【作者】 师瑞红；

【导师】 谢国生；

【作者基本信息】 华中农业大学 ， 生理学， 2007， 硕士

【摘要】 铝是酸性土壤中限制水稻生长及矿质元素吸收而导致减产的主要因子之一。本研究以IR24、金优725、两优培九为材料，结合溶液培养和铝胁迫处理，采用细胞器的分离，SDS-PAGE以及RP-HPLC等研究手段，系统研究了不同铝浓度对发芽期和四叶一心期水稻幼苗根系铝胁迫相关生理特性的影响。主要结果如下：1．水稻发芽期铝胁迫浓度梯度处理实验表明，0.075、0.1mmol／L铝处理时，金优725和两优培九的根长、单株根重和根系活力较CK小幅度下降或增加，IR24各指标有小幅度下降，各材料处理间差异不显著。0.30mmol／L-7.5mmol／L铝处理时，3个供试材料的上述指标都明显下降，品种间、处理间差异显著，尤其是IR24下降幅度最大。7.5mmol／L铝处理时3个材料都受到了严重的铝毒害。因此，0.3mmol／L是鉴定耐铝胁迫的临界浓度。金优725和两优培九的根系生长指标优于IR24，可以认为是相对耐铝性品种。2．低浓度（0.50mmol／L）和高浓度（7.5mmol／L）铝胁迫处理进一步证实3个供试材料耐铝性的差异。然后，采用SDS-PAGE技术分析了铝胁迫下地上部可溶性蛋白的变化，发现了4条分子量分别为80.8kD，78.5kD，52.9 kD和16.4kD的蛋白谱带在铝处理后表达量减少，推测与铝胁迫有关，但是本实验中没有发现特异表达的谱带。3．外源有机酸（柠檬酸、苹果酸、琥珀酸）和铝胁迫处理实验表明，1.5mmol／L铝胁迫处理抑制了水稻幼苗根系的生长，同时，根系线粒体中POD和APX活性明显增加，CAT活性变化不大；而添加有机酸后，铝胁迫得到了明显缓解，POD和APX活性呈下降趋势，CAT活性变化相对较小，表明铝胁迫引起水稻根系细胞线粒体中活性氧代谢平衡发生变化，而有机酸的加入调节了铝胁迫下根系细胞线粒体中活性氧清除酶的活性，从而部分缓解了发芽期水稻幼苗的铝毒害。4．四叶一心期水稻幼苗铝胁迫处理10d后，发现幼苗的根长、苗长、新根长、单株苗鲜重以及根系活力都下降。其中，对IR24的影响最大，各项指标下降幅度均大于金优725和两优培九。同时，测定了两种杂交稻的呼吸速率，发现随铝浓度的增加，2个杂交稻的抗氰呼吸呈现先增加后降低的趋势，但总呼吸速率的变化趋势随品种不同而不同。5．四叶一心期水稻幼苗铝胁迫处理10d后，根系线粒体中的H₂O₂含量增加，活性氧清除酶活性随品种不同而不同。随铝浓度的增加，POD呈现敏感品种中先升后降，耐性品种中上升的趋势，而且品种问和处理间差异显著。CAT在低浓度处理时增加但不明显，高浓度铝处理时各个材料中酶活性大幅度增加，敏感品种的增加幅度大于耐性品种，APX的变化趋势与POD基本一致。6．四叶一心期水稻幼苗铝胁迫处理10d后，水稻幼苗根系中有机酸含量的RP-HPLC测定结果表明，随铝胁迫处理浓度的提高，谷氨酸含量呈倒“V”字形变化；但PEP含量呈增加趋势，这可能在一定程度上刺激了抗氰呼吸速率；柠檬酸含量呈下降趋势，推测可能与它分泌到细胞外参与解铝毒作用有关，也可能是由于它的合成减少或者被分解或转化成了其它物质。通过对以上几方面的研究，认为水稻根系呼吸与耐铝性的关系密切；线粒体活性氧清除酶的活性变化对耐铝性有重要意义；有机酸代谢水平与铝胁迫适应性有关。更多还原

