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1.
The changes in the main storage protein of seeds of buckwheat ( Fagopyrum esculentum Moench cv. Shatilovskaya 5), 13S globulin, were studied during seed germination. During the first three days of germination the 13S globulin is subjected to a limited proteolysis, which consists in the splitting of some of its subunits into large polypeptide fragments. Insignificant changes in the sedimentation coefficient of the 13S globulin during the first days of germination as well as immunochemical data, indicate that the limited proteolysis of the 13S globulin does not cause any major changes in its structure.
Dormant buckwheat seeds contain a proteolytic enzyme (a metalloproteinase), which can cause limited proteolysis of 13S globulin. The proteinase hydrolyzed some subunits of the 13S globulin to high molecular weight fragments. In the presence of sodium dodecylsulphate the electrophoretic pattern of 13S globulin, isolated from 3-day-old buckwheat seedlings, was almost identical to that of 13S globulin from dormant seeds hydrolyzed with metalloproteinase. It is suggested that the proteolysis of 13S globulin observed in vitro may also take place in vivo in the course of seed germination.  相似文献   

2.
Cotyledons of dry buckwheat (Fagopyrum esculentum Moench) seeds were used to study the cellular localization of a metalloproteinase which performs in vitro the initial limited proteolysis of the main storage protein of the seed, and of its proteinaceous inhibitor. Fractions of complex protein bodies (PB 1) and of the cytoplasm and membrane material (CMM) were obtained by fractionating cotyledons in a mixture of acetone and CCl4. The greater part of the metalloproteinase activity was found to be localized in the PB 1 fraction, with a lesser amount in the CMM fraction, whereas the metalloproteinase inhibitor was localized almost entirely in the PB 1 fraction. The data obtained indicate that the complex protein bodies of dry buckwheat seeds contain the components of the proteolytic system responsible for the initial degradation of the main storage protein — the 13S globulin — of buckwheat seeds, i.e. 13S globulin, the metalloproteinase, and its inhibitor. This confirms that it is possibile for the metalloproteinase to perform a controlled proteolysis of the 13S globulin in vivo. The effect of divalent cations on the degradation of the 13S globulin was also studied. A mechanism is discussed whereby the proteolysis of 13S globulin is initiated by divalent cations released as a result of phytin decationization during seedling growth.Abbreviations CMM cytoplasm and membrane material - PAGE polyacrylamide gel electrophoresis - PB 1 complex protein bodies with globoids  相似文献   

3.
荞麦13S球蛋白是荞麦种子中的一类主要贮藏蛋白。本研究选用荞麦属植物甜荞栽培种及其野生类型、苦荞栽培种及其野生类型、毛野荞、左贡野荞、细柄野荞和硬枝万年荞6个种共44份材料,进行PCR特异性扩增、测序得到荞麦13S球蛋白基因的保守片段序列。对序列进行差异分析,结果发现44份供试材料13S球蛋白基因片段的285个排列位点中不变位点为24个,多态性位点S为261个(含简约信息位点数198个和单型可变位点63个),序列总突变位点Eta为503个。野生甜荞种内13S球蛋白基因序列差异明显高于栽培甜荞,但野生苦荞种内13S球蛋白基因序列差异仅稍高于栽培苦荞。推测其一方面可能与繁殖方式有关,另一方面可能与荞麦驯化过程中通常只有少数野生型群体被驯化有关。系统聚类分析发现栽培甜荞与野甜荞亲缘关系近,与左贡野荞亲缘关系次之;栽培苦荞与野苦荞亲缘关系近,与毛野荞亲缘关系次之;细柄野荞和硬枝万年荞的13S蛋白基因片段序列差异较小,说明其亲缘关系较近。上述结果为荞麦属种间遗传多样性与进化关系研究提供了理论依据。  相似文献   

4.
Two proteolytic enzymes, a cysteine proteinase and a carboxypeptidase, responsible for breakdown of the main storage protein, 13S globulin, were purified from buckwheat seedlings (Fagopyrum esculentum Moench) by (NH4)2SO4 fractionation, gel-filtration on Sephadex G-150, ionexchange chromatography on DEAE-Toyopearl 650 M and chromatofocusing. The cysteine proteinase was purified 74-fold. It has a pH optimum of 5.5, a pI of 4.5 and an apparent molecular mass (Mr) of 71000. The carboxypeptidase was purified 128-fold. It has a pH optimum of 5.3, a pI of 5.8 and a Mr of 78500. Cysteine proteinase hydrolyzed the modified 13S globulin only if the reaction products were eliminated from the incubation mixture by dialysis. Storage protein degradation by the proteinase increased in the presence of carboxypeptidase. We suggest that the two enzymes complete the digestion of 13S globulin after its preliminary hydrolysis by the earlier described enzyme, metalloproteinase, present in dry buckwheat seeds.Abbreviations BSA bovine serum albumin - DEAE diethylaminoethyl - Mr apparent molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate  相似文献   

