臺灣博碩士論文加值系統

植物體內小分子量熱休克蛋白質 (small heat shock protein, sHSP) 為普遍存在且具高度保留性，其分子量約為15-30 kDa，在逆境中植物常被誘導累積小分子量熱休克蛋白質，這類蛋白質具有分子伴護（molecular chaperone）的功能，可保護植物適應逆境。根據多重校對分析 (multiple sequence alignment) 可知，水稻小分子量熱休克蛋白質共可分成十四個不同的族群 (class)，目前第二族群共有兩個小分子量熱休克蛋白質。本研究係探討水稻第二族群小分子量熱休克蛋白質在經由生物及非生物性逆境處理後其誘導情形、並測試其在大腸桿菌 (Escherichia coli) 中的異源保護效果及生體外 (in vitro) 之分子伴護活性測試，此外，為瞭解實際水稻幼苗的反應，也測試了水稻幼苗於非生物性逆境之交互適應情形。由結果可知，水稻幼苗經42℃水浴、5 mM 過氧化氫及紫外光 (UV-C) 處理後，發現OsHSP19.0-CII及OsHSP18.0-CII基因皆有被誘導的情形；另外，亦想探討其對水稻病原真菌立枯絲核菌之反應，結果得知在接種水稻後第四天， OsHSP18.0-CII基因表現量會下降，而OsHSP19.0-CII基因在接種後表現量並無差異。利用大腸桿菌 [E. coli strain BL21 (DE3) ] 表現重組小分子量熱休克蛋白質OsHSP19.0-CII及OsHSP18.0-CII，在50℃水浴、5 mM過氧化氫及紫外光 (UV-C) 處理下，小分子量熱休克蛋白質OsHSP18.0-CII的重組蛋白質可對大腸桿菌提供保護效果，而小分子量熱休克蛋白質OsHSP19.0-CII的重組蛋白質只有在50℃水浴處理下可保護大腸桿菌。利用純化之OsHSP19.0-CII及OsHSP18.0-CII重組蛋白質，以檸檬酸合成酶當做基質，在1 M 過氧化氫逆境下測試其分子伴護活性，得知此二水稻第二族群小分子量熱休克蛋白質皆有分子伴護活性。水稻幼苗經42℃水浴預處理兩小時以誘導熱休克蛋白質累積，再經由5 mM之過氧化氫處理5小時後，利用氯化三苯基四氮唑染色法 (triphenyl tetrazolium chloride reduction test) 來測試水稻幼苗之細胞活性，發現經由42℃預處理之水稻幼苗可誘導表現小分子量熱休克蛋白質，並讓水稻幼苗於後續的氧化逆境中降低其細胞活性受損程度，而OsHSP19.0-CII及OsHSP18.0-CII基因表現量於42℃預處理再經過5小時的28℃水浴或過氧化氫處理後，皆比無預熱處理者來的高，且其細胞活性受損程度的降低與過氧化氫酶 (catalase) 及過氧化酶 (peroxidase) 此二抗氧化酵素無關。

The small heat shock proteins (sHSPs) of plants with a molecular mass of 15-30 kDa are ubiquitous and conserved. sHSPs are usually induced and accumulated under stresses in plants. These proteins possess molecular chaperone activities and could protect plants to accommodate stresses. According to the multiple sequence alignment analysis, the rice sHSPs could be divided to fourteen classes. Only two class II sHSPs would be found in rice so far. In this study, I would take two class II sHSPs of rice to test their expression profiles under biotic and abiotic stresses, heterologous protection in Escherichia coli, and molecular chaperone activities in vitro. Moreover, I would test the cross adaption of rice seedlings under abiotic stresses. As the results, OsHSP19.0-CII and OsHSP18.0-CII genes could be induced in rice seedlings under 42℃, 5 mM hydrogen peroxide, and ultraviolet-C (UV-C) treatments. OsHSP18.0-CII gene expression would be down-regulated at the fourth day after rice seedlings inoculated with Rhizoctonia solani, but no difference was detected in OsHSP19.0-CII gene expression. OsHSP19.0-CII and OsHSP18.0-CII genes were overexpressed as recombinant