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標題: 水稻小分子量熱休克蛋白質於不同逆境下之基因表現及其重組蛋白質對大腸桿菌於逆境下之保護功能
Expression of rice small heat shock proteins under different stresses and their protective functions in Escherichia coli
作者: 林昀聖
Lin, Yung-sheng
關鍵字: small heat shock protein
Oryza sativa
oxidative stress
biotic stress
gene expression profiles
出版社: 植物病理學系所
引用: 林奕宏。 2007。 水稻第一族與第二族小分子量熱休克蛋白(Oshsp16.9A-CI 與 Oshsp18.0-CII)於非生物逆境下之基因表現與其分子伴護子活性測試。國立 中興大學植物病理學研究所碩士論文。 共 97 頁。 黃文寬。 2004。 水稻第二族低分子量熱休克基因,Oshsp18.0-CII,於非生物逆境 下的表現情形與此重組蛋白對於大腸桿菌之保護功能分析。國立中興大學植 物病理學研究所碩士論文。 共 67 頁。 謝麗娟、張義璋、謝廷芳。 2005。 水稻白葉枯病檢定方法之改良。 台灣農業研 究 54:15-22。 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. 13:519-527. Bowler, C., van Montagu, M. V., and Inzé, D. 1992. Superoxide dismutases and stress tolerance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:83-116. 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. Chang, P. F. L., Jinn, T. L., Huang, W. K., Chen, Y., Chang, H. M., and Wang, C. W. 2007. 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S. A. 100:11777-11782. Thao, N. P., Chen, L., Nakashima, A., Hara, S. I., Umemura, K., Takahashi, A., Shirasu, K., Kawasaki, T., and Shimamoto, K. 2007. RAR1 and HSP90 form a complex with Rac/Rop GTPase and function in innate-immune responses in rice. Plant Cell. 19:4035-4045. Vierling, E. 1991. The roles of heat shock proteins in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:579-620. Volkov, R. A., Panchuk, I. I., and Schöffl, F. 2005. Small heat shock proteins are differentially regulated during pollen development and following heat stress in tobacco. Plant Mol. Biol. 57:487-502. Volkov, R. A., Panchuk, I. I., Mullineaux, P. M., and Schöffl, F. 2006. Heat stress-induced H2O2 is required for effective expression of heat shock genes in Arabidopsis. Plant Mol. Biol. 61:733-746. Wang, Q., and Fang, R. 1996. Structure and expression of a rice hsp70 gene. Sci. China Ser. C. 39:291-299. Waters, E., and Vierling, E. 1999. 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摘要: 水稻 (Oryza sativa L.) 熱休克蛋白質 (heat shock protein, HSP) 主要被描述會受熱逆境誘導而大量累積,具有分子伴護子 (molecular chaperone) 的功能,常與植物後天適應逆境的耐性有關。而在多數環境逆境與生物逆境下,常伴隨有活化氧族 (reactive oxygen species) 的累積,則會對植物細胞形成氧化逆境。故比較不同 HSPs,包含高分子量熱休克蛋白質 (high molecular mass HSP, HMM HSP)與低分子量熱休克蛋白質 (low molecular mass HSP, LMM HSP),或稱小分子量熱休克蛋白質 (small heat shock protein, sHSP),於氧化逆境與生物逆境下的基因表現與蛋白質的累積情形,來探討其相關連性;並藉由外源表達重組 sHSP,進行對大腸桿菌 (Escherichia coli) 於氧化逆境下的保護性測試。本研究以水稻台農 67 號 (O. sativa L. cv. Tainung 67, TNG67) 幼苗為材料,於氧化逆境 (過氧化氫、氯化銅及紫外光) 處理或接種水稻白葉枯病病原菌 (Xanthomonas oryzae pv. oryzae) 之生物逆境處理下,分析不同 HSP 之基因表現圖譜。結果顯示,各基因表現受到不同氧化逆境或生物逆境而有不同的影響。其中,Oshsp16.9A-CI、Oshsp16.9B-CI、Oshsp16.9C-CI、Oshsp16.9D-CI、Oshsp17.3-CI、Oshsp17.7-CI、Oshsp17.9A-CI、Oshsp18.0-CI、Oshsp18.0-CII 及 Oshsp18.6-CIII 等水稻 sHSPs 都因受到不同能量的紫外光 (UV-C) 逆境處理而誘導其基因表現增加,但其誘導 情形不盡相同。而以過氧化氫 (H2O2) 處理不同時間後,各 sHSP 基因表現於處理前後無明顯差異,但有微量變動的情況。另在氯化銅 (CuCl2) 處理中,各 sHSP 基因在處理 3 至 6 小時間其表現情形各有增減;而在處理 9 至 48 小時間,多數 sHSP 基因表現量則有先下降隨後上升的情形。上述三種氧化逆境處理後的水稻苗總量蛋白質經西方轉漬法 (western blot) 分析之結果顯示,水稻第一族 sHSPs 僅於 UV-C 逆境下可見微量蛋白質的累積。而在生物逆境下,部分第一族與第三族的 sHSPs 基因於接種後 6 小時可被誘導,但於接種後 12 至 24 小時,其表現量有下降的趨勢,而西方轉漬分析 (western blot) 的結果可見於接種後 6 至 12 小時有明顯 sHSP-CI 蛋白質的累積,而接種後 24 小時則無蛋白質的累積。進一步於大腸桿菌中表現重組之 sHSPs,Oshsp16.9D-CI、Oshsp17.3-CI 及Oshsp18.0-CII,由結果觀察到此些重組蛋白質可以增加大腸桿菌對 UV-C 與H2O2 的耐性,而增加其存活數量。根據以上結果可知,即便是同一族之各個sHSP 基因在相似的逆境環境下,不同的 sHSP 對不同逆境的誘導表現情形仍不盡相同。而於植株中對逆境有所反應的 sHSPs 基因,藉由在大腸桿菌中表達外源重組蛋白質的實驗,也發現其可以增加大腸桿菌對氧化逆境的耐性程度,顯示其於氧化逆境下具有保護的功能。