5.
A thiol proteinase was isolated from buckwheat seeds and purified 300-fold, using ammonium sulfate, acetone fractionation ion-exchange chromatography on Sephadex CM-50 and electrofocussing. The proteinase preparation obtained was found homogenous after polyacrylamide gel electrophoresis at pH 4.5. The molecular weight of the enzyme (75.000) was determined by gel-filtration through Sephadex G-100. The activation of proteinase by cysteine, 2-mercaptoethanol and dithiothreitol, its inhibition by p-chloromercurybenzoate and the absence of inhibition by diisopropyl fluorophosphate and EDTA suggest that the enzyme isolated is a thiol proteinase. The enzyme hydrolyzed many peptide bonds in the B-chain of insulin, showing high substrate specificity. The glutelin and globulin fractions of buckwheat seed proteins were hydrolyzed by the enzyme. It is assumed that the hydrolysis of reserve proteins of buckwheat seeds is the main function of the proteinase isolated.  相似文献   

6.
The role of the embryonic axis in regulation of proteolysis of the main storage protein was studied in buckwheat ( Fagopyrum esculentum ) seed. Polyacrylamide gel electrophoresis (PAGE) analysis revealed that removal of the embryonic axis had no effect on the first stage of hydrolysis, that is proteolytic modification, of 13S globulin. This modification took place in the growing seedlings also in the presence of cycloheximide, i.e. it was due to an enzyme present in dry seed. However, in isolated cotyledons the 13S globulin was not degraded completely. Incubation of isolated cotyledons with cytokinins, gibberellic acid and indoleacetic acid could not replace the excised embryonic axis. At the same time, proteolysis of the 13S globulin in the growing seedlings was strongly inhibited by casein hydrolyzate. It is suggested that a complete proteolysis of the modified storage protein is regulated by the concentration of hydrolysis products at the site of hydrolysis. The embryonic axis serves, most probably, as a site of efflux of the products of protein hydrolysis in the cotyledons during seedling growth and thus regulates the course of proteolysis.
Abscisic acid (10–100 μ M ) was without effect on modification of the 13S globulin, but suppressed the complete proteolysis of the protein by inhibiting, apparently, the synthesis of the cysteine proteinase in the growing seedlings.  相似文献   

7.
The paper describes the amino acid sequence of a 26 kDa basic subunit of 13S globulin of common buckwheat (Fagopyrum esculentum Moench). The protein has 93 and 75% sequence homology with 11S globulin of Coffea arabica and beta subunit of 11S globulin of Cucurbita pepo respectively. The subunit has the "globally conserved" N-terminal sequence consisting of Gly-Ile-Asp-Glu and the cysteine at P7' from the proteolytic processing site. A conserved 7 residue domain of Pro-His-Trp-Asn-Ile-Asn-Ala, characteristic of basic subunits of legumins from non-leguminous angiosperms, is also present in this protein. A distinguishing features of this subunit is the relatively high level of lysine and methionine.  相似文献   

8.
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。  相似文献   

9.
We used proteomics analysis to generate the profiles of proteins in the endosperm and embryo of common buckwheat grains. These differentially expressed proteins are potentially involved in seed metabolism. Extractions were done by trichloroacetic acid (TCA) precipitation. The resulting proteins were separated using SDS-PAGE coupled to LC-ESI-Q/TOF-MS/MS. This allowed us to detect and identify 67 proteins with isoforms, making this the most inclusive protein profile. The proteins were determined to be functionally involved in the central metabolic pathway of the seed, with metabolic interest being reflected in the occurrence of a tissue-specific enzyme balance. For a case in point, we found a tissue-specific and subcellular compartment-specific isoform of granule-bound starch synthase 1 in the chloroplast/amyloplast. This provided proteomic verification of the presence of a distinct regulatory mechanism for the biosynthesis of glycan and starch, which produce amylase and amylopectin. Furthermore, several previously characterized allergenic proteins such as 11S and 13S globulin seed storage protein were acknowledged in our seed samples, thus representing the potential for proteomics techniques that survey food sources for any incidence of allergens. This protein profile of common buckwheat grain is a new avenue for understanding its seed physiology in dormant stage as well as suggesting commercial applications for the buckwheat industry as buckwheat flour.  相似文献   

