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標題: | 高溫與水稻穎果基因表現及稻米品質形成相關性之研究 Study on the Relationship between High Temperature, Caryopsis Gene Expression, and Quality Formation in Rice (Oryza sativa L.) |
作者: | Shao-Kai Lin 林韶凱 |
指導教授: | 盧虎生(Huu-Sheng Lur) |
關鍵字: | 蛋白質體,水稻,高溫,逆境,穎果,品質, proteomics,rice,high temperature,stress,caryopsis,quality, |
出版年 : | 2005 |
學位: | 博士 |
摘要: | 第一章
蛋白質為影響水稻穎果發育及稻米品質形成的主要因子,溫度則為主要影響稻米品質的環境因子。本研究建立水稻穎果發育期間蛋白質表現之雙向電泳圖譜,再以液相層析串聯式質譜儀(LC-MS/MS)鑑定具表現差異的蛋白質。水稻穎果之蛋白質雙向電泳可分離出超過400個於發育期間表現的蛋白質。其中包括許多溫度處理具差異表現的蛋白質,亦有水稻品種間具差異表現的蛋白質,其中利用質譜分析已鑑定出具表現差異的蛋白質共70個,其中54個為已知功能的蛋白質;包括21個與碳水化合物生合成與代謝相關的蛋白質,14個與蛋白質生合成與累積相關的蛋白質,9個與逆境反應有關的蛋白質。Waxy蛋白質與glutelins為隨著水稻穎果發育期呈顯著增加的蛋白質。 Allergen-like proteins, pyruvate orthophosphate dikinase 及 NADH-dependent sorbitol dehydrogenase, 也在穎果發育期間表現,且扮演其重要的生理角色與功能。其中, Waxy蛋白質各isoforms的表現量與amylose 的含量成正相關。此外,本研究找出一個基因,具有高度GC比例的DNA序列,且可能與心腹白的形成有關。高溫(35/30 ℃)會降低Waxy 蛋白質、allergen-like proteins以及elongation factor 1 β的表現。然而,一群低分子量的heat shock proteins (sHSP)、glyceraldehyde-3-phosphate dehydrogenase以及prolamin等蛋白質的表現量會增加。sHSP的含量與心腹白的程度呈正相關。穎果發育期間,glutelins會發生phosphorylation與glycosylation的現象,顯示此類分子具轉譯後修飾的作用。本文亦探討數個可能與品質形成有關的蛋白質。此外,本研究亦利用RT-PCR確認相關基因在RNA層次上的表現。 第二章 稻米品質是水稻穎果發育期間一連串的生理及生化反應所累積的結果,而溫度是影響稻米品質形成的主要環境因子之一。基於高溫環境使稻米品質降低之觀點,本研究之目的為探討溫度對水稻穎果發育過程中基因表現之影響,及其與稻米品質形成之關係。結果顯示,以紅外線熱感應分析,發現水稻穎果為不良之散熱體,相對受高溫之影響較顯著。高溫環境使穗重、充實率以及千粒重減低,導致最終產量下降,且造成嚴重之心腹白外觀性狀。此外,高溫會影響水稻穎果分化及降低細胞核內DNA再複製之生理現象,同時使稉稻品種之直鏈澱粉含量減低及粗蛋白質含量下降。此外,RVA分析結果顯示高溫環境生產的稻米,其米粉之膠化溫度提高,膠體硬度增加以及黏度參數等性質的改變。 澱粉及蛋白質是決定稻米品質的主要因子,澱粉合成方面,本研究室發現高溫環境使稉稻品種之GBSS基因表現減少,而秈稻品種之starch synthase Ⅱ表現增加,雖機制不同,然而其結果都會導致直鏈澱粉含量之下降。蛋白質組成方面,高溫對貯藏性蛋白質的影響隨品種而異。高溫導致TNG67、TN1與Koshihikari的成熟穎果之prolamins含量顯著下降,亦使TNG67及TN1之glutelins含量降低,然而對Koshihikari之glutelins含量的影響並不顯著。直鏈澱粉及總蛋白質含量的降低,將導致稻米膠化溫度上升、尖峰黏度下降及膠體硬度增加。另一方面,高溫環境會提高穎果內16.9c HSP, 17.9 HSP, 18 HSP, glyceraldehyde-3- phosphate dehydrogenase及elongation factor 1 beta蛋白質之含量;降低nucleotide diphosphate kinase與allergen-like protein之表現量。綜合本研究的分析結果推論,高溫對澱粉含量與結構以及穎果內蛋白質種類及組成的總合改變,為高溫造成稻米品質降低之主因。 Chapter 1 Proteins are essential to rice caryopsis development and quality formation. High temperature is an important environmental factor which may decrease grain quality. In the present study rice caryopsis proteins were profiled by two-dimensional polyacrylamide gel electrophoresis, and differentially expressed proteins were analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Expressions of more than 400 polypeptide spots during caryopsis development, in response to temperature treatments or between varieties were monitored. Among them, more than 70 differentially expressed polypeptides were analyzed by LCMSMS and 54 proteins were identified with known functions. Of these, 21 were involved with carbohydrate metabolism, 14 with protein synthesis and sorting, and 9 with stress responses. Waxy (Wx) proteins and glutelins were the most prominous spots increased significantly during development. Allergen-like proteins, pyruvate orthophosphate dikinase and NADH-dependent sorbitol dehydrogenase, were also expressed during development, implying their physiological roles in caryopsis. Expression of large isoforms of Wx proteins was correlated with the amylose content of rice caryopsis. One protein with high GC content in its DNA sequence was correlated with the chalky trait of kernels. High