sHSPs in E. coli BL-21 (DE3). The recombinant sHSPs were used to study their protective function in E. coli under 50℃, 5 mM hydrogen peroxide, and ultraviolet-C (UV-C) treatments. Based on the survival rates of E. coli cells, accumulation of recombinant OsHSP18.0-CII protein could enhance stresses tolerance in E. coli under above stresses, but the accumulation of recombinant OsHSP19.0-CII protein could only enhance stress tolerance to 50℃ heat stress in E. coli. To test the chaperone activities of these two class II small heat shock proteins, the recombinant OsHSP19.0-CII and OsHSP18.0-CII proteins were purified and citrate synthase was used as a substrate for denaturation test under 1 M hydrogen peroxide. Both of the two class II small heat shock proteins showed chaperone activities. Rice seedlings were pre-heated under 42℃ water bath to induce HSPs accumulation to test the effects of cross adaption. After pre-heating, rice seedlings were treated with 5 mM hydrogen peroxide for five hours and then subjected to triphenyl tetrazolium chloride (TTC) reduction test to assay the cell activities of rice seedlings. As the results, the pre-heated rice seedlings could induce and accumulate more sHSPs and the pre-heating could reduce the impaired cell activities of rice seedlings under the following oxidative stress. After five hours of hydrogen peroxide or water control treatment at 28℃, the gene expression levels of OsHSP19.0-CII and OsHSP18.0-CII in pre-heated rice seedlings were higher than that in those rice seedlings without pre-heating. The reduction in the impaired cell activities of rice seedlings was not related to the antioxidation activities of catalase and peroxidase.

中文摘要 I
英文摘要 III
前言 1
材料與方法 7
1. 植物材料 7
2. 熱處理 7
3. 過氧化氫 (H2O2) 處理 7
4. 紫外光 (UV-C) 處理 7
5. 生物性逆境處理 8
6. 水稻總量RNA (核醣核酸，Ribonucleic acid) 萃取 8
7. 反轉錄-聚合酶連鎖反應 (reverse transcription-polymerase chain reaction, RT-PCR)分析 9
8. 即時定量聚合酶連鎖反應 (Real-time Quantitative PCR, qPCR) 9
9. 真菌DNA萃取 10
10. 聚合酶連鎖反應 (polymerase chain reaction, PCR) 分析 10
11. 重組小分子量熱休克蛋白質基因之構築 11
12. 重組小分子量熱休克蛋白質之誘導 11
13. 重組小分子量熱休克蛋白質對大腸桿菌於逆境下之保護性測試 12
14. 重組蛋白質之粗萃取 12
15. 水稻總量蛋白質萃取 13
16. 蛋白質之單向十二磺酸鈉聚乙烯醯胺膠體電泳分析 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) 13
17. 西方轉漬分析 (western blot) 14
18. 蛋白質純化 14
19. OsHSP19.0-CII及OsHSP18.0-CII的重組蛋白質之分子伴護活性測試 15
20. 水稻幼苗交互適應處理 15
