不同品种水稻种子贮藏蛋白质组分的差异

水稻是我国的主要粮食作物,它的蛋白质含量为6—14%。在水稻种子蛋白质中,谷蛋白占80%,球蛋白占10%,醇溶蛋白占5%,清蛋白占5%。据研究,水稻种子清蛋自主要由分子量16800的亚基组成,球蛋白由10种不同分子量的亚基通过疏水交互作用相结合,谷蛋白由分子量38000、25000和16000三种亚基通过双硫键相结合。但是,不同品种水稻种子贮藏蛋白质组分有何差异?研究报道的很少。本文简要报道不同品种水稻种子(籼稻和粳稻)贮藏蛋白质组分的差异,为     

蚕豆蛋白质亚基分析与特异种质鉴定

采用SDS-PAGE方法,对112份不同基因型蚕豆的清蛋白和球蛋白亚基的差异性进行分析。结果显示:(1)蚕豆清蛋白和球蛋白亚基的有效等位变异分别为1.750 0±0.452 3、1.545 5±0.522 2,多态性比率分别为75.00%、54.55%,清蛋白的亚基遗传多样性指数较球蛋白亚基高。(2)蚕豆清蛋白和球蛋白分别含有在不同基因型蚕豆中构成不同的12和10个亚基,其中清蛋白含有9个基本亚基,116kD、96kD、45kD为清蛋白的3个特异亚基;球蛋白含有8个基本亚基,58kD、35kD为球蛋白的2个特异亚基;研究共鉴定筛选出42个含有清蛋白特异亚基和21个含有球蛋白亚基的种质资源。(3)清蛋白的97kD、63kD基本亚基和球蛋白的97kD、56kD、47kD基本亚基存在一定的缺失现象,共鉴定出19个清蛋白亚基和21个球蛋白亚基缺失的优异种质。研究表明,蚕豆清蛋白和球蛋白亚基构成在不同种质之间具有差异性,除基本亚基外,部分种质还含有特异亚基或缺失亚基。     

小麦低分子量麦谷蛋白亚基组成研究

利用改良的两步一向SDS—PAGE(two—step one—dimensional SDS—PAGE)分析了几种小麦低分子量麦谷蛋白亚基(LMW-GS)组成。70％热乙醇提取总谷蛋白,11％分离胶进行第一步SDS—PAGE分离．电泳1h后切取顶端1cm胶条并置于巯基乙醇溶液进行还原,还原后的胶条于11%～16．5％的梯度胶进行第二步SDS—PAGE分离。结果显示。两步一向SDS—PAGE可以彻底除去清蛋白、球蛋白和醇溶蛋白对LMW—GS分离的背景干扰。提高LMW—GS的分辨率。对几种小麦低分子量麦谷蛋白亚基分析表明：LMW-GS组合比HMW—GS更为丰富,每种小麦含有2～5种B亚基,2～4种C亚基．B、C亚基的总数量为4～8种。     

甜荞不同品种不同海拔地区种子蛋白组分含量研究

该研究通过分析甜荞10个品种在4个不同海拔栽培的种子蛋白质组分(清蛋白、球蛋白、醇溶蛋白和谷蛋白)的含量变异,以揭示不同荞麦品种之间以及不同栽培地点甜荞种子蛋白组分的变异规律。结果表明:在所有甜荞品种种子蛋白组分含量中清蛋白谷蛋白球蛋白醇溶蛋白。其中,种植于海拔最低的内蒙古通辽的甜荞种子平均球蛋白含量最高(1.081%),而种植于海拔1 450 m的河北甜荞谷蛋白平均含量最高(2.805%);海拔2 620 m的青海甜荞清蛋白平均含量为4.750%,而在海拔最高的西藏日喀则收获的甜荞种子的醇溶蛋白最高(平均为0.393%)。另外,蒙0530在4个地区的平均种子清蛋白和谷蛋白含量都最高,而球蛋白含量最高的品种是赤甜荞1号,定甜荞2号的种子醇溶蛋白含量最高。双因素方差分析表明,种子清蛋白含量品种间变异达极显著水平,不同地点间的种子醇溶蛋白含量达极显著水平,而地点和品种两个因素对种子球蛋白含量和谷蛋白含量的变异都有极显著影响。相关性分析表明,赤甜荞1号的醇溶蛋白含量与海拔呈显著正相关,蒙0530的球蛋白含量与海拔呈显著负相关,其他品种蛋白组分与海拔的相关性不显著。该研究结果对于甜荞优质品种培育和栽培以及推广都有一定的指导意义。     

