臺灣博碩士論文加值系統

氧氣是植物生長的必須條件，本研究是以蛋白質的觀點探討氧氣對稻米芽鞘的影響。藉由控制氧氣的含量，讓稻米在不長根的情況下恆溫培養。等到芽鞘長到約2、3公分，將一組芽鞘通入定量空氣培養；另一組則是靜置培養，當作對照組。這樣的條件下，一直到通氣12小時都不會有長根的情形。將通氣時間不同(1、2、4、6、12和24小時)的稻米芽鞘收集起來，然後萃取芽鞘內的蛋白質。利用二維膠體電泳來研究實驗組和對照組之蛋白質，發現有新的蛋白質點出現在實驗組的二維電泳膠片中，這新的蛋白質點一為分子量約94kD在pI約為10之處，另一為分子量約14kD在pI約為3之處。LC/MS將進一步被利用來鑑定這新蛋白質點。離子交換管柱和膠體層析管柱也將被用來純化此蛋白質，再進一步研究該蛋白質在生理方面所扮演的角色(biological roles)。本研究藉由蛋白質的觀點，顯示通氣對於稻米芽鞘生長的影響。

Oxygen is requisite for plants to grow. Investigation of the effect of oxygen content on plant is then interesting. This study is to explore the effect of oxygen on the rice coleoptile in view of protein express. The rice coleoptiles were incubated in an unrooting condition by controlling the oxygen content. When the coleoptiles grew to about 2 to 3 cm, the experimental group was continued incubating with the bubbling of air, while the control group was without air bubbling. Under this condition, no roots were found for the experimental rice within 12 hours air bubbling. The coleoptiles obtained from experimental group bubbled with air for various times (1, 2, 4, 8, 12, and 24 hrs) and control group were collected and the coleoptile proteins were extracted. 2-D gel electrophoresis was employed to study the total protein differences between experimental and control coleoptile. Two additional protein spots, one with molecular weight about 94kD located at pI around 10; one with molecular weight about 14kD located at pI around 3, are found in experimental coleoptiles. These additional proteins will be further identified by LC/MS spectroscopic technique. Ion exchange and gel filtration column chromatograph also were employed to purify these proteins. It is interesting and important to further study the possible biological role for these proteins. This study has shown that air content has effects on the growth of coleoptile in terms of protein view.

目錄
中文摘要……………………………………………………………….…i
英文摘要…………………………………………………………….…...ii
誌謝……………………………………………………………………...iii
表目錄………………………………………………………………….viii
圖目錄…………………………………………………………………...ix


第一章 緒論 1
一、 稻米的基本性質…………………………………….…………….3
1. 種子………………………………………………………………3
2. 種子的萌發………………………………………………………6
3. 種子萌發的條件…………………………………………………6
4. 種子萌發的吸水過程……………………………………………7
5. 種子萌發的呼吸作用……………………………………………9
6. 萌發中的呼吸四個時期 ………………………………………10
7. 呼吸基質的供應………………………………………………..12
8. 避免沒入水面的特性…………………………………………..13
9. 通氣過程………………………………………………………. 15
二、 稻米的代謝作用…………………………………………………16
1. 種子萌發過程的有機物轉變…………………………………..16
2. 澱粉代謝的基本途徑…………………………………………..17
3. 能量之代謝……………………………………………………..18
4. ATP與化學能之轉變…………………………………………..20
5. 植物細胞如何製造ATP………………………………………..21
三、 細胞生長及細胞分化……………………………………………23
1. 生長、分化與發育之義………………………………………23
2. 細胞生長………………………………………………………25
3. 調控生長的蛋白質……………………………………………28
4. 植物細胞壁的特性……………………………………………29
5. 細胞分化……………………………………………………..35
四、 研究動機………………………………………………………...37

第二章 實驗 38
一、 儀器…………………………………………….……………...…38
二、 藥品………………………………………………………………40
三、 實驗步驟…………………………………………………………42
1. 水稻品種………………………………………………………...42
2. 水稻培養方式…………………………………………………...42
3. 氧氣調控培養.…………………………………………………..43
4. 芽鞘蛋白質的萃取……………………………………………...43
5. 蛋白質樣品的前處理…………………………………………...44
6. 稻米芽鞘蛋白質的電泳分析…………………………………...45
7. 管柱層析法分離稻米芽鞘蛋白質……………………………...46
8. 銀染法.…………………………………………………………..47

第三章 結果與討論 49
一、稻米的栽種條件………...…………………………………………49
二、透析條件的改變……………………….…………………………..54
三、TCA沈澱的收集結果………………….…………………….……55
四、電泳圖的分析結果…………….………………………..…………58
五、管柱層析圖的結果………………….……………………..……....64
1. 離子交換樹脂…………………………………………………...64
2. 膠體層析管柱…………………………………………………...64
六、結論……………………………….…………………….……..…..68

