【作者】 钟方旭；

【导师】 兰盛银；

【作者基本信息】 华中农业大学 ， 细胞生物学， 2004， 博士

【摘要】 本文以不同品质的水稻(Oryza sativa L．)为材料，研究了不同品种水稻灌浆过程中淀粉胚乳细胞内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、淀粉合成关键酶的活性动态及其与胚乳细胞去核化发育的关系；应用透射电子显微镜观察了中籼8836胚乳细胞发育中各种细胞器结构变化及其消长衍变与胚乳细胞PCD进程；应用扫描电子显微镜观察了籽粒灌浆过程中的胚乳细胞结构和淀粉质体的发育及稻米糊化前后的微观结构；采用差式扫描量热仪(DSC)和质构仪(TA)研究了不同品种稻米的糊化热力学特性及质构品质特征，同时还运用化学方法分离了不同品质稻米的直、支链淀粉和中间成分并测定了其相对含量，主要结果如下： 1．随着胚乳细胞的发育，淀粉胚乳细胞核到花后第5天开始变形，由核被膜的内外层所构成的膜间腔多处变形膨大、染色质凝聚，产生一些大小不一的絮状团块，分散在核内或粘附于核膜的内表面。到花后第6—7天，去核发育加快，核变形、解体也随之加剧。到花后第10天，去核较早的细胞已完成去核化，很难观察到核的残片。Evan’s blue染色检测表明，细胞去核发生的部位是随机的。中籼8836的淀粉胚乳组织全部细胞去核完成约在开花后第12—13天。伴随胚乳细胞去核发育的同时，一部分线粒体开始解体，内嵴断裂直至消失，最后线粒体空泡化或被淀粉粒充满。 2．水稻淀粉胚乳细胞去核过程中细胞核的解体具有一般动、植物细胞程序化死亡的共同特征，去核化发育是淀粉胚乳细胞程序化死亡的第一阶段。淀粉胚乳细胞去核化过程又表现出一些特有现象，如细胞核是最先消亡的细胞器、细胞核在细胞内被发育中的淀粉质体吸收消化、淀粉胚乳细胞核解体产生的核团块类似凋亡小体等，属于一种特殊的PCD现象。 3．胚乳细胞去核化发育阶段SOD和CAT酶活性维持在很高水平，CAT活性在品种间差异不大。高垩白度品种的SOD活性降低快，低垩白度的品种SOD活性降低慢。高垩白度品种的CAT和SOD活性在籽粒发育各阶段比低垩白度品种略低，灌浆快，成熟快。 4．水稻淀粉胚乳细胞去核化发育阶段是整个水稻胚乳发育期代谢活性最高的时期，无论是籽粒增重，还是灌浆速率都是最快的时期，淀粉合成酶活性处于很高水平。高垩白度品种的AGPase在花后9天达到活性高峰，低垩白度品种的峰值滞后3天出现，AGPase活性动态在不同直链淀粉含量类型品种间没有明显差别；各品种问SS酶活性差异较大，花后SS酶活性持续增加，除桂朝2号酶活性在花后12天达最大值外，其余品种在花后9天达到最大值，酶活性达峰值后即急剧下降；Q酶的活性变化趋势不同于AGPase、SS，后者酶活性在籽粒灌浆期变化幅度较大，Q酶活性变幅不大，处于高活性水平的时间较长，花后20天达活性高峰。不同品质水稻Q酶的活性高低与垩白度大小没有显著相关性，低直链淀粉含量品种的Q酶活性钟方旭:水稻胚乳细胞PCD中的去核化及酶活性动态与稻米品质的关系大于同时期高直链淀粉含量品种。坚白度与AGPase活性动态相关极显著、与55和SBE活性动态相关性不显著，直链淀粉含量与AGPase活性动态相关性不显著、与55和SBE活性动态相关性显著。 5.稻米奎白的形成与水稻胚乳细胞内淀粉粒的形状、大小、结构和发育进程均有密切联系。桂朝2号与明科釉的胚乳微观结构从胚乳发育开始到灌浆结束一直存在差别，明科釉虽然早期灌浆速度较桂朝2号缓慢，但其淀粉细胞和淀粉粒的发育进程却比桂朝2号快，这一特征贯穿了胚乳发育的整个过程。长柱形细胞和圆球形淀粉粒多是品质差的稻米所具有的典型特征。 6.品质不同的釉米粉，其糊化温度和烩变差别很大。T。、几、T。和△H表现出正相关，糊化起始温度高者，其峰值温度和终结温度也高，糊化过程中吸热也多。高至白度品种的糊化起始温度(T。)、峰值温度(Tp)和△H均显著大于低奎白度品种。T。、几、△H与奎白度表现出极明显的正相关，与直链淀粉含量相关性不显著;T。与奎白度、直链淀粉含量相关性都不显著。 7.至白度和直链淀粉含量对米饭的食味品质影响很大，主要影响米饭的咀嚼度、粘性、硬度、破碎度和弹性。翌白度和直链淀粉含量越高，米饭硬度越大，弹性越好，破碎度(使米饭破碎所需的压力值)越大，食味越差。 8.高至白度的稻米糊化困难，在蒸煮过程中糊化不彻底，严重影响米饭的口感和食味。同等直链淀粉含量的釉米比较，坚白度越大，长柱形细胞越多，稻米吸水率越低，糊化越困难;米质好的胚乳细胞易破裂，淀粉粒容易暴露，而米质差的胚乳细胞不易破裂，断裂一般发生在细胞间而不是细胞内部;扫描电镜观察稻米蒸煮糊化后结构揭示，长柱形细胞具有较厚细胞壁和细胞膜可能是造成高呈白度稻米糊化难的原因之一。 9.分离三种淀粉成分得到的直链淀粉含量与碘试剂显色测定结果不同，不同类型品种的变化不一。明科釉和桂朝2号的直链淀粉分离物含量较表观直链淀粉含量减小;高奎白度品种的DMSO不溶物含量高于同等直链淀粉含量的低奎白度品种，其中间成分的含量低于同等直链淀粉含量的低至白度品种。DMSO不溶物和直链淀粉含量高、中间成分含量低可能是导致同等表观直链淀粉含量釉稻品种具有不同米更多还原

