水稻悬浮细胞凋亡的诱导及其机制研究
摘要
为了更深入研究植物细胞凋亡的信号传导和作用机理，建立一个稳定而高效的植物细胞凋亡诱导体系是非常必要的。同时，为筛选植物诱抗剂提供一种确实可行的办法，也为作物育种筛选优良的抗病抗逆性品种提供新的途径。本文在水稻悬浮细胞诱导凋亡体系的建立及其细胞凋亡诱导机制等方面进行初步探讨，研究结果如下：
采用不同水稻品种、不同继代次数的愈伤组织建立了不同水稻悬浮细胞体系，比较不同悬浮细胞的生长状况，并探讨了悬浮细胞在培养过程中产生胞外POD活性的变化情况。结果表明：水稻愈伤组织分散性越好，愈伤组织的褐化程度越轻，越容易建立悬浮细胞系。随着愈伤组织继代次数的增加，早星106悬浮细胞胞外POD活性和细胞悬浮度呈先上升后下降趋势。在愈伤组织继代次数相同的情况下，威优46和浙辐802的悬浮细胞胞外POD／A_(600)比早星106和粳稻1号的要高。
外源SA、CaCl_2与H_2O_2诱导早星106的悬浮细胞所产生的胞外POD活性并不相同，3mmol／L H_2O_2严重地抑制悬浮细胞分泌所有胞外POD活性，而高浓度的SA和CaCl_2诱导悬浮细胞产生胞外POD活性，没有诱导新的POD产生。经3mmol／L H_2O_2诱导培两优288悬浮细胞后，对胞外POD活性影响不大。
3mmol／L SA、CaCl_2和H_2O_2处理早星106悬浮细胞48h后，均发生不同程度的DNA片段化，而在培两优288中只有SA能诱导产生DNA片段化。利用DAPI荧光染色也观察到细胞质浓缩、染色质凝聚和细胞核解体等凋亡细胞的形态变化。
SA能显著抑制早星106悬浮细胞过氧化氢酶活性(P＜0.01)和使可溶性蛋白质含量下降(P＜0.01)，而H_2O_2、CaCl_2却能显著地促使过氧化氢酶活性(P＜0.01)和可溶性蛋白质含量升高，SA、H_2O_2和CaCl_2都不同程度地抑制胞内POD活性。SA能显著抑制培两优288悬浮细胞过氧化氢酶活性(P＜0.01)和使其可溶性蛋白质含量下降(P＜0.01)，而H_2O_2、CaCl_2却能显著地促进过氧化氢酶活性(P＜0.01)和可溶性蛋白质的积累，SA、H_2O_2和CaCl_2都不同程度地促使胞内POD活性升高。三种外源化学信号分子处理水稻悬浮细胞后其过氧化氢酶活性与可溶性蛋白质含量是极为相关(r=0.92478)。
To inspect signal transmission and mechanism of plant apoptosis, it is necessary to establish an effective and stable induction apoptotosis system in plant cell. Because the system can provide us an effective and reliable way to select plant elicitors and a new path to elect elite varieties of high resistance and anti-adversity for crop breeding. This paper established the induction apoptotosis system of rice suspension cell and discussed the induction apoptotosis mechanism. The results as follows:
Different rice suspension cell systems were established using callus cell of different rice breeds and germintions and the growth status of different suspension cells were compared. The different extraceller POD producing abilities were discussed. The results indicated that rice suspension cell can be established easier when rice callus cell was better dispersive and more difficult to brown. Along with germintion of Zaoxingl06 callus cell, activity of extracelluar POD and suspensibility (A600) raised at first and then declined in suspension cells. In same germintion of callus cell, extracelluar POD/A600 of Weiyou46 and Jingdaol suspension cell were higher, comparing to Zaoxingl06 and Zhefu802.
The activity of extracelluar POD induced by exogenous H2O2, CaCl2 and SA were different from each other , respectively. After Zaoxingl06 suspension cell was treated with 3mmol/L H2O2 , activity of all extracelluar POD was inhibited. But activity of extracelluar POD was increased by high concentration of exogenous SA and CaCl2, and new POD isoenzyme was not produced by PAGE. While the changes of extracelluar POD activity was not obvious when Peiliangyou288 suspension cell was treated with 3mmol/L H2O2.
After Zaoxing106 suspension cell was treated with 3mmol/L SA, H2O2 and CaCl2 for 48 hours, its DNA became fragment at some degree in all treatment. But DNA fragmentation in Peiliangyou288 suspension cell was found only by treatment of 3mmol/L SA. Some changes of apoptosis cell such as the cytoplasm concentrating, chromatin aglomeration and nucleolus disjoint were also observed using the DAPI fluorescence dyes.
After Zaoxing106 suspension cell was treated with SA, the activity of celluar catalase was dramatically inhibited(P<0.01) and soluble protein content was dramatically decreased(P<0.01),but the activity of celluar catalase and soluble protein content were both increased after treated with H202 and CaCl2. After Zaoxingl06 suspension cell was treated with SA, H2O2
and CaCl2 the activities of celluar POD were all inhibited, but after treated with H2O2 the inhibition was the most remarkable. After Peiliangyou288 suspension cell was treated with SA, the activity of celluar catalase was dramatically inhibited (P<0.01) and soluble protein content was dramatically decreased (P<0.01),but the activity of celluar catalase and soluble protein content were both increased after treated with H2O2 and CaCl2. After Peiliangyou288 suspension cell was treated with SA, H2O2 and CaCl2 the activities of celluar POD were increased at different degree, respectively. After rice suspension cell was treated with signal molecules, the activity of celluar catalase and soluble protein content were highly positive correlated(r=0. 92478).