第四章 參考文獻 69


表目錄
表一、 在黑暗中萌發水稻種子化學組成變化………………………16
表二、 在充滿空氣的水中、持續通氣的溶液中，以及靜置的溶液
中，Calrose、KR 90，以及KR 56芽鞘生長的速度
比較表…………………………………………………………31
表三、 稻米芽鞘分別在靜置溶液以及通氣溶液下，生長速度和
乾重的比較圖………………………………………………...31


圖目錄
圖一、種子吸水的三個階段模式圖……………………………………8
圖二、在暗處萌發中的豌豆整粒種子及去除種皮的子葉的
呼吸進程………………………………………………………..11
圖三、稻米部分沒入水面的生長圖…………………………..………..15
圖四、澱粉降解的途徑……………………………………..…………..18
圖五、生物合成的主要步驟………………………………..…………..19
圖六、ATP與能量形式之轉換……………………………..…………..20
圖七、粒腺體ATP之合成…………………………………..………….22
圖八、細胞壁的模型圖………………………………………..………..27
圖九、稻米芽鞘長度在動力學上的改變圖……………….…………..33
圖十、稻米芽鞘的細胞壁延展性比較圖……………………….……..33
圖十一、稻米芽鞘中，每一個樹膠醛醣殘基所含的阿魏酸和
雙-阿魏酸的含量…………………………………………….34
圖十二、稻米芽鞘在好氧和厭氧條件生長的速度比較圖…………...51
圖十三、(a)圖是兩公分沒通氣的芽鞘，當作對照組；
(b)圖是兩公分，持續通氣24小時的芽鞘，
當作實驗組…………………………………………………..52
圖十四、(a)圖是2公分，持續通氣24小時的芽鞘蛋白質；
(b)圖是3公分，持續通氣24小時的芽鞘蛋白質…………53
圖十五、(a)圖是靜置培養的芽鞘蛋白質；
(b)圖是3公分，持續通氣24小時的芽鞘蛋白質…………56
圖十六、(a)圖是持續通氣6小時的芽鞘蛋白質；
(b)圖是持續通氣12小時的芽鞘蛋白質….………………...57
圖十七、持續通氣1小時的芽鞘蛋白質……………………………....60
圖十八、持續通氣2小時的芽鞘蛋白質………………….…………..60
圖十九、持續通氣4小時的芽鞘蛋白質………………………………61
圖二十、持續通氣6小時的芽鞘蛋白質…………………….………..61
圖二十一、持續通氣12小時的芽鞘蛋白質………………………….62
圖二十二、持續通氣24小時的芽鞘蛋白質………………………….62
圖二十三、通氣1~24小時的芽鞘蛋白質的一維電泳圖……………63
圖二十四、靜置培養的芽鞘蛋白質…………………………………..63
圖二十五、DEAE陰離子交換樹脂層析圖…………………………..65
圖二十六、DEAE陰離子交換樹脂層析圖…………………………..66
圖二十七、Superdex 200 膠體層析管柱圖…………………………..66
圖二十八、Superdex 200 膠體層析管柱圖……………………….….67

1. Armstrong, W.; Brandle, R.; Jackson, M. B., Mechanisms of flood tolerance in plants, Acta. Bot. Neerl., 43, 307-358, 1994

2. Aspart, L,; Got, A,; Delseny, M.; Mocquot, B.; Pradet, A., Adaptation of ribonucleic acid metabolism to anoxia in rice embryos, Plant Physiol., 72, 115-119, 1983

3. Atwell, B. J,; Waters, I.; Greenway, H., The effect of oxygen and turbulence on elongation of coleoptiles of submergence-tolerant and –intolerant rice cultivars, J. Exp. Bot., 33, 1030-1044, 1982

4. Beckett, P. M.; Armstrong, W.; Justin SHFW; Armstrong, J., On the relative importance of convective and diffusive gas-flows in plant aeration, New Phytol., 110, 463-468, 1988

5. Bewley, J. D,; Black, M., Physiology and biochemistry of seeds, Springer-Verlag, 1, 306, 1978

6. Bleecker, A. B., Schyette, J. L.; Kende, H., Anatomical analysis of growth and developmental patterns in the internode of deep water rice, Planta, 169, 490-497, 1986

7. Burstrom, H.; Uhrstrom, I.; Wurscher, R., Physiol. Plant, 47, 805, 1971

8. Carpita, N. C., Structure and biogenesis of the cell walls of grasses, Annu. Rev. Plant Physiol. Plant Mol. Biol., 47, 445-476, 1996

9. Carpita, N. C.; Gibeaut, D., Structural models of primary cell walls in flowering plants : consistency of molecular structure with the physical properties of the walls during growth, Plant J., 3, 1-30, 1993

10. Cleland, R. E., Plant Physiol., 47, 805, 1971

11. Cosgrove, D., How do plant cell walls extend ? Plant Physiol., 102, 1-6, 1993

12. Duperon, R., Acad. Sci. Paris, 241, 1817-1819, 1955

13. Ellis, M. H.; Setter, T. l., Hypoxia induces anoxia tolerance in completely submerged rice seedlings, J. Plant Physiol., 154, 219-230, 1999

14. Fry, S. C., Phenolic components of the primary cell wall and their possible role in the hormonal regulation of growth, Planta, 146, 343-351, 1979

