水稻 (Oryza sativa L.) 第三型幾丁質酶的分子研究
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2006
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我們自水稻懸浮培養細胞所分泌的蛋白質當中，鑑定出兩種第三型的幾丁質酶 (Osm30和Osm34)。本研究不但針對這兩種幾丁質酶進行生化特性與生理功能的探討，也就水稻基因組資料庫中的32個第三型幾丁質酶成員，進行親緣系統樹的分析，結果顯示Osm30及Osm34分別歸類為第IIIa和IIIb亞型。
幾丁質酶屬於pathogenesis-relative (PR) 蛋白質家族成員，常參與植物的抗病防禦機制。當水稻細胞受到四種病原真菌：Rhizoctonia solani、Sarocladium oryzae、Gibberella fujikuroi和Bipolaris oryzae的感染時，Osm30和Osm34的表現量，均會被誘導增加。此外，Osm30和Osm34也受到甲基茉莉酸 (MeJA) 處理的誘導，而水楊酸 (SA) 的處理則不影響兩者基因的表現，顯示此兩種第三型幾丁質酶可能是經由JA/ethylene-dependent訊息傳導路徑，而非以SA-dependent路徑，來參與植物的抗病機制。
本研究培養水稻細胞的過程中也發現，每當更換新培養液後，水稻細胞的Osm30和Osm34基因都會被短暫誘導而增加表現，隨後又降回基礎表現量。此暫時性表現的情形可能受到繼代培養的攪拌動作 (agitation)，及更換新舊培養液時產生的滲透壓差的正向調控；此外，水稻細胞在培養過程中，會新生成並分泌一些抑制幾丁質酶表現的成分，以調節Osm34基因的表現。當水稻細胞以與逆境有關的植物生長調節劑ABA處理時，結果顯示其對於Osm30為正向的促進表現，對Osm34卻為負向的抑制表現，顯示此兩個幾丁質酶在不同的逆境下，其基因表現的調控機制可能有所不同。
綜合上述資料，Osm30和Osm34幾丁質酶在水稻細胞內平常只有基礎量的表現，當細胞感受到環境的變化，包括滲透勢改變、受到機械傷害或病原菌感染時，可能經由JA/ethylene訊息路徑，和/或由其他未知的因子，來調控共同調控其基因的表現，當水稻細胞已適應環境或威脅去除後，水稻細胞有另外的機制來節制幾丁質酶基因的表現。
Rice is one of the most important agricultural crops; biotic stresses are limiting crop productivity worldwide, hence studies aiming at identification and characterization of novel pathogen related gene(s) is gaining momentum. Chitinases belong to the pathogenesis-related protein families and play significant role in plant defense mechanism. In the present study, we identified several chitinases in rice suspension-culture, among Osm30 and Osm34 (class III) are selected for further investigations including biochemical properties, physiological functions, gene expression and regulation for future application. The phylogenetic trees of Osm30 and Osm34 with other 30 putative class III chitinases in the rice genome defines the evolutionary relationship and are classified into subclasses IIIa and IIIb respectively, indicating diverse functions in vivo. The phylogenetic analysis of Osm30 and Osm34 with other 32 putative class III chitinases in the rice genome revealed subclasses IIIa and IIIb, respectively. The expression of Osm30 and Osm34 genes was up-regulated in the presence of common rice pathogens, such as Rhizoctonia solani, Sarocladium oryzae, Gibberella fujikuroi and Bipolaris oryzae, ethylene and methyl jasmonate (MeJA), but not salicylic acid (SA). These results indicate that the expressions of these chitinases are JA/ethylene dependent and may participate in SA independent signal transduction pathway in the plant defense mechanism. Upon transfer of rice cells into fresh culture medium, Osm30 and Osm34 transcripts displayed a transient expression pattern in rice cells and subsequently reduce to basal expression level. This phenomenon may be due to mechanical agitation and osmotic change in the culture medium. In addition, rice cells secreted some inhibitory factors that regulated the Osm30 and Osm34 gene expression during culturing. It was interesting to note that ABA positively regulated the expression of Osm30 gene and inhibited Osm34. Taken together, the expression of Osm30 and Osm34 was basal level under normal culture conditions, the changes in the expression level of chitinase genes was observed after transferring into fresh culture medium. Thus rice cells sense the environmental alternation such as osmotic shock, agitation and pathogen infection probably via JA/ethylene-dependent pathway and/or other unidentified factors, to augment cellular defense mechanism.
Rice is one of the most important agricultural crops; biotic stresses are limiting crop productivity worldwide, hence studies aiming at identification and characterization of novel pathogen related gene(s) is gaining momentum. Chitinases belong to the pathogenesis-related protein families and play significant role in plant defense mechanism. In the present study, we identified several chitinases in rice suspension-culture, among Osm30 and Osm34 (class III) are selected for further investigations including biochemical properties, physiological functions, gene expression and regulation for future application. The phylogenetic trees of Osm30 and Osm34 with other 30 putative class III chitinases in the rice genome defines the evolutionary relationship and are classified into subclasses IIIa and IIIb respectively, indicating diverse functions in vivo. The phylogenetic analysis of Osm30 and Osm34 with other 32 putative class III chitinases in the rice genome revealed subclasses IIIa and IIIb, respectively. The expression of Osm30 and Osm34 genes was up-regulated in the presence of common rice pathogens, such as Rhizoctonia solani, Sarocladium oryzae, Gibberella fujikuroi and Bipolaris oryzae, ethylene and methyl jasmonate (MeJA), but not salicylic acid (SA). These results indicate that the expressions of these chitinases are JA/ethylene dependent and may participate in SA independent signal transduction pathway in the plant defense mechanism. Upon transfer of rice cells into fresh culture medium, Osm30 and Osm34 transcripts displayed a transient expression pattern in rice cells and subsequently reduce to basal expression level. This phenomenon may be due to mechanical agitation and osmotic change in the culture medium. In addition, rice cells secreted some inhibitory factors that regulated the Osm30 and Osm34 gene expression during culturing. It was interesting to note that ABA positively regulated the expression of Osm30 gene and inhibited Osm34. Taken together, the expression of Osm30 and Osm34 was basal level under normal culture conditions, the changes in the expression level of chitinase genes was observed after transferring into fresh culture medium. Thus rice cells sense the environmental alternation such as osmotic shock, agitation and pathogen infection probably via JA/ethylene-dependent pathway and/or other unidentified factors, to augment cellular defense mechanism.
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水稻, 幾丁質酶, 病原相關基因, 茉莉酸, 離層酸, rice, chitinase, pathogenesis-related gene, jasmonate, ABA