乳鐵蛋白為哺乳動物乳汁中的一種多功能的醣蛋白，其具有結合鐵離子、抑菌、增進免疫力和消除自由基的能力。本研究以轉入豬乳鐵蛋白基因之水稻為材料進行分析。由於轉入豬乳鐵蛋白基因之轉殖水稻第一代不是同質品系，基因仍會分離；因此，本研究利用PCR和西方墨點法來篩選轉殖水稻，以獲得轉殖水稻同質品系。目前在水稻胚特有基因起動子-721LF轉殖水稻獲得10個轉殖水稻同質品系，而利用CaMV35S起動子之35LF轉殖水稻獲得2 個轉殖水稻同質品系，其中721LF轉殖水稻已栽培至第六代，而35LF轉殖水稻則栽培至第五代，南方墨漬法分析發現這些同質品系的豬乳鐵蛋白基因分別嵌入水稻染色體的不同位置。由西方墨漬法的結果顯示不同起動子之轉殖水稻所表現的豬乳鐵蛋白含量的確有差異，而來自不同世代之相同轉殖水稻所表現的豬乳鐵蛋白含量則沒有差異。由研究結果初步推測在721LF轉殖水稻糙米、米糠中之豬乳鐵蛋白佔總蛋白質之百分比分別為0.04%、0.06%，而在35LF轉殖水稻之糙米、米糠、精白米中之豬乳鐵蛋白佔總蛋白質之百分比分別為0.07%、0.04%、0.06%。利用組織免疫墨漬法(Tissue immunoblots) 來分析在不同起動子所起動的豬乳鐵蛋白在水稻種子組織的位置，結果顯示在705LF、721LF、731LF轉殖水稻主要表現在種子胚和糊粉層之中，而在持續表現起動子轉殖水稻種子中則整粒都有表現。利用Heparin-sepharose純化轉殖水稻同質品系中之豬乳鐵蛋白，結果發現豬乳鐵蛋白會在0.6 M NaCl和0.8 M NaCl淋洗出來，同時亦發現由水稻所表現的豬乳鐵蛋白能和鐵離子結合及抑制大腸桿菌DH5α生長的功能。此外，本研究的結果顯示轉殖水稻的農藝性狀不會因為有外來的基因嵌入而有明顯的改變。

Lactoferrin is a multifunctional glycoprotein, found mostly in milk and involved in iron absorption, antibacterial activity, immune response and free-radical scavenging. The analysis of lactoferrin gene expression in transgenic rice was performed in this study. Because the porcine lactoferrin gene was segregated in the first generation (T1), the homozygous lines were screened with the medium containing hygromycin, and further detected by PCR and Western blot assay. In the present, we obtained ten homozygous lines of Ose721 promoter and two of CaMV35S promoter. The homozugous line of Ose721 promoter was cultivated to the sixth (T6) generation, whereas the homozygous line of CaMV35S promoter was cultivated to fifth (T5) generation. The amount of porcine lactoferrin detected in brown rice and bran of transgenic rice of Ose721 promoter were 0.04% and 0.06% of total proteins, whereas the amount in brown rice, bran and milled rice of transgenic rice of CaMV35S promoter were 0.07%, 0.04% and 0.06% of total proteins respectively. Results of tissue immunoblots showed that porcine lactoferrin gene driven by Ose705, Ose721and Ose731 promoters, porcine lactoferrin was detected in embryo and aleurone cells, whereas the gene driven by actin and CaMV35S promoters, porcine lactoferrin was observed in whole seeds. Porcine lactoferrin purified by heparin-sepharose column from transgenic homozygous line was eluted with 0.6M and 0.8M NaCl, while the iron absorption and the growth inhibition of E. Coli-DH5α was observed in this study. Besides, the alternations of agronomic trait of transgenic rice were not significant due to the insertion of foreign gene.

ABSTRACT 1
中文摘要 2
壹、前言 3
貳、前人研究 5
一、利用植物生產外源蛋白之研究現況 5
1. 起動子之選擇 6
2. 外源蛋白之表現 8
3. 轉殖植物的同質品系篩選 9
二、關於乳鐵蛋白的研究 11
三、轉殖植物表現乳鐵蛋白的研究 13
參、材料與方法 15
(一)材料 15
(二)方法 15
1. 篩選轉殖水稻同質品系 15
2. 少量質體DNA之萃取 16
3. DNA 片段之純化回收 16
4. 水稻total DNA之抽取 18
5. 轉殖植物之確認 18
6. 雜交探針之製備 19
7. 南方墨點分析 19
8. 自動放射顯影 20
9. 利用不同蛋白萃取液分析水稻中的蛋白質 20
10. 蛋白質膠體電泳分析 21
11. 西方墨點分析 21
12. 豬乳鐵蛋白於轉殖水稻中之表現分析 22
13. 轉殖水稻種子中豬乳鐵蛋白的含量 22
14. 豬乳鐵蛋白在轉殖水稻組織中之位置 23
15. 以Heparin-sepharose管柱純化豬乳鐵蛋白 23
16. 抑菌活性分析 25
17. 鐵離子結合分析 25
18. 轉殖水稻的農藝性狀調查 25
肆、結果 26
一、 同質轉殖水稻品系之篩選 26
1. 利用含抗生素的培養基篩選 26
2. 聚合酵素鏈鎖反應分析 27
3. 同質轉殖水稻品系之確認分析:西方墨點法 27
4. 同質轉殖水稻品系之南方墨點分析 27
二、 轉殖水稻生產豬乳鐵蛋白之純化 28
1. 不同蛋白質萃取液之應用 28
2. 利用硫銨沈澱法部分純化豬乳鐵蛋白 29
3. 利用heparin-sepharose 管柱純化豬乳鐵蛋白 29
三、轉殖水稻種子中豬乳鐵蛋白之表現 29
1. 水稻種子之糙米、米糠及精白米之蛋白質種類之分布 30
2. 豬乳鐵蛋白在721LF轉殖水稻種子之含量百分比 30
3. 豬乳鐵蛋白在35LF轉殖水稻種子之含量百分比 30
4. 不同轉殖水稻植株之豬乳鐵蛋白表現 31
5. 轉殖水稻不同世代之豬乳鐵蛋白表現 31
6. 利用組織免疫墨漬法分析豬乳鐵蛋白在轉殖水稻種子中的分布 32
四、 豬乳鐵蛋白之活性分析 32
1. 豬乳鐵蛋白與鐵離子結合分析 32
2. 抑菌活性分析 33
五、轉殖水稻之農藝性狀調查 33
伍、討論 35
一、轉殖水稻同質品系之篩選 35
二、水稻生產豬乳鐵蛋白之純化 36
三、轉殖水稻種子中豬乳鐵蛋白之表現 37
四、豬乳鐵蛋白之活性分析 40
1. 豬乳鐵蛋白與鐵離子結合分析 40
2. 抑菌活性分析 40
五、轉殖水稻之農藝性狀調查 41
陸、參考文獻 42
圖表 49

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