水稻种子发育过程中种子蛋白四组分的差别合成（简报）

~3H-亮氯酸标记实验表明,水稻种子蛋白的合成主要发生在开花后16天以前。开花后7～10天之间清蛋白、球蛋白、醇溶蛋白和谷蛋白4个组分都有很高的合成活力。谷蛋白的合成高峰在开花后7天,而其余组分在开花后10天时合成最活跃。     

薏苡仁蛋白质组分研究

目的:对药食两用功能的薏苡仁蛋白质四类组分和氨基酸含量进行分析。方法:采用旋光法、索氏提取法、烘干法、马弗炉法分别进行淀粉、粗脂肪、水分和灰分的测定;采用顺序抽提法依次进行清蛋白、球蛋白、醇溶蛋白和谷蛋白的提取,用Brandford和凯式定氮法进行蛋白质含量分析;采用氨基酸分析仪进行氨基酸含量测定。结果:薏苡仁总蛋白含量为14.17%,其中清蛋白、球蛋白、醇溶蛋白和谷蛋白含量分别为0.20、0.88、6.34和5.30 mg/100mg鲜重,分别占总蛋白质含量的1.43%、6.20%、44.74%和37.38%;薏苡仁粉经酸水解后共检测到15种氨基酸,除Trp外,人体必需氨基酸和半必需氨基酸均有检测到;各氨基酸含量也存在着差异,含量最高的为Glu(3.59 mg/100mg),含量最低的为Me(t0.17 mg/100mg)。结论:薏苡仁蛋白中醇溶蛋白和谷蛋白含量较丰富,为今后进一步开发薏苡仁功能食品提供了理论数据。     

水稻57H突变体系列的胚乳储藏蛋白质表型多样性及其分类(英文)

种子储藏蛋白质主要由谷蛋白、醇溶谷蛋白和球蛋白组成。这些蛋白质在种子发育期被合成后,经过区室化过程,被蓄积在胚乳中。本研究系统分析了水稻57H突变体的表型多样性,旨在为胚乳储藏蛋白质的遗传调节机制的全面解明展示一个新的前景。胚乳蛋白质的SDS-PAGE分析和免疫印迹分析显示了高量含有57kD谷蛋白前体的水稻57H突变体系列具有多样的储藏蛋白质表型。基于其表型的多样性,57H突变体系列被分成了三种类型。与野生型水稻品种相比,所有的类型Ⅰ突变体(glup4,glup6,Glup5,esp2)不仅高量蓄积谷蛋白前体、少量蓄积成熟型谷蛋白的40kD酸性亚基和20kD碱性亚基,而且显著减少了13kD醇溶谷蛋白b组分和26kDa球蛋白的蓄积;类型Ⅱ突变体(Glup1,glup2)不仅高量蓄积谷蛋白前体,还减少了26kDa球蛋白的蓄积;类型Ⅲ突变体(glup3)除了高量蓄积谷蛋白前体、少量蓄积成熟型谷蛋白亚基之外,并没有减少其它储藏蛋白质的蓄积。结果指出了57H变异系列对储藏蛋白的蓄积具有多样的影响,实质上是关于储藏蛋白质区室化的遗传体系。并就该遗传体系对储藏蛋白质的翻译后区室化及其营养性状的可能的影响进行了讨论。     

灌浆期不同阶段遮光对小麦籽粒蛋白质组分含量和加工品质的影响

选用强筋小麦济麦20、中筋小麦泰山23和弱筋小麦宁麦9号3个小麦品种,设置了灌浆期不同阶段遮光处理:开花后不遮光(S0)、0～11 d遮光(S1)、12 ～23 d遮光(S2)、24～35 d遮光(S3),研究了其对不同小麦品种籽粒蛋白质组分含量和加工品质的影响.结果表明:3个小麦品种的籽粒清蛋白+球蛋白含量遮光处理间无显著差异;遮光均显著提高了济麦20和泰山23的高分子量谷蛋白亚基、低分子量谷蛋白亚基、谷蛋白、醇溶蛋白和总蛋白含量,其中灌浆中期遮光(S2)处理提高幅度高于其他处理;灌浆中期(S2)和后期(S3)遮光处理显著提高了宁麦9号各蛋白质组分含量.遮光显著降低了小麦籽粒产量,提高了籽粒面团形成时间、面团稳定时间和沉降值,其中灌浆中期遮光处理更为显著,表明籽粒品质的形成与灌浆中期的光照条件更为密切.总体上灌浆期遮光对3个小麦品种籽粒产量、蛋白质组分含量及加工品质指标的调节幅度为济麦20＞泰山23＞宁麦9号.     

