【作者】 李海霞；

【导师】 曾汉来；

【作者基本信息】 华中农业大学 ， 生理学， 2007， 硕士

【摘要】 温度条件是影响植物生长发育的主要生态因子之一，温度的变化引起植物产生复杂的生理响应。探讨水稻对高温适应性的遗传生理基础和全球气温升高的克服对策是当前水稻研究的重要热点之一。此外，光温敏核不育水稻的育性受到温度变化的精确调控，研究温度对育性的调控中的基因表达对阐明光温敏不育水稻的育性稳定性基础、丰富我国两系杂交水稻的理论基础有重要意义。本研究针对上述两方面的内容进行了初步探讨。1．光合作用高温稳定性生理基础研究植物感受温度变化的主要器官是叶片，光合作用是叶片的基本功能，叶片光合作用的高温度稳定性是评价高温适应性的重要指标。本研究在已对几十份不同水稻品种材料进行光合作用高温稳定性鉴定并获得高温稳定的代表品种蜀恢527和高温敏感型品种特籼占的基础上，对这两个材料进行了高温反应下的系列生理差异分析。在对维管束显微结构与气孔特性观察、功能叶片蛋白质表达差异、光合作用关键酶Rubisco大、小亚基和蛋白含量热稳定性与生理指标变化等方面的比较生理学分析后获得初步结果，为分析水稻在高温胁迫下的生理和表达差异特征并进一步研究抗(耐)高温机理积累了基础。主要结果如下：(1)光合作用高温稳定型品种蜀恢527表现出稳定的生理特性，高温下，能维持较稳定的细胞膜结构和较高的脯氨酸水平，增强光合器官耐受高温逆境的能力，以维持相对稳定的光合作用。(2)经双向电泳分析，高温胁迫下产生的热激蛋白(HSP)种类不同，光合作用高温敏感型品种特籼占产生4种HSP，且都为大分子热激蛋白laHSP，分子量和等电点主要分布于63.5-76.8 kD和PI 4.74-5.31之间。高温稳定型品种蜀恢527也产生了4种HSP，但都为小分子热激蛋白smHSP，分子量和等电点主要分布于16.3-23.3kD和PI 4.80-5.91之间，并且除组成型热激蛋白，还产生了1种诱导型热激蛋白。(3)光合作用高温稳定型品种蜀恢527剑叶的Rubisco大、小亚基和蛋白含量的50℃热稳定性均高于敏感型品种特籼占。(4)与敏感型品种特籼占相比，光合作用高温稳定型品种蜀恢527剑叶中脉和第2节间茎秆维管束发达，剑叶表皮气孔密度大，并表现出独特的气孔特性。2．光温敏核不育水稻育性温度敏感性差异分子机理研究在影响光温敏不育水稻育性的光照和温度两个重要因子中，自然光照长度是有规律变化的生态因子，而自然温度的变化则较复杂。不育系的不育临界温度是反映不育系不育性稳定性的关键指标。目前生产上的一个突出问题是经过鉴定的不育系临界温度值存在不稳定性，会逐代升高，从而使该不育系逐渐丧失实用性能。临界不育温度升高后，在遇到低于临界温度的自然条件下造成可育，这是两系杂交稻发展过程中存在的关键技术障碍。本研究以不育临界温度值分别为23℃和28℃的培矮64S近等基因不育系P2364S和P2864S为材料，对在相同温度下处于育性转换温度敏感期的幼穗进行表达谱基因芯片分析，在转录水平上比较近等基因不育系间的生理遗传表达差异，探讨光温敏核不育水稻育性温度敏感性差异的分子机理。所用基因表达芯片为GeneChip Rice Genome Array，包括57381个水稻籼粳亚种的表达序列探针组。结果表明：得到差异表达2倍以上的探针组1334个，为总探针组数的2.32％，对其中差异表达16倍以上的73个探针组进行生物信息学分析，其中54个有推测功能，主要为涉及电子和质子传递、蛋白翻译、细胞生长发育、抗性、信号传导、物质代谢、转座和转录等相关功能的基因。经分析后认为，抗性相关基因、信号传导相关基因和转座相关基因可能与光温敏核不育水稻的育性温度敏感性有关，其中主要功能基因分析与验证有待深入研究。更多还原

