臺灣博碩士論文加值系統

先前實驗室偶然發現一株水稻 T-DNA 轉殖株，其穀粒的長和寬相較於野生株 (TNG67) 明顯增加，分別達 19% 與 15% ，將之命名為 Large Spikelet 1 (LS1)。雖然 LS1高度不稔，結實率僅有 3~6%，但其自交子代 LS1-3、LS1-4、LS1-5所結的少數榖粒的重量仍顯著高於 TNG67。我們希望探討 LS1 性狀的分子機制，故利用反向聚合酶鏈反應 (inverse PCR) 尋找 T-DNA 的插入位置，發現其位於Hemolysin III (HlyIII) 和Sucrose Phosphate Synthase (SPS) 兩個基因之間，我們使用 genomic PCR鑑定LS1和其子代之分離率，從11株LS1其子代鑑定結果中，未得到無T-DNA之分離株，因此無法確認T-DNA與大穀粒的關係。由於 T-DNA 位於SPS基因的下游，SPS蛋白為植物蔗糖合成的關鍵酵素，會影響光合產物澱粉的轉運與累積，然而初步 Real-time PCR 檢查發現 T-DNA 插入並未影響 SPS 基因的表現量，因此排除其相關性。HylIII 蛋白則為一個具有七次跨膜區的膜蛋白，屬於 Progestin and AdipoQ Receptors (PAQR) 蛋白質家族的一員。研究指出阿拉伯芥的HlyIII會受到鹽和滲透逆境誘導影響表現量，HlyIII 突變株對於滲透逆境和 ABA 有高度敏感性，推測 HlyIII 在逆境訊號中扮演負調控的功能，然而在水稻中尚無研究報導。水稻中共有六個 PAQR 成員蛋白，但 HlyIII與其他成員蛋白相似度均小於 58%，推測可能具有獨特之功能。查詢 TIGR 的資料庫得知 HlyIII基因雖普遍表現於水稻各組織器官，但在25天的胚乳中含量最豐富，而初步進行 RT-PCR 分析，發現 HlyIII基因在 LS1 和其自交子代的轉錄量均較 TNG67 為高，我們懷疑HlyIII的表現量可能造成LS1的外表型。利用構築 HlyIII基因靜默轉殖水稻 (pHL2)、 HlyIII基因大量表現轉殖水稻 (pHL4) 及阿拉伯芥HlyIII 基因剔除株來探討此基因之功能，結果發現 HlyIII基因表現與LS1的外表型都沒有相關性。由於前人研究發現，HlyIII在酵母菌Saccharomyces cerevisiae磷代謝中扮演重要的角色，因此我們比較LS1和TNG67的磷含量，結果發現不論於葉片、葉鞘或穀粒樣品中，LS1之磷含量均顯著高於TNG67，但是在轉殖水稻中，並沒有發現HlyIII基因與磷含量的相關性。由實驗結果得知，大穀粒的特徵與HlyIII基因並沒有關係，LS1可能是由轉殖水稻的過程中經常發生的本體突變(somaclonal mutation)所導致。

A T-DNA mutant, named as Large Spikelet (LS1), was noticed previously due to its increased length, by 19%, and width, by 15%, in grains, compared to the wild type rice (TNG67). The selfed progenies LS1-3, LS1-4 and LS1-5 still produced heavier grains than TNG67, though the fertility rate of LS1 was severely decreased to 3~6%. In order to investigate the molecular mechanism of LS1, inverse PCR was performed and the result indicates an integration site of T-DNA located between the Hemolysin-III (HlyIII) and Sucrose Phosphate Synthase (SPS) genes. Genomic PCR on LS1 and its progenies further confirmed the result. Unfortunately, no segregation line without T-DNA insertion was identified among the tested progenies; the genetic linkage between the T-DNA insertion and the Large Spikelet phenotype was therefore un-confirmed. SPS encodes a key enzyme involved in starch and sucrose metabolism. As T-DNA is located downstream of the SPS gene, the expressions of SPS was unchanged in LS1 according to Real-time PCR quantitation. This result rules-out the possible role of SPS in LS1 phenotype. HlyIII encodes an integral membrane receptors, belonging to the Progestin and AdipoQ Receptors (PAQR) family, with seven transmembrane domains. Disruption of HlyIII in Arabidopsis renders plants with hypersensitivity to ABA/osmotic stress; therefore, HlyIII may play a negative role in ABA/osmotic signaling. Rice has six PAQR members. As HlyIII share at most 58% similarity in protein sequence to the other members, HlyIII may play a different function from its kin in rice. According to transcriptome information from the TIGR databank, HlyIII was highly expressed in the endosperm 25 days after pollination. Moreover, expression of HlyIII was higher in LS1 and its selfed progenies than in the TNG67, suggesting an effect of T-DNA insertion. We suspected that over expressions of HlyIII may be responsible the LS1 phenotype. However, further examinations using the HlyIII-overexpressed and HlyIII-silenced transgenic rice, together with the HlyIII T-DNA-tagged Arabidopsis, did not correlate the LS1 phenotype with the HlyIII gene expression or gene integrity. HlyIII was also known to play an important role in phosphate metabolism in Saccharomyces cerevisiae; therefore, we compared the phosphate content between LS1 and TNG67 plants. Surprisingly, both LS1 and its selfed progenies exhibited higher phosphate content than TNG67, especially in the leaf sheath. Unfortunately, the correlation between the HlyIII gene and phosphate content was not found in the HlyIII transgenic plants. In conclusion, the Large Spikelet phenotype is not related to the HlyIII gene but may be caused by somatic mutations that happened accidently during the previous transformation process of TNG67.