21. 氯化三苯基四氮唑染色法 (TTC reduction test) 及其定量 16
22. 測試抗氧化相關酵素之蛋白質萃取 16
23. 過氧化氫酶 (catalase) 活性測試 16
24. 過氧化酶 (peroxidase) 活性測試 17
25. 水稻回復生長 17
26. 統計分析 17
結果 18
(一) 水稻第二族群小分子量熱休克蛋白質於非生物及生物性逆境處理下的基因表現圖譜 18
1. 熱處理 18
2. 過氧化氫 (H2O2) 處理 18
3. 紫外光 (UV-C) 處理 19
4. 生物性逆境處理 19
(二) 於大腸桿菌 [E. coli BL21 (DE3)] 表現OsHSP19.0-CII、OsHSP18.0-CII、OsHSP17.4-CI及OsHSP16.9D-CI的重組蛋白質對氧化及熱逆境的耐受性測試 20
1. 大腸桿菌菌株之生長曲線與總量蛋白質粗萃取 20
2. 耐熱測試 21
3. 過氧化氫 (H2O2) 耐性測試 21
4. 紫外光 (UV-C) 耐性測試 22
(三) OsHSP19.0-CII及OsHSP18.0-CII的重組蛋白質之分子伴護活性測試 ...........23
(四) 水稻幼苗於氧化逆境處理下之細胞活性及交互適應測試 ...24
討論 27
參考文獻 36
圖表 42
表一、本研究中使用之各專一性引子對及其 PCR或qPCR相關資料 42
圖一、水稻幼苗於42℃水浴處理後第二族群小分子量熱休克蛋白質基因表現情形 43
圖二、水稻幼苗於5 mM過氧化氫處理後第二族群小分子量熱休克蛋白質基因表現情形 ....45
圖三、水稻幼苗於150,000 μJ能量之UV-C處理下，於28℃恢復後之第二族群小分子量熱休克蛋白質基因表現情形 47
圖四、水稻接種立枯絲核菌 (Rhizoctonia solani) 不同天數之病徵情形 ....49
圖五、即時定量聚合酶連鎖反應之各引子對對目標水稻基因的專一性測試 .....51
圖六、水稻植株接種立枯絲核菌 (Rhizoctonia solani) 後其第二族群小分子量熱休克蛋白質基因表現情形 .......................................53
圖七、各大腸桿菌 (Escherichia coli) 供試菌株生長情形 54
圖八、累積個別水稻小分子量熱休克蛋白質之重組蛋白質 56
圖九、累積個別水稻小分子量熱休克蛋白質之重組蛋白質之大腸桿菌於熱處理下的存活率 58
圖十、累積個別水稻小分子量熱休克蛋白質之重組蛋白質之大腸桿菌於 5 mM過氧化氫處理下的存活率 59
圖十一、累積個別水稻小分子量熱休克蛋白質之重組蛋白質之大腸桿菌於UV-C處理下的存活率… 60
圖十二、檸檬酸合成酶在不同過氧化氫濃度下之不正常聚合情形 .61
圖十三、檸檬酸合成酶在1 M過氧化氫處理不同時間後之不正常聚合情形 ..62
圖十四、在大腸桿菌中誘導表現之水稻小分子量熱休克蛋白質OsHSP19.0-CII及OsHSP18.0-CII的重組蛋白質之純化及其西方轉漬分析結果 ..63
圖十五、純化之水稻小分子量熱休克蛋白質OsHSP19.0-CII及OsHSP18.0-CII的重組蛋白質之分子伴護活性測試 ..65
圖十六、水稻幼苗經不同濃度之過氧化氫處理後的細胞活性情形 ..66
圖十七、以triphenyl tetrazolium chloride (TTC) 染色量化水稻幼苗經不同濃度的過氧化氫處理後之細胞活性 ..67
圖十八、以triphenyl tetrazolium chloride (TTC) 染色量化水稻幼苗經熱處理後之細胞活性及其蛋白質電泳和小分子量熱休克蛋白質之累積情形... 68
圖十九、預熱處理的水稻幼苗在過氧化氫 (H2O2) 處理下，其triphenyl tetrazolium chloride (TTC) 染色量化水稻幼苗之細胞活性的結果 70
圖二十、預熱處理的水稻幼苗在過氧化氫 (H2O2) 處理下，其第二族群小分子量熱休克蛋白質基因的表現情形 ....71
圖二十一、預熱處理的水稻幼苗在過氧化氫 (H2O2) 處理下，其過氧化氫酶及過氧化酶的活性 ....73
圖二十二、預熱處理的水稻幼苗在過氧化氫 (H2O2) 處理後，回復生長的結果 .....75
附圖一、UBQ5、qPCR-sHSP19.0-CII及qPCR-sHSP18.0-CII引子對之 qPCR產物的melting curve結果 .....76
附圖二、利用qPCR-sHSP19.0-CII引子對可增幅出OsHSP19.0-CII基因片段，其NCBI (National Center for Biotechnology Information) 基因庫中的目標基因序列經序列比對結果 .....77
附圖三、利用qPCR-sHSP18.0-CII引子對可增幅出預期的OsHSP18.0-CII基因片段，其NCBI基因庫中的目標基因序列經序列比對結果 .....78
附圖四、利用UBQ5引子對可增幅出預期的水稻Ubiquitin 5基因片段，其NCBI基因庫中的目標基因序列經序列比對結果 .....80

Banzet, N., Richaud, C., Deveaux, Y., Kazmaier, M., Gagnon, J., and Triantaphylides, C. 1998. Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells. Plant J. 13 (4):519-527.