【Abstract】 Temperature is one of the main ecological factors that effect plant growth and development, the change of which will arose the plant complicated physiological responses. At present, discussion on rice genetic and physiological basis in high temperature adaptation and the countermeasures of the global temperature rising is one of the important hotspots that research rice. Moreover, fertility of photo-thermo-sensitive genic male sterile (PTGMS) rice is accurately regulated by temperature changes. So, research on the gene expression that regulates fertility by temperature has an important meaning to illustrate the basis in fertility stability of PTGMS rice and enrich the theoretical foundation of two line hybrid rice in China. Two aspects mentioned above were discussed in this paper.1. Study on the physiological basis of high temperature stability of photosynthesis (HTSP)The leaves are the major organ that accepts temperature changes, the basic function of which is photosynthesis. And HTSP of leaves is the important index that evaluates high temperature stability. In this study, Shuhui527-high temperature tolerant representative and Texianzhan-high temperature susceptible representative were screened on the basis of identification of HTSP in tens of different rice varieties, were used, and a series of physiological differences under high temperature were analyzed. The vascular bundle’s microstructure and stomata characteristics investigated, temperature response of differential proteins expression, stability of synthetic contents of protein in enzyme-extracting solution and large and small subunit of Rubisco, physiological indexes determinated were analyzed by comparative physiological methods, and some primary results were obtained. These could accumulate foundation for physiological and differently expressed characteristics under high temperature stress and further study on the resistant/tolerant mechanism to high temperature. The results were as follows:(1) High temperature tolerant cultivar of photosynthesis-Shuhui527 (T- Shuhui527) has the stable physiological characteristics. Because it could maintain the more stable cell membrane structure and higher proline level, resulting in photosynthetic organs’ better suitable ability of high temperature stress, as a result, T-Shuhui527 could keep more stable photosynthesis.(2) T-Shuhui527 and high temperature susceptible cultivar of photosynthesis-Texianzhan (S-Texianzhan) expressed the different kinds of heat shock proteins (HSP) under high temperature stress by two-dimensional gel electrophoresis method. S-Texianzhan expressed four kins of HSP, all classified to large HSP (laHSP), with molecular weights (MW) ranging from 63.5 kD to 76.8 kD and isoelectric points (pI) from 4.74 to 5.31. And T-Shuhui527 also expressed four kins of HSP, but all classified to small HSP (smHSP), with molecular weights (MW) ranging from 16.3 kD to 23.3 kD and isoelectric points (pI) from 4.80 to 5.91. It is remarkable that only one kind of HSP was induced completely in T-Shuhui527, others were the heat shock cognate proteins (HSC) with the differentially expressed levels.(3) The contents of protein in enzyme-extracting solution and large and small subunit of Rubisco in flag leaves of T-Shuhui527 indicated much higher thermal stability when compared to S-Texianzhan.(4) Compared to S-Texianzhan, more developed vascular bundles of main vein of flag leaf and the second internode, and higher stomatal density on epidermis and particular stomatal character of flag leaf were observed in T-Shuhui527.2. Research on molecular mechanism of different temperature sensitivity of male fertility of PTGMS riceThe natural illumination and temperature are two important factors that influced the male fertility of PTGMS rice. The former variation is well-regulated, but the latter is untraceable. The critical sterility temperature point (CSTP) of PTGMS lines is the crucial index that evaluates the stability of male sterility. Badly, CSTP of almost all PTGMS lines identified presents unstability, which seem to have the tendency to gradually increase their CSTP and thus producing fertile pollens under the previous sterile conditions. Some originally qualified PTGMS lines therefore have gradually become ineligible after several generations of propagation. This is the key technical obstacle present in two- line hybrid rice development.In this study, P2364S and P2864S with CSTP of 23℃and 28℃respectively, near isogenic lines of PTGMS rice Peiai 64S were used. The gene expression of spikelet during thermo-sensitive period of fertility transition was analyzed using expression profile genechip technology. The physiology and differential genetic expression at transcriptional level were compared between P2364S and P2864S, and, molecular mechanism of temperature sensitivity of male fertility of PTGMS rice was discussed. GeneChip Rice Genome Array was used, which contained 57381 probe sets representing the expressed sequences of rice japonica and indica cultivars. The results showed that: 1334 probe sets were up- or down-regulated at least 2 fold, accounted for 2.32% of overall probe sets numbers. 73 probe sets up- or down-regulated at least 16 fold expressed differentially were analyzed using bioinformatic methods through the public databases. Among these, there were 55 probe sets having putative functions, which related to electron and proton transport, translation, cell growth and development, resistance, signal transduction, material metabolism, transposon and transcription. After analyzed, the genes related to resistance, signal transduction and transposon may have the relationship with temperature sensitivity of male fertility of PTGMS rice. The analysis and validation of major functional genes need further study.更多还原