摘要
本研究以超级杂交水稻“两优培九”为实验材料,运用蛋白质组学方法和技术研究分析水稻生育后期不同氮素处理条件下叶片和籽粒蛋白质组变化,并鉴定分析了这些差异蛋白点的生物功能,试图从氮素营养方面来探讨氮素营养对杂交水稻灌浆过程中叶片及籽粒灌浆生理影响的分子机制,初步阐明制约生育后期弱势粒灌浆不足,充实度差的分子机制。
本研究在水稻整个生育期设置不同的氮素处理(即正常供氮水平的1/2,正常供氮水平,正常供氮水平的2倍),并于开花期以及开花后25天分别提取不同处理叶片蛋白,通过双向电泳(2—DE)和图谱分析,叶片共发现17个蛋白质发生差异表达,这些蛋白质点经过质谱分析和数据库检索,其中有16个得到鉴定。根据其功能,分为与植物光合作用有关的(有6个)分别为核酮糖—1,5—二磷酸活化酶、碳酸酐酶、核酮糖—1,5—二磷酸羧化酶/加氧酶大亚基、光系统工,RieskeFe—s蛋白以及Cytb6/f;与植物体能量代谢途径相关(有2个)分别推定为叶绿体磷酸甘油酸激酶、核苷二磷酸激酶工;与清除自由基有关的(有3个)推定为超氧化物歧化酶铜—锌复合体、抗坏血酸过氧化物酶和Peroxiredoxin(PRX)家族蛋白;与蛋白质合成、贮藏蛋白及蛋白互作有关的酶(有3个)推定为叶绿体核糖体蛋白L1、Cupin家族、锚蛋白重复序列家族蛋白;与氮素同化有关的(有1个)为谷氨酰胺合成酶;其他功能类型蛋白(1个)其编号为0s07g0212200(推测为具有催化活性的辅酶结合蛋白)。于开花后25天分别提取不同氮素处理的强弱势粒蛋白,弱势粒共发现10个蛋白质发生差异表达,有9个蛋白得到鉴定。强势粒发现5个蛋白质发生差异表达,其中有4个蛋白得到鉴定。
通过对叶片进行的差异蛋白质点鉴定结果的分析,研究发现与植物光合作用有关的蛋白占的比例比较大,而且表达量大,说明氮素营养对于光合的影响是通过影响与光合有关酶的活化、CO_2的活化、光系统单位和电子传递链构成来影响和调节光合作用。同时氮素还影响了能量的合成和植物体内氧自由基的产生和清除系统,在能量产生和氧自由基的清除方面起生理功能的调节作用。另外,氮素还影响到蛋白质的合成储藏及互作。在本研究中,一个与氮素同化有关的蛋白——谷氨酰胺合成酶被鉴定出,随着氮素水平的提高其表达量上调说明氮素直接促进了氮素代谢的同化酶GS的合成,从而影响氮素的同化和代谢。
通过对弱势粒进行的差异蛋白质点鉴定结果的分析,发现ATP合酶、精氨(基)琥珀酸合酶、琥珀酰辅酶A合成酶、吲哚乙酸酰胺水解酶,苹果酸脱氢酶,二氢硫辛酸乙酰(基)转移酶,Ran GTP酶在二倍氮素处理下表达量上调,弱势粒中这些上调蛋白分别集中在:(1)能量合成的相关酶蛋白;(2)加速产能丰富的TCA循环;(3)促进生长和细胞分裂的生长素的释放。结果说明能量和促生长是弱势粒灌浆的主题。作为分子伴侣在籽粒灌浆中起调控作用的α-晶状体球蛋白类型热休克蛋白在正常氮素和2倍氮素处理时稳定表达。通过对强势粒进行的差异蛋白质点鉴定结果的分析,发现作为水稻储藏蛋白的谷蛋白表达量随着氮素水平的提高而提高,蓖麻蛋白B凝集素以及果糖二磷酸醛缩酶细胞质的同工酶与高低氮两种处理相比,正常氮素水平表达量最高。葡糖-1-磷酸腺苷酰基转移酶主要与籽粒中淀粉的形成有关,二倍氮素处理与正常氮素相比,其表达量反而下降。这些点的表达量变化说明了强弱势粒不同的灌浆阶段其代谢特点和中心具有明显区别,这时氮素影响的是强势粒相关蛋白的贮藏和碳水化合物的代谢以及对于其呼吸作用的调控,这一阶段的高氮水平对强势粒淀粉的合成并非积极因素。
氮素是植物的生命元素,氮素营养对于植物生命活动的影响和调节具有重要作用,水稻生育后期合理运用氮素营养对于提高剑叶叶片的光合性能,增强源的供应能力和抗早衰能力等具有积极的作用;从结果来看能量和激素水平是制约弱势粒发育、充实的主要因素之一,生育后期充足的氮素供应对于弱势粒的充实具有重要的意义,虽然对于强势粒的灌浆影响没有弱势粒显著,但是充足、正常的氮素水平对于其贮藏物质的累积以及碳水化合物代谢等同样具有重要作用。
Proteomics was used as a tool to study changes in leaf and grain proteome at different Nitrogen treatments during the late growth stage of liangyoupeijiu which is the super cross rice,and then identified and analyzed the biological function of these differential proteins,attempted to discuss the molecular mechanism from Nitrogen on leaf and grain during grain filling periods of the cross rice,and clarify the molecular mechanism of the weak grain lack of filling and poor adequacy preliminarily.