Basha, E., Jones, C., Wysocki, V., and Vierling, E. 2010. Mechanistic differences between two conserved classes of small heat shock proteins found in the plant cytosol. J. Biol. Chem. 285 (15):11489-11497.
Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.
Caspers, G. J., Leunissen, J. A. M., and Jong, W. W. 1995. The expanding small heat-shock protein family, and structure predictions of the conserved “α-crystallin domain”. J. Mol. Evol. 40 (3):238-248.
Chang, P.-F. L., Jinn, T.-L., Huang, W.-K., Chen, Y., Chang, H.-M., and Wang, C.-W. 2007. Induction of a cDNA clone from rice encoding a class II small heat shock protein by heat stress, mechanical injury, and salicylic acid. Plant Sci. 172 (1):64-75.
Chen, C., Belanger, R. R., Benhamou, N., and Paulitz, T. 2000. Defense enzymes induced in cucumber roots by treatment with plant growth-promoting rhizobacteria (PGPR) and Pythium aphanidermatum. Physiol. Mol. Plant P. 56:13-23.
COA. 2012. Agriculture and Food Agency. Council of Agriculture, Executive Yuan. Taipei, Taiwan.
DeRocher, A. E., and Vierling, E. 1994. Developmental control of small heat shock protein expression during pea seed maturation. Plant J. 5 (1):93-102.
Diffey, B. 2004. Climate change, ozone depletion and the impact on ultraviolet exposure of human skin. Phys. Med. Biol. 49:R1-R11.
Giglio, S., Monis, P. T., and Saint, C. P. 2003. Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR. Nucleic Acids Res. 31 (22):e136.
Gill, S. S., and Tuteja, N. 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Bioch. 48 (12):909-930.
Gong, M., Chen, B., Li, Z. G., and Guo, L. H. 2001. Heat-shock-induced cross adaptation to heat, chilling, drought and salt stress in maize seedlings and involvement of H2O2. J. Plant Physiol. 158 (9):1125-1130.
Guan, J. C., Jinn, T. L., Yeh, C. H., Feng, S. P., Chen, Y. M., and Lin, C. Y. 2004. Characterization of the genomic structures and selective expression profiles of nine class I small heat shock protein genes clustered on two chromosomes in rice (Oryza sativa L.). Plant Mol. Biol. 56 (5):795-809.
Haslbeck, M., Franzmann, T., Weinfurtner, D., and Buchner, J. 2005. Some like it hot: the structure and function of small heat-shock proteins. Nat. Struct. Mol. Biol. 12:842-846.
Jaenicke, R., and Rudolph, R. 1989. Folding proteins. In Protein Structure: A Pratical Approach (Creighton, T. E., ed) pp. 191-223, IRL Press Oxford.
Johanson, A., Turner, H. C., Mckay, G. J., and Brown, A. E. 1998. A PCR-based method to distinguish fungi of the rice sheath-blight complex, Rhizoctonia solani, R. oryzae and R. oryzae-sativae. FEMS Microbiol. Lett. 162:289-294.
Keller, E., Steffen, K. L. 1995. Increased chilling tolerance and altered carbon metabolism in tomato leaves following application of mechanical stress. Physiol. Plant. 93:519-525.
