【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， 2011， 博士

【摘要】 全球气候变暖已经成为不可逆转的变化趋势,给生态环境和粮食安全生产带来了重大影响,并且这种气温升高的潜在影响将进一步加剧。目前气候变暖对农作物生产的影响倍受各国科学家和政府的重视。温室气体所导致的气候变化使平均气温升高,且夜间温度上升幅度更为明显。水稻生长期特别是开花及灌浆期的高温热害导致结实率大幅降低甚至绝收,同时影响稻米品质。近数十年来,国内外关于高温对水稻产量和米质影响的研究较多,水稻高温遗传与生理基础研究已成为热点方向。研究籽粒形成过程中光期和暗期的高温影响差异,将对充分认识和评估气温升高对水稻产量和品质的影响有一定参考意义。本研究选取高温适应性不同的4个中籼稻品种9311、蜀恢527、N22和特籼占,利用人工气候箱在灌浆期进行不同的温度处理：光期高温(35℃/27℃)、暗期高温(27℃/35℃)和对照(28℃/20℃),时间为受精后持续20 d,对灌浆期不同温度处理对稻米品质和籽粒蛋白表达谱的差异进行分析,旨在为揭示灌浆期光期高温和暗期高温胁迫下引起的水稻籽粒形成和品质变化的差异分子生态机理积累一定基础。主要结果如下：1.高温处理下各品种水稻籽粒都比对照灌浆速率大且成熟早,暗期高温对籽粒鲜重的影响大于光期高温；直链淀粉的积累在灌浆前期受高温促进,而后期受高温抑制,两种高温处理对灌浆期直链淀粉量的差异影响主要发生在灌浆早期,各品种间差异表现不一；高温处理降低了粒重、糙米率、精米率、整精米率、直链淀粉量和胶稠度,使垩白度增加,不同高温处理间、品种间的差异只是这些特征的变化幅度不同,其中整精米率和垩白度的变化幅度较大；与光期高温相比,暗期高温对粒重和精米率的影响较大,对整精米率的影响较小；两种高温处理对糙米率、垩白度和直链淀粉含量的差异影响在各品种间表现不一。2.用双向电泳技术对不同灌浆时期不同高温处理下籽粒的蛋白表达谱进行分析,确定了81个高温处理差异表达蛋白点,经质谱分析后成功鉴定出66个差异蛋白,将其按功能分成8类：碳水化合物代谢、氨基酸代谢、胁迫与防御蛋白、信号转导、蛋白合成与定位、多功能、功能未知和其它分类蛋白；同时对这66个差异表达蛋白点响应不同高温处理的表达变化进行了分析,暗期高温和光期高温间呈现出5种不同的蛋白表达变化类型；不同品种中,暗期高温和光期高温间的差异表达蛋白的变化类型具有相同点和特异性。3.在成功鉴定的所有蛋白中,11个蛋白质拥有2-5个同源异构体,对它们在两个品种的4个灌浆阶段中的表达变化进行了分析,获得水稻籽粒中包括PPDK和支链淀粉酶在内的同源异构体响应不同高温胁迫的动态表达谱,大部分同源异构体及其在9311和特籼占两个品种间的表达变化类型不同；通过分析发现cyPPDKB同源异构体的表达量与垩白度的大小负相关,可能由于cyPPDKB的表达量受到不同程度的抑制而导致暗期高温和光期高温处理间垩白度的不同。4.水稻灌浆期高温胁迫激发了水稻灌浆籽粒中包括h型硫氧还蛋白、过氧化物氧还酶、Cu/ZnSOD、抗坏血酸过氧化物酶、热激蛋白和乙二醛酶Ⅰ在内的一些胁迫与防御蛋白的差异表达,其在光期高温和暗期高温下的表达变化类型不同；利用实时荧光定量RT-PCR对5个胁迫与防御蛋白的编码基因Trx h、APX、2-Cys Prx、Cu/ZnSOD和HSP70进行转录分析,发现大多数蛋白的蛋白水平与其转录水平的表达变化并不一致,但可以肯定的是水稻灌浆期间参与暗期高温和光期高温抗性的分子机制不同,在耐高温性不同的品种中发挥作用的胁迫与防御蛋白有差异。5.在水稻籽粒品质形成中灌浆期高温适应性强的品种具有的特点为：cyPPDKB的表达量较高,其在高温胁迫下受抑制程度较小；高温会促进各时期大部分支链淀粉酶同源异构体的表达,并产生对光期高温和暗期高温响应较快的同源异构体；含stress responsive alpha-beta (A/B) barrel结构域蛋白表达量较高,且同源异构体蛋白点60在早期有表达。本研究为揭示灌浆期光期高温和暗期高温胁迫下引起的水稻籽粒形成和品质变化的差异分子生态机理提供了进一步研究的基本数据和线索。更多还原

【Abstract】 Global warming has become an irreversible trend and had a major impact to the ecological environment and food safety, and, the potential impact of rising temperature would be further exacerbated. At present, the effect of climate warming on crop production gets much of the attention of scientists and governments in the world. The daily mean temperature is elevated owing to the climate change caused by atmospheric greenhouse gas, and the night temperature rise is more evident. The seed setting rate significantly reduced or even crop failure and quality declined were caused by heat stress in rice growing season, especially at the flowering and grain-filling stages. In recent decades, there were more researches on the high temperature on rice yield and quality at home and abroad. Studies on the rice genetic and physiological basis of high temperature have become a hot spot direction. Researches on the different effects of day and night high temperature during grain formation will have a certain reference value for fully understanding and assessing the impact of temperature increasing on rice yield and quality.In this study, four middle-season indica rice varieties ’9311’, Shuhui527, N22 and Texianzhan with different high temperature adaptation were selected. During rice grain-filling stage, the treatment temperatures of the control, night high temperature (NHT), and day high temperature (DHT) were 28℃/20℃,27℃/35℃, and 35℃/27℃, respectively, and, all the treatments were carried out in plant incubators and maintained for 20 days after fertilization. The different influences of NHT and DHT on rice quality and seed proteins expression profiles during grain filling were studied, to accumulate a certain foundation for revealing the different molecular ecological mechanisms of variations in rice grain formation and quality induced by DHT and NHT stress during grain filling. The main results were as follows:1. Compared with the control, the grain-filling rate and maturation of the grains from plants of all varieties exposed to high temperature were faster, and, the effect of NHT on the fresh weights was more serious than that of DHT. The accumulation of amylose was increased and suppressed by heat treatment at the early and late stage, respectively. The different influences of DHT and NHT on the amylose content during grain filling stage mainly occurred at the early grain-filling stage, which among