鸡冠花种子蛋白质的提纯及分析

鸡冠花种子干燥后用石油醚脱酯,用凯氏定氮法测定蛋白质含量。按Osbern系统分别提取白蛋白、球蛋白、醇溶蛋白、谷蛋白;用Folin一酚试剂法测定各自相对含量,并用单向和双向SDS—PAGE方法分析种子总蛋白和4类不同溶性蛋白质的组成成分及这些组份的热稳定性。实验发现:鸡冠花种子蛋白质含量达26.04％,白蛋白、球蛋白、醇溶蛋白、谷蛋白的相对含量分别为11.5％、72％、4.6％、12％。SDS—PAGE表明;其种子蛋白中20KD球蛋白成份含量最高,其它一些次要组分为63KD、61KD、15KD、13KD白蛋白成份,58KD、37KD、23KD球蛋白成份,39KD、34KD、25KD谷蛋白成份及26KD醇溶蛋白成份,其中58KD由37KD和20KD两亚基经二硫键连接而成,39KD组份由24.5KD和18KD两亚基通过二硫键连接而成,23KD球蛋白组份遇热沉淀。     

豆球蛋白β亚基的分离提纯及其完整一级结构

豆球蛋白(Legumin)是豌豆、蚕豆、大豆等豆科植物种子中一类主要贮藏蛋白质。豆球蛋白是由6对亚基组成的六聚体,每对亚基是由分子量40000的酸性亚基(α亚基)和分子量20000的碱性亚基(β亚基)通过双硫键相结合。α亚基和β亚基又是由分子量60000的豆球蛋白原前体多肽(Prolegumin precursorpolypeptide)通过翻译后蛋白质水解作用(Post-translational proteolysis)而生成。一些研究者还发现,水稻、燕麦等禾本科植物种     

The purification and partial amino acid sequence of a polypeptide from the glutelin fraction of rice grains; homology to pea legumin

The glutelin fraction was extracted from grain meals of rice (Oryzea sativa) with 50 mM Tris-HCl buffer (pH 8.8) containing 6 M urea and 10 mM 2-mercaptoethanol. Polypeptides of glutelin were separated and purified by ion-exchange chromatography under denaturing conditions. Analysis by two-dimensional gel electrophoresis showed that 2 major polypeptides of the rice glutelin fraction, M_r 36 000 and 22 000, were linked in disulphide bonded pairs containing one M_r 36 000 and one M_r 22 000 subunit. A partial amino acid sequence of the purified M_r 22 000 glutelin subunit showed it to be homologous to the β-subunit of pea legumin, a storage protein which also contains disulphide-linked subunit pairs (M_r 38 000 and M_r 22 000). It is therefore proposed that the major component of rice glutelin is a legumin-like protein.     