The research set different Nitrogen treatments during the whole rice growth period (half of normal nitrogen, normal nitrogen and doubleness of normal Nitrogen) and total leaf proteins of different treatments were extracted at 0 days,25 days after anthesis..By two-dimensional gel electrophoresis(2-DE) and image analysis,it was found that 17 leaf proteins expressed differentially in the three Nitrogen treatments,and 16 of them were identified by MALDI-TOF-TOF/MS and database searching.According to their function,those proteins were divided into these categories: about plant photosynthesis(the number was 6),they were ribulose-1,5-bisphosphate carboxylase activase, Carbonic Anhydrase, rubisco large subunit, photosystem I subunit VII,Rieske Fe—S protein,and Cytb6/f;about plant energy metabolic pathways(the number was 2)they were putative chloroplast phosphoglycerate kinase and Nucleoside diphosphate kinase I;about radical Scavenging(the number was 3)they were putative superoxide dismutase [Cu-Zn], Ascorbate peroxidase,and Peroxiredoxin (PRX) family;about protein synthesis , storage and protein-protein interaction (the number was 3)they were Putative chloroplast ribosomal protein L1, Cupin family and Ankyrin repeat domain protein;about Nitrogen assimilation(the number was 1),that was Glutamine synthetase , and others(the number was 1) that was No.Os07g0212200(.Speculate for the coenzyme binding protein with catalytic activity).Total strong and weak grain proteins of different treatments were extracted respectively 25 days after anthesis. It was found that 10 weak grain proteins expressed differentially in the three Nitrogen treatments and 9 were identified; it was found that 5 strong grain proteins expressed differentially in the three Nitrogen treatments and 4 were identified.
By analysis of the identification results of the different leaf proteins,it was found that proteins about plant photosynthesis had the largest proportion and their expressional quantity were large,which showed Nitrogen affected and regulated photosynthesis by its impact on activating the photosynthetic enzyme,activating CO_2,PS unit and electronic transfer chain. Nitrogen also impacted energy synthesis,the formation and scavenging synstem of oxygen free radicals in plants,and regulated producing energy and scavenging oxygen free radicals as physiological function.Another,Nitrogen affected protein synthesis,storage and interaction..Duringmy research,a protein about Nitrogen assimilation——Glutamine synthetase was identified.Andwith the increasing of Nitrogen, its expressional quantity increased,which showed Nitrogen promoted the synthesis of GS which about Nitrogen metabolism, and affected Nitrogen assimilation and metabolism.
By analysis of the identification results of the different weak grain proteins, it was found that the expressional quantity of ATP synthase, Argininosuccinate synthase, succinyl-CoA synthetase beta subunit, IAA amidohydrolase, malate dehydrogenase , dihydrolipoamide acetyltransferase and Ran GTPase were increased at doubleness Nitrogen treatment .And these increased proteins of the weak grain focused in these areas :(1)Proteins about synthesis energy;(2) Accelerate TCA cycle of producing energy;(3) Promote growth and cell division in the release of IAA.The results showed energy and growth were the theme of weak grain filling. alpha-crystallin-type heat shock proteins had stable expression at normal and doubleness Nitrogen treatments as molecular chaperone regulating graining filling.By analysis of the identification results of the different strong grain proteins,it was found with the increase of the Nitrogen, the expressional quantity of Glutelin as storage protein increased, the expressional quantity of Ricin B lectin and Fructose-bisphosphate aldolase cytoplasmic isozyme were the most at normal Nitrogen treatment compared with the high and the low Nitrogen treatments. The expressional quantity of glucose-1-phosphate adenylyltransferase which was about the formation of starch was decreased at doubleness Nitrogen treatments instead compared with the normal Nitrogen .Those changes of expressional quantity showed the strong and weak grain had clear distinctions in metabolic characteristics and center at different filling stages. Then Nitrogen affected the storage of related protein and carbohydrate metabolism and respiration regulation of strong grain, and the high Nitrogen level of this phase was not positive factor for starch synthesis of strong grain.
Nitrogen is the life element in plant, and Nitrogen nutrition plays an important role in impacting and regulating plant life activity. Reasonable use of the Nitrogen nutrition has an important role for raising the flag leaf photosynthetic , enhancing supply capacity of source and the ability of preventing aging. From the results , the energy and hormone level is one of the main factors restricting development and filling of weak grain. Adequate supply of Nitrogen has great significance for weak grain filling at the late stage. Although the impact for strong grain filling is not significant compared with weak grain, adequate and normal level of Nitrogen also has an important role in cumulating storage material and carbohydrate metabolism .
引文
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