10.
Amaranth is a dicotyledonous plant whose major seed storage proteins are globulins and glutelins. An unique feature of amaranth seeds is the presence of a fraction named albumin-2, that is extractable with water only after an exhaustive extraction of globulins and albumin-1. In this work, we tested the hypothesis that albumin-2 fraction could be constituted by a non-processed 11S globulin (proglobulin). To this end, the gene encoding the amaranth 11S subunit was cloned and expressed in Escherichia coli. Subsequently, the recombinant proglobulin and albumin-2 purified from seeds were treated with a sunflower vacuolar processing enzyme (VPE). A 55 kDa component of albumin-2 was specifically cleaved into 38 and 17-15 kDa polypeptides, as a consequence of this endoproteolytic cleavage a change of the oligomeric state from trimeric to hexameric was observed. Amaranth 11S globulin fraction was not modified under these proteolysis conditions. Using VPE-specific antibodies, it was shown that amaranth expresses a 57 kDa VPE, and that both developing and mature amaranth seeds have VPE activity, although the increase of this activity during amaranth seed development is higher than that observed for sunflower seeds. These results confirm the presence of unprocessed 11S precursors in mature amaranth seeds; this phenomenon cannot, however, be attributed to low VPE activity during developing of amaranth seeds.  相似文献   

11.
为探究荞麦新品种的营养保健价值,该文对自交可育甜荞、金苦荞、米苦荞共56个不同品系荞麦种子的粗蛋白、总黄酮、蛋白组分含量及其果实性状的变异进行了研究。结果表明:(1)甜荞、金苦荞、米苦荞种子中粗蛋白含量平均值分别为13.19%、15.44%、11.75%,总黄酮含量平均值分别为0.14%、2.50%、2.09%,清蛋白含量的平均值分别为5.22%、6.13%、4.56%,球蛋白含量的平均值分别为1.29%、1.15%、0.91%,醇溶蛋白含量的平均值分别为0.42%、0.58%、0.55%,谷蛋白含量的平均值分别为2.66%、3.36%、2.80%,三种荞麦的蛋白组分均符合清蛋白>谷蛋白>球蛋白>醇溶蛋白。(2)果实性状中,甜荞果实千粒重、果实面积、果实直径的变异系数最大,米苦荞果实周长、果实长宽比、果实长、果实宽和50 mL容重的变异系数最大。(3)相关分析表明甜荞种子粗蛋白含量与果实长宽比、果实长,金苦荞种子粗蛋白含量与果实周长、果实长,米苦荞种子粗蛋白含量与果实宽、总黄酮含量与果实面积、果实宽、果实直径、50 mL容重的相关性均达到了显著或极显著水平。(4)该研究筛选出甜荞(1808-166贵甜2号优系)、金苦荞(多苦74、多苦78)、米苦荞(1906-136黑米荞麦、43-2)等高蛋白含量或高黄酮含量的荞麦品系。该研究结果对荞麦优良品种的选育和荞麦新产品的开发具有一定指导意义。  相似文献   

12.
选取生菜(Lactuca sativa)种子作为试材,外源添加蛋白酶抑制剂2-硝基苯甲酸[5,5'-Dithiobis-(2-nitrobenzoic acid),DTNB]对种子吸胀处理,通过程序降温,分析2-硝基苯甲酸对低温下种子发芽率及生理活性的影响。结果表明,低温下含水生菜种子的致死温度为–20 ℃;外源添加2-硝基苯甲酸2 mmol·L–1时种子发芽率最高,即对种子活性的保护效果显著;在此浓度下种子内SOD活性比对照提高1.38倍,羟基自由基清除能力提高1.17倍;与对照组相比产生两种新的蛋白11 S种子贮藏球蛋白Jug r4和11 S种子贮藏球蛋白2,均属于球蛋白家族,可提高含水种子的耐冻性;低温下种子内积累更小分子量的球蛋白多肽,对种子具有低温保护效果。综上,低温条件下生菜种子产生一定的抗冷反应,外源添加2 mmol·L–1 2-硝基苯甲酸可提高含水种子发芽率及生理活性,产生抗冻蛋白,积累更小分子量的球蛋白多肽进而提高种子抗冻性。  相似文献   