Extraction and composition of rice endosperm glutelin

“Crude” glutelin was prepared from milled rice (Oryza sativa) flour by sequential extraction of the albumin-globulin fraction with 0.5 M NaCl and prolamin with 70% ethanol-0.6% β-mercaptoethanol. The solvent, 0.5% sodium dodecyl sulphate (SDS)-0.6% β-mercaptoethanol, extracted 91% of the endosperm glutelin without gelatinizing starch granules, whereas chaotropic solvents such as urea and guanidine caused extensive gelatinization. The S-cyanoethyl glutelin (Ce-glutelin) prepared by SDS extraction of the “crude” glutelin (9.5% protein) of IR480-5-9 rice gave three major subunits with MW 38000, 25000 and 16000 in the ratio 2:1:1 as determined by SDS polyacrylamide gel electrophoresis. A similar preparation from “crude” glutelin of a lower protein containing rice had the corresponding subunits in the ratio of 16:3:1. The MW 38000 subunit was unique to glutelin and was not present in C3-albumin-globulin or prolamin; the subunits were only partially purified by SDS Sephadex G-150 gel-filtration. The C3-glutelin was also prepared from a crude glutelin-prolamin preparation from IR480-5-9 by NaOH extractions followed by precipitation at pH 10 and ethanol extraction of the precipitate (C3-glutelin). This preparation had the same three major subunits and in the same ratio as C3-glutelin prepared by the SDS method. The subunits of the former preparation were separated by carboxymethyl Sephadex C-50 chromatography; the MW 38000 subunit eluted between pH 6.2–8.5, the MW 25000 in an impure state at pH values above 9, and the MW 16000 subunit was eluted at pH 8.6—9.2. Amino acid composition of the Ce-glutelin preparations were similar to each other. The MW 38000 and 16000 subunits had lower lysine contents than whole C3-glutelin, whereas the MW 25000 subunit had a higher lysine content.     

Properties of glutelin from mature and developing rice grain

Aminograms and SDS-polyacrylamide electrophoresis of milled rice glutelin of 12 Oryza sativa samples showed similar composition and ratio of 1 : 1 : 1 for subunits with MW 38 000:25 000: 16 000, indicating little possibility of finding variants of rice glutelins. Fractionation of S-cyanoethyl glutelin of 3 rices on polyacrylamide-agarose gels gave MW subunits differing in amino acid analysis of which the subunits with MW > 38 000 had the highest lysine content. Of the solubility fractions of endosperm glutelin, the fraction extracted by 0.5 M NaCl-0.6 % β-mercapto-ethanol-0.5% SDS was closest to glutelin in properties. In the developing grain of two varieties, appearance of protein bodies and rapid synthesis of glutelin from 7 days after flowering onward coincided with a drop in lysine content and appearance of MW 38 000 and 25 000 of crude glutelin. The MW 38 000 subunit is thus unique to endo-sperm glutelin.     

Molecular Species in the Protein Body II (PB-II) of Developing Rice Endosperm

The molecular weight and subunit composition of glutelin, the major storage protein of rice, in the major type of protein bodies of developing rice seeds was examined by gradient and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Glutelin in the protein body was the assembled from heterogeneous subunits, and the molecular weights were estimated to be 64, 140, 240, 320, 380, and 500 k by gradient SDS-PAGE. High molecular weight proteins (larger than 2,000 k) were also observed.

The two-dimensional SDS-PAGE under reduced conditions showed, that the glutelin in the protein body was composed of two groups of polypeptides, 22~23 and 37~39k, bound by disulfide linkages.     

Immunological relationships among the major seed proteins of cereals

The structural relationships among the major seed proteins of cereals was evaluated by Western blot analyses using antibodies raised against the wheat gliadin, rice glutelin acidic and basic subunits, and rice prolamine polypeptide. Consitent with the conservation of the primary sequences of these proteins, antibodies to the acidic and basic glutelin subunits cross-reacted with homologous polypeptides from oat as well as pea. The rice glutelin antibodies did not react with the major seed proteins from barley, rye, maize and sorghum. Antibodies raised against the acidic glutelin subunit reacted with the wheat glutenins but antibodies to the basic glutelin subunit did not. A comparison of the published primary sequences of a high molecular weight glutenin and rice glutelin showed little similarity except for a conserved peptide with the motif arg-gln-leu-gln-cys. The possible significance of this conserved element shared by these widely different proteins is discussed. Similar studies with the wheat gliadin antibody showed immunologically related components in plants of the subfamily Festucoideae except for rice. Antibodies raised against the rice prolamine recognized only the rice prolamine, indicating that this polypeptide was structurally distinct from other cereal prolamines. Overall, these results support and help clarify the evolutionary relationship of the cereals.     

Studies on Rice Glutelin

The complete amino acid analysis of the whole glutelin preparation from rice endosperm was performed. The recoveries were 101.59% for amino acid residues and 101.68% for nitrogen, and the standard deviations for four determinations on the 22 and 70 hr hydrolyzates were very small. The features of the amino acid composition of the protein were as follows; (1) the high contents of dicarboxylic amino acids, particularly glutamic acid, (2) about 60% of these dicarboxylic amino acids was in the amide form, and (3) the significantly low contents of tryptophan, methionine and half cystine. The amino acid analyses of the two kinds of the subunits of glutelin, the neutral major one and the basic minor one, were also carried out. There were some significant differences between the two subunits, for instance, in the contents of glutamic acid, tryptophan, glycine, half cystine, methionine and lysine. However, the composition of whole glutelin seemed to be settled predominantly by that of the major subunit.     

