【作者】 刘晓璐；

【导师】 万建民；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2010， 博士

【摘要】 垩白是指稻米胚乳中白色不透明的部分。垩白现象的出现直接影响稻米的外观品质和蒸煮品质,这是因为高垩白的稻谷在精碾加工过程中极易破碎、整精米率低,并且在蒸煮后米饭蓬松中空、蒸煮食味品质较差。研究表明,稻米垩白性状属于复杂的数量性状,受多基因控制,目前对于垩白形成的分子机制尚不清楚,这在很大程度上阻碍了稻米品质的遗传改良。本研究以Asominori为遗传背景、IR24为供体的高垩白染色体片段置换系(CSSL)和低垩白品系Asominori为材料,通过生理生化分析和cDNA微阵列分析,研究稻米垩白形成的酶学特性及相关基因的表达调控。研究结果表明,稻米垩白的形成具有复杂的内在生理环境,与灌浆期水稻籽粒细胞内的碳代谢(淀粉合成和降解;纤维、半纤维素和果胶生物合成)、信号传递、细胞防御、抗氧化平衡等基因功能类群和调控网络途径密切相关。此外,研究中同时发现高垩白CSSL50中的氧化-还原体系被激活,表明ROS信号传导途径与稻米垩白形成存在密切的相关性。主要研究结果如下:1.稻米垩白的形成与淀粉合成酶动力学变化、较快的籽粒灌浆速率,以及淀粉粒的精细结构相关。生理生化分析研究结果发现,高垩白置换系CSSL50与低垩白品系Asominori胚乳细胞中淀粉体的形态存在差异,Asominori淀粉体为多边形、排列紧密、颗粒间基本没有空隙、透光性好;然而,CSSL50籽粒胚乳垩白部位的淀粉体为椭圆形、排列疏松、颗粒间存在较大空隙、透光性差;与Asominori相比,CSSL50中聚合度为8和9的短链比例显著偏高,聚合度为12的中长链,以及聚合度为17的长链所占比例显著偏低;高垩白CSSL50的SuSy、AGPase、SBE和DBE酶的活性显著高于低垩白Asominori;在灌浆后10-15 d时,CSSL50籽粒灌浆速率明显高于Asominori。上述结果表明CSSL50高垩白性状的形成与淀粉合成酶动力学变化,较快的籽粒灌浆速率,以及淀粉粒的精细结构密切相关。2.稻米胚乳垩白的形成可能与灌浆期籽粒细胞内的信号传递、细胞防御、抗氧化平衡、碳代谢、转录调控和蛋白降解等基因功能类群和调控网络途径密切相关。基因表达谱分析发现,以低垩白背景亲本Asominori为参比,高垩白置换系CSSL50中极显著上调或下调(P < 0.01)的基因623个,其中上调的有324个,下调的有299个。结合差异基因的相关功能注释,将此623个差异表达基因分为18个功能类别,其中6个主要功能类别基因群(信号传递、细胞防御、抗氧化平衡、碳代谢、转录调控和蛋白降解)占差异显著基因总量的68.8%,说明稻米胚乳垩白的形成可能与灌浆期籽粒细胞内的信号传递、细胞防御、抗氧化平衡、碳代谢、转录调控和蛋白降解等基因功能类群和调控网络途径密切相关3.稻米垩白的形成与多糖合成关系密切。结合基因差异表达、碳代谢相关酶活性和物质含量变化等研究结果,发现稻米垩白的形成与增强的淀粉代谢和减弱的纤维、半纤维素及果胶生物合成密切相关。基于生理生化测定结果和基因表达谱分析结果对碳代谢相关差异表达基因进行pathway构建,发现水稻碳代谢影响稻米垩白形成的机制表现出一致性,即在形成稻米高垩白的生理环境中,淀粉合成能力增强,降解程度减弱,与此相反,在低垩白环境下胚乳细胞中纤维素和半纤维素的合成能力减弱,降解程度增强。4.基于本研究中基因芯片、RT-PCR和过氧化氢含量变化的研究结果,可发现高垩白近等基因系CSSL50中抗氧化体系被激活。本研究发现高垩白CSSL50的过氧化氢含量显著地高于低垩白背景亲本Asominori。同时,SOD、APX、GPX、MDAR和PrxR等抗氧化基因在高垩白CSSL50中的表达水平均显著高于Asominori,说明高垩白CSSL50籽粒胚乳中的抗氧化体系被激活,清除多余氧化自由基(如:过氧化氢等)的能力高于Asominori。此外,GST、Glx和Trx基因在高垩白CSSL50中的表达水平也均显著高于Asominori;而CSSL50的LOX基因表达水平显著地低于Asominori;RT-PCR结果进一步证明了这一结果。因此,CSSL50籽粒胚乳中的抗氧化修复体系的功能明显高于Asominori,说明稻米胚乳垩白产生是在被激活(提高)的抗氧化水平的环境下进行的,且通过改变碳水化合物代谢而实现的。5.综合上述研究结果得出稻米垩白的形成同时受特定基因型和特定生理环境互作决定。稻米垩白形成的内在生理环境特征表现为,蛋白质合成和降解能力均增强、淀粉合成能力增强,但是纤维素和半纤维素的合成能力减弱;基因表达谱等分子研究结果显示,在稻米垩白形成过程同时由多种激素、逆境信号途径的参与和互作;其中,ROS信号传导途径与稻米垩白形成密切关联,而氧化-还原体系被激活程度及其消除胚乳生境内ROS的能力可能将在一定程度上决定了稻米垩白的形成度。至此,本研究在基因水平上发现了CSSL50产生垩白的原因/机理与经典遗传和生理研究结果“外界环境胁迫(如:高温、光照条件等)条件导致稻米垩白产生”相一致。更多还原