【Abstract】 Using different varieties with varied quality as materials, dynamics of enzyme activity of SOD, CAT and key enzymes of starch synthesis in starchy endosperm cell and relation between them and denucleation development of endosperm cell during filling stage were studied; Structural variation and development of PCD process of endosperm cell during endosperm developing stage of an indica rice variety zhongxian 8836 were observed with transmission electron microscope (TEM); Endosperm cell structures, development of starch plastid and microstructures before and after gelatinization during grain filling process were observed with scanning electron microscope (SEM);Thermodynamic property of gelatinization and feature of texture quality of different varieties were studied with differential scanning calorimetry (DSC) and texture analyzer(TA),the amylose, amylopectin and intermediate fraction of rice with varied quality were isolated by chemical methods, whose relative contents were measured, the main results were as follows:1. Starchy endosperm nucleus begin to deform on the fifth day after blooming with the developing of endosperm cells, intermembranous space composed of intrinsic-extrinsic layer of nuclear envelope deform and enlarge in many positions, the chromatin condense and produce some flocculent masses of different sizes, which disperse in cellular internalization or stick to the internal surface of nuclear membrance. On the sixth to seventh day after blooming, the denucleation development accelerate, nuclear deformation and disintegration also aggravate. On the 10th day after blooming, it is impossible to see the nuclear relic in the cells denucleated earlier which have finished their denucleation. Through Evan’s blue staining method, we found the occurrence of cellular denucleation is random in position. All cells in the tissue of starchy endosperm of the variety Zhongxian 8836 have finished their denucleation on the 12th to 13th day or so after blooming. At the same time of denucleation development of endosperm cell, a part of mitochondrions begin to disintegrate, the internal cristae collapse and disappear slowly, the interior of mitochondrion change into bubble and are full of starch grains.2. Nuclear disintegration in rice starchy endosperm cell during denucleation process has the common feature with the PCD of general plant and animal, denucleation is the first period of PCD in starchy endosperm cell, denucleation process of starchy endosperm cell present some special phenomena, for example, cell nucleus is the first cell organ dying out, nucleus is absorbed and digested by starchy plastid in the cell during developing stage, nuclear aggregates produced in the process of starchy endosperm cellnuclear disintegration are similar to the apoptosis body. Which belong to special PCD phenomena.3. Enzyme activities of SOD and CAT are higher in endosperm cell during denucleation developing stage, the difference of enzyme activity of CAT between different varieties is not notable. The bigger the chalkiness of the variety is, the faster the enzyme activity of SOD decreases, if the condition is reverse, the result is reverse. Enzyme activities of SOD and CAT in the varieties of higher chalkiness are slightly lower than those of lower chalkiness, whose grain age shorter, the ripening speed faster.4. The metabolic activity of rice starchy endosperm cellular denucleation developing stage is the highest in whole developing stage, no matter grain weight increase or filling rate are both the fastest, enzyme activity of starch synthetase is higher. Enzyme activity of AGPase in the varieties of higher chalkiness will reach peak value on the 9th day after blooming, while that of lower chalkiness will delay 3 days, the differences are not obvious comparing the dynamics of enzyme activity of AGPase between the varieties of different content of amylose; enzyme activity variance of SS in different varieties is great, enzyme activity of SS will keep increasing after blooming, enzyme activity of the va更多还原