【作者】 王妮妍；

【导师】 蒋德安； 翁晓燕；

【作者基本信息】 浙江大学 ， 植物生理学， 2003， 硕士

【摘要】 本研究通过对水稻叶片中Rubisco及其活化酶进行细胞器定位，同时结合水稻光合日变化规律，再利用免疫扩散定量分析法，研究这两种酶的分布、含量以及活力在一天中的动态变化，深入探讨了光合关键酶Rubisco和RCA活性在中午下降的原因；同时，通过对剑叶衰老期间光合及光合关键酶动态的观察以及对10叶期水稻全株不同叶位叶片和整个衰老期间单个叶片(第11叶和剑叶)衰老相关生理生化变化关系的研究，为光合“午休”理论的深入、Rubisco活化酶生理功能的进一步明确以及延缓光合下降，提高水稻光合生产率等方面提供了理论依据，也为进一步利用分子生物学手段研究衰老调控做些基础性工作。 免疫胶体金标记结果显示，Rubisco主要分布于叶绿体中，而Rubisco活化酶可分布于叶绿体和线粒体中；Rubisco初始活力和RCA活力与光合日变化密切相关；在光照最强的13时，出现光合“午休”，叶绿体中Rubisco的密度有一定程度降低，而全叶的总Rubisco保持稳定，Rubisco初始活力也有明显的“午休”，这意味着体内Rubisco的活力除受RCA调节外，可能还与叶绿体中Rubisco的分布有关。RCA活力变化与叶绿体中RCA含量变化较为一致，表明RCA在叶绿体中的分布对调节其本身活力和Rubisco活性有重要作用。 水稻剑叶抽穗后，其光合速率持续下降，但下降有缓降和速降期，缓降期(抽穗后巧天内)内剑叶的光合速率变化不大，抽穗后7天达最大值:速降发生在第14一21天。剑叶光合速率的缓降和速降与Rubisco初始活力和RCA活力与含量的缓降和速降有关。不同叶位的叶片，其叶绿素含量、Hill活力、可溶性蛋白含量和SOD活力都呈现从上到下先升后降的变化，但不溶性蛋白含量变化很小，MDA含量则是持续上升:n叶和剑叶从出叶到近死亡过程中，叶片叶绿素含量、Hill活力、可溶性蛋白含量以及SOD活力也都是先升后降，MDA含量持续上升，不溶性蛋白含量的变化幅度相对较小。对全株从上到下的叶片与第11叶出叶后5天、剑叶出叶后5天各项生理生化指标每隔5天的值对应进行回归分析，结果表明，Hill活力、可溶性蛋白含量、MDA含量和SOD活力，均有显著正相关。Hili活力变化上，不同叶位叶片的变化与第11叶和剑叶的变化相关系数分别为0.78和0.8695;可溶性蛋白含量变化上，相关系数分别为0.558和0.6188;MDA含量变化上，相关系数分别为0.6936和0.6368;SOD活力变化上，不同叶位叶片与第11叶的变化相关系数为0.6603，与剑叶没有相关性;而在叶绿素含量和不溶性蛋白含量变化上都没有相关性或相关系数很小(梦<0.3)。这表明除叶绿素和不溶性蛋白含量外，全株从上到下每个叶位叶片的生理生化指标相当于n叶或剑叶从展开起到死亡每隔5天的生理生化指标。更多还原

【Abstract】 In this paper, we studied the cellular localization of Rubisco and Rubisco activase (RCA) in rice (Oryza sativa subsp. indica cv. Zhenong 952) leaf with immunogold-labeled electron microscope techniques on the basis of determining the diurnal changes of net photosynthetic rate (Pn), Rubisco and RCA activities, and quantifying two enzyme contents in the leaf with immuno-diffusion method in order to understand why Rubisco and RCA activities decreased in the midday when their contents were high. Meanwhile, we investigated the Pn and dynamic changes of Rubisco and RCA of flag leaf during the senescence, surveyed the physiological and biochemical characteristics of the different leaves in a whole rice plant, the 11th leaf and flag leaf from their extending to dying, respectively. Moreover, we analyzed the physiological relationships between the kinetic changes of 11th leaf or flag leaf during that time and the positional changes of the leaves for the whole plant at 10-leaf stage. These studies can help us to understand further the physiological and biochemical characteristics or the regulatory mechanisms of senescence in rice leaves.Our results showed that peaks of Pn, initial Rubisco carboxylation activities (IRA) and RCA activities appeared around 8:00 and 14:00, respectively, and the midday-depressions occurred around 13:00. Comparing the changes of Pn and initial Rubisco activity, the highest peak of RCA was around 14:00 rather than around 8:00. The results of quantification of Rubisco and RCA using immuno-diffusion method showed a trend that contents of Rubisco and RCA were not abruptly fluctuated during a day compared with their activities. Rubisco content increased from 6:00 to 8:00, and it kept rather constant thereafter. Immunogold-labeled electron microscope techniques indicated that Rubisco mainly located in chloroplast, and RCA were found both in chloroplasts and mitochondria. The decline of Rubisco in chloroplast as well as Rubisco activity at noon can be one of good reasons to explain the photosynthetic midday depression in leaf. The density of RCA in chloroplasts reached the maximum at 14:00 and a valley at 11:00. The result was much coincided with the activity of RCA in leaf. In mitochondria, the density of RCA changed abruptly in one day with the highest at 13:00, and itcan well elucidate why the activities of Rubisco declined at noon when its amount was increasing. Therefore the cellular localization and/or distribution of Rubisco and RCA during a day is much important for Pn, Rubisco and RCA activity.The decreasing of Pn in flag leaf can be obviously divided into two phases, one was a slow decline occurred during 7-14d after heading, the other was a rapid one initiated at 15d after heading. The falling pattern of Pn corresponded with those of the initial Rubisco and RCA activities as well as RCA content.In different leaves of the whole rice plant, the chlorophyll content, Hill reaction activities, soluble protein contents and SOD activities increased first and then declined, while the insoluble protein contents kept steady, and the malondialdehyde (MDA) contents increased continually as lowering of leaf position. The decreasing of those physiological and biochemical characteristics was also found in the 11th leaf or flag leaf during their senescence.The analysis of the relationship showed a positive correlation between the senescence patterns for all characteristics except for the contents of chlorophyll and insoluble protein in the 11th leaf or flag leaf and the positional leaves of a whole plant at 10-leaf stage. The R2 for the Hill reaction activities was the highest (0.78-0.8695), the R2 for MDA contents was second (0.6368-0.6936), and the following were soluble protein contents (0.558-0.6188). For SOD activities, there was no positive correlation between the flag leaf and the positional leaves, but the determinant coefficient between the 11th leaf and the positional leaves of a whole plant was high (0.6603).It intimated that the senescence-related changes of different leaves更多还原