temperature (35/30 ℃) decreased expression of Wx proteins, allergen-like proteins and elongation factor 1 β, but increased expression of small heat shock proteins (sHSP), glyceraldehyde-3-phosphate dehydrogenase and prolamin. The level of sHSP was positively correlated with the appearance of chalky kernels. During development, glutelins were phosphorylated and glycosylated, indicating these molecules were post-translationally modified. Possible functions of the expression of candidate proteins on grain quality are discussed. In addition, the expression of related genes at the RNA level were also monitored by RT-PCR. Chapter 2 Temperature is a major environmental factor on grains quality formation. High temperature may reduce rice grains quality. The components of starch and proteins are the other factors to influence the grain quality of rice. Under temperature influences, the final grain quality of caryopsis is determined by the physiological responses of biochemical processes and dry mass accumulation during caryopsis development. The present study focused on the relationship between the effects of high temperature on gene expression profilings and grain quality formation during developmental stages in rice caryopsis. The result of thermo-images analysis showed that rice panicles or grains dissipated heat slowly as compared to other organs, implying that rice panicles may be more sensitive and vulnerable to high-temperature environment. High temperature leads to decrease in panicle weight, filled spikelets rate, thousand spikelets weight and grains yield; produced sever chalky appearance kernels on panicles. High temperature influenced endosperm cell differentiation and reduced DNA endoreduplication. In japonica type rice, high temperature also resulted in lower amylose and protein contents. In grain physical chemical properties, high temperature decreased viscosity but increased pasting temperature and gel consistency of brown rice flour. At molecular basis, high temperature reduced the expression of GBSS in Japonica rice varieties; as TNG67, however, high temperature enhanced starch synthase II gene expression in Indica rice varieties, as TN1. Either the decrease of GBSS or increase of starch synthase II would lead to a lower amylase ratio in rice grains. Prolamins contents declined in mature rice grains of all three cultivars grown at high temperature. However, the effects of high temperature on storage proteins were different with varieties. The changing in amylose and protein contents may cause the observed negative effect of physical chemical characteristics of the rice flour. In addition, high temperature could enhance the proteins expression of 16.9c HSP, 17.9 HSP, 18 HSP, glyceraldehyde-3- phosphate dehydrogenase and elongation factor 1 beta, while reduced the expression of nucleotide diphosphate kinase and allergen-like proteins. Based on the present results, high temperature caused a complex physiological and metabolic responses related to starch and protein accumulation and packing in rice grains. Thus resulted in defective quality of rice grain. A schematic model has been proposed to depict the influence of high temperature on grain quality formation in rice. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/36231 |
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