采用不同水稻品种、不同继代次数的愈伤组织建立了不同水稻悬浮细胞体系，比较不同悬浮细胞的生长状况，并探讨了悬浮细胞在培养过程中产生胞外POD活性的变化情况。结果表明：水稻愈伤组织分散性越好，愈伤组织的褐化程度越轻，越容易建立悬浮细胞系。随着愈伤组织继代次数的增加，早星106悬浮细胞胞外POD活性和细胞悬浮度呈先上升后下降趋势。在愈伤组织继代次数相同的情况下，威优46和浙辐802的悬浮细胞胞外POD／A_(600)比早星106和粳稻1号的要高。
外源SA、CaCl_2与H_2O_2诱导早星106的悬浮细胞所产生的胞外POD活性并不相同，3mmol／L H_2O_2严重地抑制悬浮细胞分泌所有胞外POD活性，而高浓度的SA和CaCl_2诱导悬浮细胞产生胞外POD活性，没有诱导新的POD产生。经3mmol／L H_2O_2诱导培两优288悬浮细胞后，对胞外POD活性影响不大。
3mmol／L SA、CaCl_2和H_2O_2处理早星106悬浮细胞48h后，均发生不同程度的DNA片段化，而在培两优288中只有SA能诱导产生DNA片段化。利用DAPI荧光染色也观察到细胞质浓缩、染色质凝聚和细胞核解体等凋亡细胞的形态变化。
SA能显著抑制早星106悬浮细胞过氧化氢酶活性(P＜0.01)和使可溶性蛋白质含量下降(P＜0.01)，而H_2O_2、CaCl_2却能显著地促使过氧化氢酶活性(P＜0.01)和可溶性蛋白质含量升高，SA、H_2O_2和CaCl_2都不同程度地抑制胞内POD活性。SA能显著抑制培两优288悬浮细胞过氧化氢酶活性(P＜0.01)和使其可溶性蛋白质含量下降(P＜0.01)，而H_2O_2、CaCl_2却能显著地促进过氧化氢酶活性(P＜0.01)和可溶性蛋白质的积累，SA、H_2O_2和CaCl_2都不同程度地促使胞内POD活性升高。三种外源化学信号分子处理水稻悬浮细胞后其过氧化氢酶活性与可溶性蛋白质含量是极为相关(r=0.92478)。
To inspect signal transmission and mechanism of plant apoptosis, it is necessary to establish an effective and stable induction apoptotosis system in plant cell. Because the system can provide us an effective and reliable way to select plant elicitors and a new path to elect elite varieties of high resistance and anti-adversity for crop breeding. This paper established the induction apoptotosis system of rice suspension cell and discussed the induction apoptotosis mechanism. The results as follows:
Different rice suspension cell systems were established using callus cell of different rice breeds and germintions and the growth status of different suspension cells were compared. The different extraceller POD producing abilities were discussed. The results indicated that rice suspension cell can be established easier when rice callus cell was better dispersive and more difficult to brown. Along with germintion of Zaoxingl06 callus cell, activity of extracelluar POD and suspensibility (A600) raised at first and then declined in suspension cells. In same germintion of callus cell, extracelluar POD/A600 of Weiyou46 and Jingdaol suspension cell were higher, comparing to Zaoxingl06 and Zhefu802.
The activity of extracelluar POD induced by exogenous H2O2, CaCl2 and SA were different from each other , respectively. After Zaoxingl06 suspension cell was treated with 3mmol/L H2O2 , activity of all extracelluar POD was inhibited. But activity of extracelluar POD was increased by high concentration of exogenous SA and CaCl2, and new POD isoenzyme was not produced by PAGE. While the changes of extracelluar POD activity was not obvious when Peiliangyou288 suspension cell was treated with 3mmol/L H2O2.
After Zaoxing106 suspension cell was treated with 3mmol/L SA, H2O2 and CaCl2 for 48 hours, its DNA became fragment at some degree in all treatment. But DNA fragmentation in Peiliangyou288 suspension cell was found only by treatment of 3mmol/L SA. Some changes of apoptosis cell such as the cytoplasm concentrating, chromatin aglomeration and nucleolus disjoint were also observed using the DAPI fluorescence dyes.
After Zaoxing106 suspension cell was treated with SA, the activity of celluar catalase was dramatically inhibited(P<0.01) and soluble protein content was dramatically decreased(P<0.01),but the activity of celluar catalase and soluble protein content were both increased after treated with H202 and CaCl2. After Zaoxingl06 suspension cell was treated with SA, H2O2
and CaCl2 the activities of celluar POD were all inhibited, but after treated with H2O2 the inhibition was the most remarkable. After Peiliangyou288 suspension cell was treated with SA, the activity of celluar catalase was dramatically inhibited (P<0.01) and soluble protein content was dramatically decreased (P<0.01),but the activity of celluar catalase and soluble protein content were both increased after treated with H2O2 and CaCl2. After Peiliangyou288 suspension cell was treated with SA, H2O2 and CaCl2 the activities of celluar POD were increased at different degree, respectively. After rice suspension cell was treated with signal molecules, the activity of celluar catalase and soluble protein content were highly positive correlated(r=0. 92478).
引文
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