Kim, H., and Ahn, Y. J. 2009. Expression of a gene encoding the carrot HSP17.7 in Escherichia coli enhances cell viability and protein solubility under heat stress. HortScience. 44 (3):866-869.
Kim, K. H., Alam, I., Kim, Y. G., Sharmin, S. A., Lee, K. W., Lee, S. H., Lee, B. H. 2012. Overexpression of a chloroplast-localized small heat shock protein OsHSP26 confers enhanced tolerance against oxidative and heat stresses in tall fescue. Biotechnol. Lett. 34:371-377.
Kumar, K. K., Poovannan, K., Nandakumar, R., Thamilarasi, K., Geetha, C., Jayashree, N., Kokiladevi, E., Raja, A. J., Samiyappan R., Sudhakar D., and Balasubramanian, P. 2003. A high throughput functional expression assay system for a defence gene conferring transgenic resistance on rice against the sheath blight pathogen, Rhizoctonia solani. Plant Sci. 165:969-976.
Lee, G. J., Pokala, N., and Vierling, E. 1995. Structure and in vitro molecular chaperone activity of cytosolic small heat shock proteins from pea. J. Biol. Chem. 270 (18):10432-10438.
Lee, G. J., Roseman, A. M., Saibil, H. R., and Vierling, E. 1997. A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state. EMBO J. 16 (3):659-671.
Linquist, S., and Craig, E. A. 1988. The heat-shock proteins. Annu. Rev. Genet. 22:631-677.
Lin., Y. S. 2010. Expression of rice small heat shock proteins under different stresses and their protective functions in Escherichia coli. National Chung Hsing University Department of Plant Pathology Master Thesis. 48 pages.
Livak, K. J., and Schmittgen, T. D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method. Methods. 25:402-408.
Maimbo, M., Ohnishi, K., Hikichi, Y., Yoshioka, H., and Kiba, A. 2007. Induction of a small heat shock protein and its functional roles in Nicotiana plants in the defense response against Ralstonia solanacearum. Plant Physiol. 145 (4):1588-1599.
Madronich, S., McKenzie, R. L., Bjorn L. O., and Caldwell M. M. 1998. Changes in biologically active ultraviolet radiation reaching the earth’s surface. J. Photochem. Photobiol., B. 46:5-19.
Matsumura, T., Tabayashi, N., Kamagata, Y., Souma, C., and Saruyama, H. 2002. Wheat catalase expressed in transgenic rice can improve tolerance against low temperature stress. Physiol. Plant. 116:317-327.
Moller, I. M., Jensen, P. E., and Hansson, A. 2007. Oxidative modifications to cellular components in plants. Annu. Rev. Plant Biol. 58:459-481.
Murakami, T., Matsuba, S., Funatsuki, H., Kawaguchi, K., Saruyama, H., Tanida, M., and Sato, Y. 2004. Over-expression of a small heat shock protein, sHSP17.7, confers both heat tolerance and UV-B resistance to rice plants. Mol. Breed. 13 (2):165-175.
Quan, L. J., Zhang, B., Shi, W. W., and Li, H. Y., 2008. Hydrogen peroxide in plants: a versatile molecule of the reactive oxygen species network. J. Integr. Plant Biol. 50 (1):2-18.
Sabehat, A., Weiss, D., Lurie, S., 1998. Heat-shock proteins and cross-tolerance in plants. Physiol. Plant. 103:437-441.
Sarkar, N. K., Kim, Y. K., and Grover, A. 2009. Rice sHsp genes: genomic organization and expression profiling under stress and development. BMC Genomics. 10:393.
Scharf, K. D., Siddique, M., and Vierling, E. 2001. The expanding family of Arabidopsis thaliana small heat stress proteins and a new family of proteins containing α-crystallin domains (Acd proteins). Cell Stress Chaperon. 6:225-237.