【Abstract】 Aluminum is one of the major constraints limiting rice productivity in acidic soil, which inhibits growth and minerals absorption of rice root. In the research, IR24, Jinyou 725, Liang-you-pei-jiu at germination and four-leaf stage were employed to carry out hydroponical culture coupled with aluminum stress, and then the physiological characteristics of rice seedlings responding to aluminum stress had been explored, using organelle preparation, SDS-PAGE （sodium dodecyl sulfate-polyacrylamide gel electroph- oresis） and RP-HPLC （reverse phase high performance liquid chromatography） techniques. The main results obtained are as follows:1. The experiment was conducted with the germinated rice seedlings exposed to 0, 0.075mmol/L, 0.10mmol/L, 0.30mmol/L, 1.5mmol/L, 3.0mmol/L and 7.5mmol/L aluminum stress. It demonstrated that, under the lower concentration aluminum treatment such as 0.075 and 0.10mmol/L, there was little increase or decrease in the root length, fresh root weight of single plant and root activities ofjinyou 725 and liang-you-pei-jiu, and a little decrease in that of IR24 compared with their respective control, showing no significant difference between different treatments. Under such higher concentration treatment as 0.30mmol/L, 1.5mmol/L, 3.0mmol/L and 7.5mmol/L, the three indices above of the three cultivars were decreased significantly, especially that of IR24 did. The growths of the three varieties tested were all arrested under 7.5mmol/L aluminum stress. Therefore, it suggested that 0.30mmol/L was the critical aluminum concentration to evaluate the rice germplasm to aluminum tolerance. Finally, jinyou 725 and liang-you-pei -jiu were considered to be the more tolerant cultivars; whose relative growth indices were better than that of IR24.2. On the basis of tolerance difference confirmation of the three culitivars during germination after the stress treatment of 0.50mmot/L and 7.5mmol/L aluminum, the effect of aluminum on soluble protein in rice shoots was screened by SDS-PAGE, four polypeptides with molecular weight of about 80.8kD, 78.5kD, 52.9kD and 16.4kD had been found to decline under aluminum treatment, suggesting that they were probably associated with aluminum stress, but there was no novel polypeptide induced under aluminum stress.3. Aluminum stress experiment was done with the addition of exogenous organic acids such as citrate, malic acid and succinic acid to the culture solution. It showed that, the growth of rice roots was inhibited by 1.5 mmol/L aluminum, the activities of POD （peroxidase） and APX （ascorbate peroxidase） in mitochondria of rice roots were increased remarkably and that of CAT （catalase） changed little. After the addition of exogenous organic acids, aluminum toxicity was obviously alleviated. The activities of POD and APX tumed out to be declined and that of CAT changed little, suggesting that the metabolism homeostasis of ROS （reactive oxygen species） was changed by aluminum stress and the activities of antioxidant enzymes in mitochondria of the germinated rice root were regulated by exogenous organic acids and thus aluminum toxicity was then alleviated.4. Such morphological characters as root length, shoot length, new root length, flesh shoot weight of single plant and root activity were all decreased after aluminum treatment for ten days on rice seedlings of four-leaf stage. IR24 showed obvious decline in the above five indices than the other two Al-tolerant cultivars jinyou725 and liang-you-pei-jiu, that is to say, IR24 was injured more badly than the other two cultivars. Meanwhile the respiration rate of the two hybrid rice cultivars was determined and we found that the cyanide-resistant respiration rate of them had a rise-fall tendency as aluminum increased, while the total respiration rate varied from each other.5. After the rice seedlings of four-leaf stage had been treated with aluminum for ten days, H₂O₂ （hydrogen peroxide） content in mitochondria increased when the concentration of aluminum were increased and the activities of antioxidant enzymes in mitochondria varied differently. The activities of POD increased first and then decreased in Al-sensitive cultivar but increased all the way in Al-tolerant cultivar as the concentration of aluminnum increased, and the differences between them under different aluminum stress were found to be significant. The change of APX showed a similar tendency to that of POD. The activities of CAT changed slightly under 0.50mmol/L and increased sharply under 7.5mmol/L aluminum stress. Meanwhile, the activities of CAT increased more obviously in Al-sensitive cultivar than Al-tolerant one.6. The content of organic acids were measured by RP-HPLC in rice root of four-leaf stage after ten days’ aluminum stress. The content of glutamic acid was increased under 0.50mmol/L and decreased under 7.Smmol/L aluminum stress, showing a upside-down "V" shape. The content of phosphoenolpyruvate （PEP） was increased as aluminum concentration increased and it was probably involved in the stimulation of the cyanide-resistant respiration to some extent. However, the content of citrate in rice roots showed a tendency of being decreased as the concertration of aluminum increased, which was probably due to its exudation for the detoxification of aluminum externally, reduction of its synthesis, decomposition or transformation into other substance. By research on the items above, we can draw a conclusion that there was a close relationship between respiration and aluminum tolerance of rice, the dynamic change of the activities of antioxidant enzymes in mitochondria of rice root played an important role in aluminum tolerance of plants and the metabolism of organic acids was associated with the adaption of rice to aluminum stress.更多还原