13.
Among thiamin-binding proteins that ubiquitously occur in plant seeds, that of common buckwheat became a model of extensive studies of the chemical mechanism of ligand-protein interaction. In this work, the polypeptide components of buckwheat seed thiamin-binding protein (BSTBP) are identified and characterized. We suggest that BSTBP is probably a fraction of major storage 13 S globulin (legumin), has an average molecular mass of 235 kDa and comprises hexamers of 57-kDa and 38-kDa subunits in variable combinations. Each subunit is a pair of disulfide-linked polypeptide chains, 36 kDa plus 24 kDa and two-times 22 kDa, respectively. The N-terminal sequences of 22-kDa and 24-kDa components show strict homology with those reported for basic subunits of buckwheat legumin. By photoaffinity labeling of BSTBP with 4-azido-2-nitrobenzoylthiamine, it is shown that the 36-kDa chain plays the major role in thiamin binding, but the other chains may also be variably involved. Putative thiamin-binding fragments are identified and sequenced.  相似文献   

14.
Hydrolysis of 13S globulin, the main storage protein in grains of common buckwheat (Fagopyrum esculentum Moench), proceeds in at least two phases during germination. The first stage, involving a limited proteolytic cleavage of the protein, is associated with increased activity of proteases having maximum activity at pH 7.6. The second stage, involving further hydrolysis of the partially cleaved protein, starts after 12 h of imbibition. During this phase, activity of proteases increased and activity maximum shifted to pH 5.6. Nevertheless, 13S globulin retains its antigenic identity till the emergence of radicle and plumule. Thus, it may not be the major source of amino acids utilized by the germinating seed during the initial stages of imbibition. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

15.
Approximately 40% of defatted perilla seeds consists of proteins which are primarily composed of globulin (84%). The amino acid profile of perilla proteins demonstrated balanced amounts of all essential amino acids, except for lysine. The molecular mass of the predominant globulin was estimated to be 340 kDa by gel filtration. This globulin was separated into three intermediary subunits (54, 57 and 59 kDa) by SDS-PAGE. It is suggested from these results that the globulin exists as a hexamer. A treatment with 50 mM dithiothreitol enabled the intermediary subunits to be separated into three acidic subunits (31-34 kDa) and four basic subunits (23-25 kDa). It is interesting that this subunit structure is the same as that of sesame α-globulin, despite them coming from different families. Compared to sesame α-globulin, the heat-induced gel of perilla globulin had better water-holding ability, despite it displaying the same degree of gel hardness.  相似文献   

16.
Imsande  John  Schmidt  Jean M. 《Plant and Soil》1998,202(1):41-47
During pod filling, a grain legume remobilizes vegetative nitrogen and sulfur to its developing fruit. This study was conducted to determine whether different nitrogen sources affected N and S assimilation and remobilization during pod filling. Well-nodulated plants fed 1.0 mM KNO3, 0.5 mM urea, or 2.5 mM urea assimilated 0%, 37%, or 114% more N, respectively, and 25%, 46%, or 56% more S, respectively, than did the average non-nodulated control plant fed 5.0 mM KNO3. Thus, N source during pod filling greatly affected both N and S assimilation. Depending upon N source, plant N concentration during pod filling decreased from 2.96% to between 1.36% and 1.82%. Non-nodulated control plants fed 5.0 mM KNO3 had the highest residual N at harvest. During the same treatments, plant S concentration decreased from 0.246% to a relatively uniform 0.215%. Thus, during pod filling, vegetative N was seemingly remobilized more efficiently (38–54%) than was S (13%). N source also affected seed yield and seed quality. Non-nodulated control plants fed 5.0 mM KNO3 produced the lowest yield (21.1 g seeds plant-1), whereas well nodulated plants fed 1.0 mM KNO3, 0.5 mM urea, or 2.5 mM urea produced yields of 26.2 g, 31.8 g, and 36.7 g seeds plant-1, respectively. Non-nodulated plants fed 2.5 mM urea yielded 28.6 g of seeds plant-1. Seed N concentrations of non-nodulated plants and nodulated plants fed 2.5 mM urea were high, 6.30% and 6.11% N, respectively, whereas their seed S concentrations were low, 0.348% and 0.330% S, respectively. N sources that produced both a relatively high seed yield and seed N concentration (i.e., a relatively high total seed N plant-1) produced a proportionately smaller increase in total seed sulfur. Consequently, seed quality, as judged solely by seed S concentration, was lowered.  相似文献   

