Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31969
標題: 水稻第二族群小分子量熱休克蛋白質OsHSP19.0-CII及OsHSP18.0-CII於逆境下之表現、保護效果及其分子伴護活性
The expression of rice class II small heat shock proteins, OsHSP19.0-CII and OsHSP18.0-CII, their protection effects and molecular chaperone activities under stresses
作者: 陳建志
Chen, Chien-Chih
關鍵字: 小分子量熱休克蛋白質
small heat shock protein
水稻
分子伴護活性
生物逆境
交互適應
交互保護
非生物逆境
異源保護
Oryza sativa
molecular chaperone activity
biotic stress
cross adaption
cross protection
abiotic stress
heterologous protection
出版社: 植物病理學系所
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摘要: 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.
URI: http://hdl.handle.net/11455/31969
其他識別: U0005-1208201320482500
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1208201320482500
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