varieties were inconsistent. High temperature treatments decreased grain weight, brown rice rate, milled rice rate, head rice rate, amylose content and gel consistency, and increased chalkiness degree. The differences between NHT and DHT or among rice varieties were just the distinct change extents of the above symptoms, and the changing amplitudes of head rice rate and chalkiness degree were larger among them. Compared with DHT, NHT exerted more serious effects on the grain weight and milled rice rate and less effect on head rice rate, while, the different effects of DHT and NHT on brown rice rate, chalkiness degree and amylose content among varieties were not consistent.2. After analyzing protein expression profiles of rice filling grains at different grain filling stages under different heat treatments by two-dimensional gel electrophoresis (2-DE),81 differentially expressed proteins in response to high temperature were determined. Through mass spectrometry analysis,66 out of 81 differentially expressed proteins were successfully identified, which were classified into eight groups according to their functions:carbohydrate metabolism, amino acid metabolism, stress and defense proteins, signal transduction, protein synthesis and destination, miscellaneous, unknown function and others function. After the expression changes in 66 differentially expressed proteins in response to different high temperature treatments analyzed, five distinct expression change patterns between NHT and DHT could be revealed. And, there were the same and specific protein expression change patterns between NHT and DHT in different varieties.3. Among all of the successfully identified proteins,11 proteins possessed 2-5 isoforms, whose expression variations were monitored at four grain filling stages in two varieties (’9311’ and Texianzhan). The dynamic expression profiles of isoforms, including PPDK and pullulanase, in response to different high temperature (DHT and NHT) were displayed by proteomic approach in rice filling grains. Most of the isoforms identified displayed different expression change patterns between NHT and DHT in ’9311’ and Texianzhan. There were negative correlation between the expression amount of cyPPDKB isoforms and grain chalkiness degree, and it may be because of different suppressed extent of expression amount of cyPPDKB resulted in different grain chalkiness between NHT and DHT.4. High temperature stress during grain filling stimulated the differential expression of some stress and defense proteins in rice filling grains, including h-type thioredoxin (Trx h), peroxiredoxins (Prxs), Cu/Zn superoxide dismutase (Cu/ZnSOD), ascorbate peroxidase (APX), heat shock proteins (HSPs) and glyoxalasesⅠ. The expression change patterns of stress and defense proteins induced by DHT and NHT treatments were distinct. Five genes encoding stress and defense proteins including Trx h, APX,2-Cys Prx, Cu/ZnSOD and HSP70 were selected for transcripts analysis by real-time quantitative RT-PCR (qRT-PCR). The results showed that not all the protein levels were correlated with their corresponding transcript levels. However, the molecular mechanism of seed proteins involved in NHT and DHT tolerance during grain filling should be different, and the distinct stress and defense proteins played main role in rice varieties with different high temperature tolerance.5. In rice quality formation, the characteristics of high temperature adaptable rice varieties during grain-filling stage included:the expression amount of cyPPDKB was higher and suppressed extent under high temperature stress was smaller; high temperature would increase the protein expression of most of pullulanase isoforms at most grain filling stages, and, there was isoforms with more faster responses to high temperature; the expression amount of stress responsive A/B barrel domain containing protein was higher, and its isoform protein spot 60 expressed at early stage.The results in this study provided the basic data and clues for further research on revealing the different molecular ecological mechanisms of variations in rice grain formation and quality induced by DHT and NHT stress during grain filling.更多还原