壹、 前言 1
貳、 前人研究 2
一、 水稻穀粒影響產量之因素 2
二、 有芒水稻之簡介 2
三、 大榖粒水稻與其調控之基因的簡介 2
四、 Hemolysin III蛋白之簡介 3
五、 Hemolysin III於酵母菌之功能 3
六、 Hemolysin III於阿拉伯芥扮演之角色 3
參、 材料與方法 5
一、 實驗材料-水稻 5
(一) 台農67號 (Oryza sativa L. ssp. Japonica, cv. Tainung 67; TNG67) 5
(二) 大穀粒1 (LS1) 5
二、 實驗材料-阿拉伯芥 5
(一) 阿拉伯芥 (Arabidopsis thaliana) Columbia 生態型 5
(二) 阿拉伯芥HlyIII 基因T-DNA 插入株 5
三、 聚合酶鏈鎖反應 6
(一) 反向聚合酶鏈鎖反應 (Inverse PCR) 6
(二) 反轉錄聚合酶鏈鎖反應 (Reverse Transcription PCR) 6
(三) 即時定量聚合酶鏈鎖反應 (Real-time PCR) 7
四、 水稻轉殖與轉殖株之篩選 7
五、 萃取水稻葉子之genomic DNA 8
六、 萃取水稻葉子之RNA 9
七、 萃取水稻授粉後15天之穀粒之RNA 9
八、 水稻之性狀觀察 10
(一) 穀粒長度與寬度計算 10
(二) 穀粒千粒重量計算 10
(三) 稔實率計算 10
(四) 花粉形態染色觀察 10
(五) 花粉活性染色觀察 10
(六) 花粉大小測量 11
九、 水稻T-DNA遺傳率 11
十、 水稻葉片磷含量檢測 11
十一、 阿拉伯芥之栽種 12
十二、 阿拉伯芥T-DNA插入位置 13
十三、 阿拉伯芥磷含量檢測 13
十四、 阿拉伯芥性狀分析 13
(一)種子長度與寬度測量 13
(二)果莢長度測量 13
肆、 結果 14
一、 LS1 性狀之分析 14
(一) 榖粒外表型 14
(二) 花粉觀察 14
(三) 稔實率 14
(四) 千粒重 15
(五) 確認LS1為TNG67 15
(六) LS1之T-DNA 插入數 15
二、 選殖 LS1 T-DNA 插入之染色體區塊 15
(一) Inverse PCR 結果 15
(二) 確認 T-DNA 插入位置無誤 15
(三) 檢測 LS1 與其自交子代之基因型 16
三、 LS1 T-DNA 插入位置附近之基因表現量分析 16
(一) SPS基因 16
(二) HlyIII 基因 17
(三) 比較水稻各組織 17
四、 HlyIII 基因靜默或大量表現水稻轉殖株之分析 17
(一) HlyIII 基因表現量 17
1. HlyIII 基因靜默之水稻轉殖株 (pHL2) 17
2. HlyIII 基因大量表現之水稻轉殖株 (pHL4) 18
(二) HlyIII 水稻轉殖株性狀 (穀粒大小、稔實率、千粒重) 18
1. HlyIII 基因靜默之水稻轉殖株 (pHL2) 18
2. HlyIII 基因大量表現之水稻轉殖株 (pHL4) 18
五、 阿拉伯芥 HlyIII 基因剔除株之分析 19
(一) 確認 T-DNA 插入之位置 19
(二) HlyIII 基因剔除株之外表型分析 19
(三) HlyIII 基因剔除株子代植株之基因型與外表型相關性分析 19
1. 基因型檢測 19
2. 植株種子大小 19
六、 探討 HlyIII 基因與植株磷含量之相關性 20
(一) 水稻 LS1 與其子代之磷含量分析 20
1. 葉片、葉鞘、花穗 20
(二) HlyIII 基因靜默或大量表現水稻轉殖株之磷含量分析 20
1. HlyIII 基因靜默之水稻轉殖株 (pHL2) 20
2. HlyIII 基因大量表現之水稻轉殖株 (pHL4) 20
(三) 阿拉伯芥 HlyIII 基因剔除株 (SALK_056174) 幼苗之磷含量分析 20
1. 生長於無養分培養基 (water agar) 20
2. 生長於1/2 MS培養基 20
伍、討論 22
一、 LS1其子代之基因型鑑定 22
二、 LS1與脂質代謝和磷含量相關性 22
三、 LS1與轉殖水稻的phenotype探討 22
陸、參考文獻 24

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