17.
The proteolytic activities of α-chymotrypsin, trypsin, pepsin, bromelain, and an extract from germinating pumpkin seeds (Cucurbita moschata) were determined by their ability to effect the release of 1-anilino-8-naphthalenesulfonate bound to internal hydrophobic sites in intact protein substrates. Casein, glyceraldehyde-3-P dehydrogenase, urease, catalase, pumpkin seed globulin, and bovine serum albumin enhanced the fluorescence of 1-anilino-8-naphthalenesulfonate sufficiently to be used as proteolytic substrates. Chymotrypsin, trypsin, pepsin, and bromelain exhibited activity against all or almost all of the protein substrates. The activity of 1 μg of α-chymotrypsin or trypsin and 100 ng of pepsin could be easily detected by this method of assay within 4 to 5 minutes depending upon the substrate. The enzyme extracted from 3-day germinated pumpkin seeds exhibited strong activity only against pumpkin seed globulin, weak activity against the globulins of squash and cucumber and casein, and no activity against the other protein substrates. Activity against pumpkin globulin was maximal at pH 7.4. When assayed by an increase in ninhydrin-positive products, the enzyme extract from pumpkin seeds also showed strong activity against pumpkin globulin and weak activity against casein. The 1-anilino-8-naphthalenesulfonate-fluorescence method was at least 20 times more sensitive than the ninhydrin method and was 10 to 20 times more rapid.  相似文献   

18.
19.
以苦荞(Fagopyrum tataricum(L.)Gaertn)全基因组数据为平台,采用生物信息学方法,挖掘出9个11S种子储藏蛋白基因,并对其定位、蛋白结构、系统发育及表达模式进行了分析。结果表明,苦荞9个11S种子储藏蛋白基因编码的蛋白长度为189~914 aa,等电点位于5.18~9.82之间,分子量为21.27~103.33 kD;定位分析结果显示,这些成员位于苦荞基因组的6条连锁群上(Megascaffold2/5以及scaffold77/344/395/861);序列比对分析发现,除了1个11S种子储藏蛋白sample1_00009513-RA具有1个cupin保守结构域外,其余8个都含有2个cupin结构域,并且在cupin保守结构域中,苦荞和拟南芥(Arabidopsis thaliana(L.)Heynh)共有14个保守的氨基酸残基;蛋白结构预测表明,苦荞11S种子储藏蛋白的结构具有2种类型;苦荞与其它6个物种[拟南芥、花生(Arachis hypogaea Linn.)、大豆(Glycine max(Linn.)Merr.)、杏仁(Armeniaca vulgaris Lam.)、胡桃(Juglans regia L.)和芝麻(Sesamum indicum Linn.)]11S种子储藏蛋白以及苦荞过敏蛋白(TBb和TBt)系统发育分析结果表明,这些蛋白可以分为3类,共具有4对旁系同源蛋白和3对直系同源蛋白;与已报道的苦荞过敏性储藏蛋白以及其它5个物种(花生、大豆、杏仁、胡桃和芝麻)的11S过敏蛋白比较发现,5个11S种子储藏蛋白(sample1_00013128-RA、sample1_00013130-RA、sample1_00021677-RA、sample1_00021668-RA和sample1_00021674-RA)与苦荞2个过敏蛋白的同源性较高,同时它们与胡桃11S过敏蛋白的同源性最高,但尚需进一步实验来确定这5个成员是否为食物过敏原;RNA-Seq转录组数据显示,4个基因(sample1_00018411-RA、sample1_00026786-RA、sample1_00021674-RA、sample1_00022718-RA)在2种荞麦属植物的灌浆期种子中表达水平较高,且在‘大苦1号’中的表达水平要高于‘大甜1号’。  相似文献   

20.
Sugar uptake by cotton tissues: leaf disc versus cultured roots   总被引:1,自引:1,他引:0       下载免费PDF全文
The synthesis, transport, and posttranslational processing of reserve globulin in Avena sativa L. seeds were studied by pulse-chase labeling. Developing oat seeds were labeled with radioactive sulfate and tissue homogenates were used for globulin extraction.

Two globulin precursors (58-62 kilodaltons) were labeled after 1 hour pulse. The α and β globulin subunits appeared between 2 and 10 hours later, while simultaneously the 58 to 62 kilodaltons polypeptides gradually disappeared. This confirmed a precursor-product relationship. In a second pulse-chase experiment, the tissue extracts were fractionated on a sucrose gradient. The major portion of radioactivity was initially (1 hour pulse) associated with the endoplasmic reticulum. However, a significant amount of radioactivity shifted from the endoplasmic reticulum to protein bodies after 20 hours chase, suggesting the transport of the newly synthesized proteins. Protein bodies isolated from pulse-chased seeds were analyzed for the arrival of the newly synthesized globulin. Labeled precursors were detected after 2 hours chase and gradually disappeared. The α and β subunits appeared during the same chase period and assembled into a 12S oligomer.

The data indicated that oat globulin was synthesized as two large precursors which were transported from endoplasmic reticulum into protein bodies where they were processed to the α and β subunits forming a 12S oligomer.

  相似文献   

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