Song, N. H., and Ahn, Y. J. 2010. DcHsp17.7, a small heat shock protein from carrot, is upregulated under cold stress and enhances cold tolerance by functioning as a molecular chaperone. HortScience. 45 (3):469-474.
Song, N. H., and Ahn, Y. J. 2011. DcHsp17.7, a small heat shock protein in carrot, is tissue-specifically expressed under salt stress and confers tolerance to salinity. N. Biotechnol. 28 (6):698-704.
Steponkus, P. L., and Lanphear, F. O. 1967. Refinement of the triphenyl tetrazolium chloride method of determining cold injury. Plant Physiol. 42:1423-1426.
Sun, L., Liu, Y., Kong, X., Zhang, D., Pan, J., Zhou, Y., Wang, L., Li, D., and Yang, X. 2012. ZmHSP16.9, a cytosolic class I small heat shock protein in maize (Zea mays), confers heat tolerance in transgenic tobacco. Plant Cell Rep. 31 (8):1473-1484.
Sun, W., Bernard, C., Van De Cotte, B., Van Montagu, M., and Verbruggen, N. 2001. At‐HSP17.6A, encoding a small heat‐shock protein in Arabidopsis, can enhance osmotolerance upon overexpression. Plant J. 27 (5):407-415.
Sun, W., Van Montagu, M., and Verbruggen, N. 2002. Small heat shock proteins and stress tolerance in plants. Biochim. Biophys. Acta. 1577 (1):1.
Towill, L. E., and Mazur, P. 1974. Studies on the reduction of 2,3,5-triphenyl-tetrazolium chloride as a viability assay for plant tissue cultures. Can. J. Bot. 53:1097-1102.
Van Camp, W., Van Montagu, M., Inze, D. 1998. H2O2 and NO: redox signals in disease resistance. Trends Plant Sci. 3:330-334.
Van Montfort, R. L. M., Basha, E., Friedrich, K. L., Slingsby, C., and Vierling, E. 2001. Crystal structure and assembly of a eukaryotic small heat shock protein. Nat. Struct. Mol. Biol. 8 (12):1025-1030.
Van Ooijen, G., Lukasik, E., Van Den Burg, H. A., Vossen, J. H., Cornelissen, B. J., and Takken, F. L. 2010. The small heat shock protein 20 RSI2 interacts with and is required for stability and function of tomato resistance protein I-2. Plant J. 63 (4):563-572.
Van Rajan, V. B., and D’Silva, P. 2009. Arabidopsis thaliana J-class heat shock proteins: cellular stress sensors. Funct. Integr. Genomics. 9 (4):433-446.
Vierling, E. 1991. The roles of heat shock proteins in plants. Annu. Rev. Plant Biol. 42 (1):579-620.
Wang, W., Vinocur, B., Shoseyov, O., and Altman, A. 2004. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci. 9 (5):244-252.
Waters, E. R., Lee, G. J., and Vierling, E. 1996. Evolution, structure and function of the small heat shock proteins in plants. J. Exp. Bot. 47 (3):325-338.
Xiong L., Schumaker K. S., Zhu J. K. 2002. Cell signaling during cold, drought, and salt stress. Plant Cell. 14 (Suppl):S165-S18.
Yeh, C. H., Chang, P. F. L., Yeh, K. W., Lin, W. C., Chen, Y. M., and Lin, C. Y. 1997. Expression of a gene encoding a 16.9-kDa heat-shock protein, Oshsp16.9, in Escherichia coli enhances thermotolerance. Proc. Natl. Acad. Sci. USA. 94 (20): 10967-10972.
Zhu J. K. 2001. Plant salt tolerance. Trends Plant Sci. 6 (2):66-71.
Zhu J. K. 2002. Salt and drought stress signal transduction in plants. Annu. Rev. Plant Biol. 53:247-273.
Zou, J., Liu, C., Liu, A., Zou, D., and Chen, Xinbo. 2012. Overexpression of OsHsp17.0 and OsHsp23.7 enhances drought and salt tolerance in rice. J. Plant Physiol. 169:628-635.