subunit 3的表現量亦會明顯上升，已知OsAPL3在水稻中扮演澱粉與糖原合成的限速酶。我們進一步分析臺農67號水稻OsAPL3的表現，結果發現在乳熟期到成熟期的階段，OsHSP16.9A與OsAPL3皆有逐步上升的趨勢。利用比例式雙螢光分子互補系統觀察OsHSP16.9A與OsAPL3之交互作用，結果顯示它們在正常溫度下會有交互作用的形成。為深入探討相關結果，我們繼續建構了抑制表現OsHSP16.9A及過量表現OsAPL3，另亦發現在成熟種子中，WT、OsHSP16.9A-OEs、OsAPL3-OE米粒大小沒有明顯差異，但OsHSP16.9A-OEs種子的蛋白質與澱粉含量較WT高，OsHSP16.9A-RNAi則較OsHSP16.9A-OEs、OsAPL3-OE與WT種子小，澱粉含量亦較三者為低。米粒完整性比較，結果顯示，OsHSP16.9A-OE碎粒比較WT低，表示OsHSP16.9A的累積可以提高米粒的完整性，OsAPL3-OE碎粒比亦較WT低。利用掃描式電子顯微鏡觀察澱粉粒排列，結果顯示OsHSP16.9A-OEs種子，米心排列較鬆散，而米心周圍較WT更為緊密。綜合上述觀察，OsHSP16.9A能保護或幫助OsAPL3來完成其功用，當缺乏OsHSP16.9A時，會造成澱粉粒無法正常累積與排列，所以從結果得知OsHSP16.9A對米質有一定的影響力。

摘要(英)

Small molecular weight heat shock proteins (sHSP) are commonly found in eukaryotes, where they bind to denatured proteins to prevent their accumulation and further cellular damage. Previous laboratory studies revealed that the expression of ADP-glucose pyrophosphorylase large subunit 3 (OsAPL3) was significantly increased in the DNA microarray of the over-expressed OsHSP16.9A transgenic strain (OsHSP16.9A-OE), which is known to play a role in rice as a rate-limiting enzyme for amylin and glycogen synthesis. We further analyzed the performance of OsAPL3 in TNG67 rice (Wild type; WT) and found that both OsHSP16.9A and OsAPL3 showed a gradual increase during the milky to mature stage. The results showed that both OsHSP16.9A and OsAPL3 expression level showed a gradual increasing trend from milking to maturity. The interaction between OsHSP16.9A and OsAPL3 was observed using a ratiometric bimolecular fluorescence complementation (rBiFC) system, and the results showed that they interacted to form at normal temperature. To further investigate the related results, we continued to construct the suppression expression of OsHSP16.9A (OsHSP16.9A-RNAi) and over-expression of OsAPL3 (OsAPL3-OE), and also found that in mature seeds, there was no significant difference between WT, OsHSP16.9A-OEs, and OsAPL3-OEs rice grain size, but the OsHSP16.9 A-OEs had higher protein and starch content than WT, while OsHSP16.9A-RNAi was smaller than OsHSP16.9A-OEs, OsAPL3-OEs and WT, and had lower starch content than all three. The results showed that OsHSP16.9A-OE had a lower grain fragmentation ratio than WT, indicating that the accumulation of OsHSP16.9A could improve the integrity of rice grains, and OsAPL3-OEs had a lower grain fragmentation ratio than WT. Scanning Electron Microscope (SEM) was used to observe the starch grain arrangement, and the results showed that the OsHSP16.9A-OEs seeds had a looser arrangement of rice cores and a tighter arrangement around the rice cores than WT. In summary, OsHSP16.9A can protect or help OsAPL3 to fulfill its function. When OsHSP16.9A is lacking, the starch grains cannot accumulate and arrange properly, so it is known from the results that OsHSP16.9A has some influence on rice quality.

關鍵字(中)

★ 小分子熱休克蛋白

關鍵字(英)

★ small heat shock protein

論文目次

摘要 i
Abstract iii
誌謝 iv
目錄 v
圖目錄 vi
附錄目錄 vii
縮寫對照表 viii
序論 1
研究起源與目的 6
材料與方法 9
結果 46
1. 水稻充填時期OsHSP16.9A與OsAPL3之mRNA表現情形 46
2. OsAPL3-OEs篩選與鑑定 46
3. 轉殖株種子的OsHSP16.9A與OsAPL3之mRNA表現情形 47
4. OsHSP16.9A與OsAPL3之間的蛋白質交互作用分析 47
5. 植株性狀表現 48
6. 水稻完整性表現情形 48
7. 水稻白堊質表現情形 49
8. 成熟種子之大小與重量表現情形 49
9. 水稻蛋白質組成份之分析 49
10. 水稻澱粉組成份之分析 50
11. 水稻直鏈澱粉佔比分析 50
12. 澱粉粒排列之情形 50
13. 澱粉合成相關基因之分析 51
討論 52
參考文獻 57
圖表 62
附錄 77

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指導教授

葉靖輝(Ching-Hui Yeh)

審核日期

2022-9-22

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