【Abstract】 Grain endosperm chalkiness of rice is a varietal physical characteristic that negatively affects not only the appearance and milling properties but also the cooking texture and palatability of cooked rice. Chalky grains have a lower density of starch granules compared to vitreous ones, and are therefore more prone to breakage during milling. In many rice-producing areas, high chalkiness represents one of the most serious problems that decrease grain quality. However, chalky grain is a complicated quantitative trait and the molecular mechanisms underlying its formation are still poorly understood. In this study, we performed a comparative transcriptome analysis of the caryopses of a pair of near-isogenic lines, CSSL50 with high chalkiness and its low chalkiness parental line Asominori. Corroborated with the phenotypic and physico-biochemical observations, our genome-wide transcription analysis supports the notion that rice grain endosperm development is controlled by delicate, but complex genetic networks. Our results revealed that grain endosperm chalkiness of rice formation entails an intricate gene network, including genes involved in carbon metabolism, starch biosynthesis and degradation, metabolism of cellulose, hemicellulose and pectin, signal transduction and most interestingly, we found that ROS signaling and homeostasis are closely related to the formation of rice grain endosperm chalkiness. The important results were as follows:1.The results of analyses at the phenotypic, physiological and biochemical levels showed that the occurrence of endosperm chalkiness in rice might associate with the changes of enzyme activities, the increased of grain-filling rate and the structure of starch granule. Asominori amyloid is of the form of polygon, tightly arranged, few inter-space among the granules, and good transparency; however, the amyloid on the chalky portion of CSSL50 seed granule endosperm is of the form of ellipse, loosely arranged, big interspace among the granules, and poor transparency. Besides, the SuSy, AGPase, SBE, and DBE for high chalkiness CSSL50 is remarkably higher than that for low chalkiness Asominori. What is more, on the 10-15th day after grain filling, the seed grain filling speed for CSSL50 is remarkably higher than that for Asominori. These results show that the high chalkiness of CSSL50 is most likely due to the dynamic change of starch-composed enzyme and high seed grain filling speed, and to the fine structures of starch granules.2.Based on the results of gene expression profiles between Asominori and CSSL50, we found the pathways of signal transduction, cell rescue/defense, transcription, protein degradation, carbohydrate metabolism and redox homeostasis might be play a significant role in the formation of chalkiness in rice. the occurrence of endosperm chalkiness in rice might be closely related to these functional and regulatory pathways. For the vast majority of probes, expression appeared unchanged between Asominori and CSSL50. Using the selection criteria of P value<1%, we identified a total of 623 probe sets with a significant change. Of these, expression of 324 was up-regulated and 299 down-regulated. Based on the bioinformatics tools such as Gene ontology and JAFA , and the metabolic and functional features of rice, a total of 623 differentially expressed genes were classified into 18 major categories. 68.8% of these identified genes were implicated in the first six functional groups (signal transduction, cell rescue/defense, transcription, protein degradation, carbohydrate metabolism and redox homeostasis), suggesting that the occurrence of endosperm chalkiness in rice might be closely related to these functional and regulatory pathways.3.From three aspects of expression of gene difference, carbon- metabolization-related enzyme activities and substance content, we found that there exists a close relationship between the biosynthesis/hydrolysis of starch and non-starch polysaccharide and the formation of endosperm chalkiness, namely, the enhanced starch biosynthesis and the decreased biosynthesis of non-starch polysaccharide may result in the occurrence of chalkiness in rice endosperm.4.Based on the results in this study on gene chips, RT-PCR, and physiochemical data (eg., H2O2 content), it is found that the antioxidation system in high chalkiness isogen CSSL50 is activized. It is also found that the hydrogen peroxide content of high chalkiness CSSL50 is remarkably higher than that of low chalkiness background parent strain Asominori. Meanwhile, the expression level of antioxidation genes such as SOD, APX, GPX, MDAR,and PrxR in high chalkiness CSSL50 is remarkably higher than that in Asominori, which shows that the antioxidation system in high chalkiness CSSL50 seed granule endosperm is activized and the ability of eliminating extra oxidation free radicals (such as H2O2, etc.) in CSSL50 is higher than that in Asominori. Besides, the expression level of GST, Glx, and Trx genes in high chalkiness CSSL50 is also remarkably higher than that in Asominori and the expression level of LOX gene in CSSL50 is remarkably lower than that in Asominori,which is further testified by the RT-PCR result.Then it is concluded that the function of antioxidation repair system in CSSL50 seed granule endosperm is remarkably higher than that in Asominori, which shows that the emergence process of the rice endosperm chalkiness begins in the environment of activized (increased) antioxidation level, and is fulfilled through changing carbohydrate metabolization.5 . To conclude, exterior stressful conditions are likely to further influence the carbohydrate metabolization of rice by changing the oxidation-reduction balance in the plant, finally leading to the emergence of chalkiness, which agrees with the conclusion of a dissertation by Wan et al (2008)—“The stresses of coldness, drought, salt, heavy metal,etc can lead to the fact that H2O2 content in the rice is increased and oxidation-reduction system in the rice plant is activized, finally affecting the carbohydrate metabolization.”As for the high chalkiness CSSL50, the cause for forming its chalkiness is similar to the case in which a general low chalkiness variety can produce high chalkiness in coercive environment.更多还原