The accumulation of rice (Oryza sativa L.) heat shock protein (HSP) was induced by heat stress. HSPs act as molecular chaperones, and were related to acquired stress tolerance of plants. Most biotic and abiotic stresses lead plants to generate reactive oxygen specises, such as hydrogen peroxide (H2O2) and superoxide (O2-•). The accumulation of ROS caused oxidative stress to plant cells. The gene expression profiles and the accumulation of heat shock proteins, including high molecular mass HSPs (HMM HSPs) and low molecular mass HSPs (LMM HSPs, also called sHSPs), under oxidative stresses and biotic stress were compared to study their correlation to stress adaptation. According to the gene expression profiles under UV-C treatments, some sHSP genes were selected and tested for the protective functions of these sHSPs in Escherichia coli under oxidative stress treatments. Ten-day-old rice (O. sativa L. cv. Tainung 67, TNG67) seedlings were treated with oxidative stresses (UV-C, H2O2, and CuCl2) and biotic stress (Xanthomonas oryzae pv. oryzae, Xoo). The gene expression profiles were analyzed by reverse transcription-polymerase chain reaction (RT-PCR), and western blotting was used for detection of proteins accumulation. Oshsp16.9A-CI, Oshsp16.9B-CI, Oshsp16.9C-CI, Oshsp16.9D-CI, Oshsp17.3-CI, Oshsp17.7-CI, Oshsp17.9A-CI, Oshsp18.0-CI, Oshsp18.0-CII, and Oshsp18.6-CIII were induced by ultra violet-C (UV-C) treatments with different expression patterns. Expression of the tested genes did not obviously change under hydrogen peroxide (H2O2) treatments, but the expression level with some influenced. The expression of most of the tested sHSP genes were influenced by treatment with copper chloride (CuCl2) 3 to 6 h, and expression of the most sHSP genes were decreased and tended to increase after 9 to 48 h treatment with CuCl2. The accumulation of sHSP-CI under UV-C treatments was only slightly detected by western blot analysis. Some of the tested sHSP genes were induced 6 h post Xoo inoculation, but the gene expression tended to decrease at 12 to 24 h post Xoo inoculation. The accumulation of sHSP-CI was obviously detected at 6 and 12 h, but not at 48 h, post inoculation with Xoo. Furthermore, we used E. coli transformed with the recombinant sHSPs was used to study their protective function under oxidative stresses. According to the results of the bacterial cell survival rates under certain tested stress treatments, three recombinant sHSPs, Oshsp16.9D-CI, Oshsp17.3-CI, and Oshsp18.0-CII, could enhance tolerance of E. coli to UV-C and hydrogen peroxide. These observations imply that sHSPs may play different roles in abiotic and biotic stress responses. sHSPs may have protective function under oxidative stress.
其他識別: U0005-2008201014150800
Appears in Collections:植物病理學系



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