Specific subunit pairs of legumin from Vicia faba

Four pairs of disulphide-linked acidic (α) and basic (β) subunits were isolated from legumin of Vicia faba. Pairing between α- and β-subunits is nonrandom, supporting the view that each subunit pair arises from a common precursor polypeptide, already containing intramolecular disulphide bonds, when cleavage to the subunit pair takes place. The subunit pairs belong to two structural types: type A contains Met, whereas type B lacks Met. In addition to these four subunit pairs, at least two more pairs are present in legumin in minor amounts.     

The Processing of a 57-kDa Precursor Peptide to Subunits of Rice Glutelin

The processing of a 57-kDa peptide into 37- and 22-kDa subunitsof glutelin, a major storage protein of rice, was confirmedby the immunological compatibility between the precursor andglutelin subunits. The 57-kDa peptide reacted with the antiseraraised against purified 37-kDa and 22-kDa subunits of glutelin.The processing was further confirmed by alteration of an invivo protein synthesis by monensin, a sodium ionophore whichinhibits the intracellular transport of secretory and membraneproteins. Infusion of monensin into developing rice grains resultedin suppressed formation of mature glutelin subunits with concomitantaccumulation of the 57-kDa peptide. The present results indicatethat both subunits of rice glutelin were produced by post-translationalcleavage of the 57-kDa peptide. (Received July 9, 1986; Accepted October 1, 1986)     

不同倍性水稻胚乳蛋白的差异表达研究

以4份同源四倍体水稻和4份相应的二倍体水稻为研究材料, 对其种子内清、球蛋白(清蛋白和球蛋白)、醇溶蛋白和谷蛋白的含量进行了测定, 利用聚丙烯酰胺凝胶电泳(SDS-PAGE)技术分析了其特异性。结果表明: 同源四倍体水稻胚乳蛋白各组分含量与相应的二倍体相比, 大部分呈增加趋势; 同源四倍体水稻胚乳蛋白的亚基类型与相应的二倍体水稻基本一致, 仅在全蛋白电泳和清、球蛋白电泳中各发现1条差异条带。研究结果认为: 二倍体水稻经过染色体组加倍之后, 同源四倍体水稻重复基因组在蛋白质水平上的表达结果趋于“二倍化”, 即与二倍体水稻表现出相似的特点, 但在蛋白质表达量上前者往往高于后者。基因组多倍化对同源四倍体水稻重复基因组进化最主要的影响并不是体现在蛋白质结构上的差异, 而是体现在蛋白质表达量上的差异。     

The amino-acid sequence of the 2S sulphur-rich proteins from seeds of Brazil nut (Bertholletia excelsa H.B.K.)

Storage proteins of the albumin solubility fraction from seeds of Bertholletia excelsa H.B.K. were separated by reversed-phase high-performance liquid chromatography and their primary structures were determined by gas-phase sequencing on intact polypeptides and on the overlapping tryptic and thermolysin peptides. The 2S storage proteins consist of two subunits linked by disulphide bridges. The large subunit (8.5 kDa) is expressed in at least six different isoforms while the small subunit (3.6 kDa) consists of only one form. These proteins are extremely rich in glutamine, glutamic acid, arginine and the sulphur-containing amino acids cysteine and methionine. One of the variants even contains a sequence of six methionine residues in a row. Comparison with known sequences of 2S proteins of other dicotyledonous plants shows limited but distinct sequence homology. In particular, the positions of the cysteine residues relative to each other appear to be completely conserved, suggesting that tertiary structure constraints imposed by disulphide bridges dominate sequence conservation. It has been proposed that the two subunits of a related protein (the Brassica napus storage protein) is cleaved from a precursor polypeptide [Crouch, M. L., Tenbarge, K. M., Simon, A. E. & Ferl, R. (1983) J. Mol. Appl. Genet. 2,273-283]. The amino acid sequence homology of the Brazil nut protein with the former suggests that a similar protein processing event could occur.