15. Fukui, T.; Nikuni, Z., J. Biochem. Japan, 43, 33, 1956

16. Heyn, A. N., J. Rec. Trav. Bot. Neerl., 28, 113, 1931

17. Horton, R. F., The effect of ethylene and other regulators on coleoptile growth of rice under anoxia, Plant Sci., 79, 57-62, 1991

18. Justin SHFW; Armstrong, W., The anatomical characteristics of roots and plant response to soil flooding, New Phytol, 106, 465- 495, 1987

19. Kolloffel, C., Acta, Bot. Neel, 16, 111-122, 1967

20. Kordan, H. A., Oxygen as an environmental factor in influencing normal morphogenetic development in germinating rice seedlings, J. Exp. Bot., 27, 947-952, 1976

21. Kordan, H. A., Mitotic activity in rice seedlings germinating under oxygen deficiency, J. Cell Sci., 20, 57-59, 1976a

22. Kordan, H. A., Adventitious root initiation and growth in relation to oxygen supply in germinating rice seedlings, New Phytol., 76, 81-86, 1976b

23. Ku, H. S.; Suge, H.; Rappaport, L., Pratt, H. K., Stimulation of rice coleoptile growth by ethylene, Planta, 90, 333-339, 1970

24. Kutschera, U.; Kende, H., The biophysical basis of elongation growth in internodes of deepwater rice, Plant Physiol., 88, 361-366, 1788
25. Lorbiecke, R.; Sauter, M., Induction of cell growth and cell division in the intercalary meristem of submerged deepwater rice, Planta, 204, 140-145, 1998

26. Lorbiecke, R.; Sauter, M., Adventitious root growth and cell-cycle induction in deepwater rice, Plant Physiol., 119, 21-29, 1999

27. Macleod, A. M., New Phytol., 56, 210-220, 1957

28. Mayer, A. M.; Poljakoff-Mayber, A., The germination of seed 2nd ed, Pergamon, 1975

29. McQueen-Mason, S.; Cosgrove, D. J., Disruption of hydrogen bonding between plant cell wall polymers by proteins that induce wall extension, Proc. Natl. Acad. Sci. USA, 91, 6574-6578, 1994

30. McQueen-Mason, S.; Durachko, D.; Cosgrove, D., Two endogenous proteins that induce cell wall extension in plants, Plant Cell, 4, 1425-1433, 1992

31. Metraux, J. P.; Kende, H., The cellular basis of the elongation response in submerged deepwater rice, Planta, 160, 73-77, 1984

32. Mocquot, B.; Pradet, A.; Livak, S., DNA synthesis and anoxia in rice coleoptile, Plant Sci. Lett., 9, 365-371, 1977

33. Normile, D., Yangtze seen as earliest rice site, Science, 275, 309, 1997

34. Ohwaki, Y., Growth of rice coleoptiles in relation to oxygen concentrations, Sci. Rep. Tohoku. Univ. Ser. Ⅳ, 33, 1-5, 1967

35. Opik, H., Effect of anaerobiosis on respiratory rate, cytochrome oxidase activity and mitochondrial structures in coleoptile of rice, J. Cell Sci., 12, 725-739, 1973


36. Palmiano, E. P.; Juliano, B. O., Plant Physiol., 49, 751-756, 1972

37. Plaxton, W. C., The organization and regulation of plant glycolysis, Annu. Rev. Plant Physiol. Plant Mol. Biol., 47, 185- 214, 1996

38. Raskin, I.; Kende, H., How does deep water rice solve its aeration problem, Plant Physiol., 72, 447-454, 1983

39. Raskin, I.; Kende, H., Mechanism of aeration in rice, Science, 228, 327-329, 1985

40. Remo, R.; Hochkoeppler, A.; Bertani, A., Polyamines and anaerobic elongation of rice coleoptile, Plant Cell Physiol., 30, 893-898, 1989

41. Satler, S. O.; Kende, H., Ethylene and the growth of rice seedlings, Plant Physiol, 79,194-198, 1985

42. Swain, R. R.; Deckker, E. E., Biochem. Biophys. Acta., 122, 87-100, 1966

43. Tan, K. S.; Hosen, T.; Masuda, Y.; Kamisaka, S., Correlation between cell wall extensibility and the content of diferulic and ferulic acids in cell walls of Oryza sativa coleoptiles grown under water and in air, Physiologia Plantarum, 83, 397-403, 1991

44. Waters, I.; Morrell, S.; Greenway, H.; Colmer, T. D., Effect of anoxia on wheat seedlings, J. Exp. Bot., 42, 1437-1447, 1991

45. Webb, J.; Jackson, M. B., A transmission and cryo-scanning electron microscopy study of the formation of aerenchyma (cortical gas-filled space) in adventitious roots of rice, J. Exp. Bot., 37, 832-841, 1986

46. Zarra, I.; Masuda, Y., Growth and cell wall changes in rice coleoptiles growing under different conditions. I. Changes in turgor pressure and cell wall polysaccharides during intact growth, Plant Cell Physiol., 20, 1117-1124, 1979