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1.
The polypeptide subunits present in SDS-unextractable glutenin, the glutenin macropolymer (GMP) and the 70% (v/v) ethanol unextractable protein, the Osborne glutenin fraction, of various cultivars were separated by RP–HPLC and capillary electrophoresis (CE) under denaturing (urea and SDS, respectively) and reducing conditions. In addition, the SDS-extractable protein was separated by CE. HighMrglutenin subunits were well separated by CE, while the separation of lowMrglutenin subunits was better by RP–HPLC. HighMrglutenin subunits separated by RP–HPLC were collected and separated by CE. The subunits were identified unequivocally using the combined information from these two techniques and from SDS–PAGE patterns using the cvs. Spring and Troy Spring. By both RP–HPLC and CE it could be demonstrated for flour from three wheat cvs. (Camp Remy, Obelisk and Rektor) and a blend of flour from two of those cvs. (Camp Remy/Obelisk) that the highMrglutenin subunit content of the GMP was 29–31%. In contrast, the SDS-extractable protein consisted of 4–6% highMrglutenin subunits, which accounted for 14–23% of the highMrglutenin subunits in flour. Interestingly, the SDS-extractable highMrglutenin subunits consisted mainly (90–96%) of x-type subunits whereas, in the GMP, only 70–75% of the highMrsubunits were x-type subunits. Although the SDS extractable protein was not separated by RP–HPLC, results similar to those obtained by CE could be inferred from the subtraction of the contents of glutenin subunits of the GMP from the contents in the Osborne glutenin fraction. The results suggest that x- and y-type highMrglutenin subunits may have a different role in the structure (size and composition) of glutenin polymers.  相似文献   

2.
The effect of lowMrwheat protein addition on the amount and composition of the glutenin macropolymer (GMP) of dough was investigated for the three wheat cultivars Obelisk (weak), Camp Remy (medium strong) and Rektor (strong). During mixing, the amounts of high and lowMrglutenin subunit classes, and of the individual subunits decreased. The proportion of highMrglutenin subunits decreased and that of lowMrglutenin subunits increased, indicating an inhomogeneous distribution of the two subunit classes within the polymers present in GMP. During resting, the amounts of the glutenin subunit classes and of individual subunits increased. Meanwhile, the proportion of highMrglutenin subunits in GMP increased. LowMrwheat protein addition retarded re-polymerisation in that the amounts of glutenin subunit classes and of individual highMrglutenin subunits in GMP increased less than without addition. The proportion of highMrglutenin subunits in GMP directly after mixing was also decreased by lowMrwheat protein addition, and the proportion increased faster during dough resting, compared with the GMP in dough without lowMrwheat protein addition. Eventually, after 90 or 135 min resting, no differences existed in the proportions in GMP from doughs with and without lowMrwheat protein addition. LowMrwheat protein addition had no specific effect on individual highMrglutenin subunits, nor on the x-type/y-type subunit ratio in the GMP. In contrast, with increasing lowMrwheat protein addition, a highly significant reduction in the subunit 10 or 12/subunit 9 ratio in GMP was observed. This finding is in line with the decrease in this ratio directly after mixing in GMP of the dough without lowMrwheat protein addition. Since no specific effects were observed, it can be concluded that the lowMrwheat protein acts rather unspecifically on the GMP of dough.  相似文献   

3.
Wheat flour was washed with Tris-HCl buffer containing 4% Triton X114 before extracting the residual gluten with 70% ethanol. The glutenin extraction with 50% ethanol was performed at various ratios of DTT/protein; a minimum ratio of 0·1 g/g was needed to solubilise the maximum amount of glutenin. An experimental design was used to optimise the extraction conditions to obtain the best yield and purity of lowMrand highMrglutenin subunits. The purity of each glutenin subunit fraction was measured by RP-HPLC analysis after reduction and alkylation. Both temperature and protein concentration had an effect on the preparation of these fractions. An increase in the protein concentration enhanced the yield of the highMrglutenin fraction and simultaneously decreased that of the lowMrglutenin. Using the Deringer desirability function, conditions giving the optimum separation were determined. The procedure was scaled up and permitted the preparation of 0·96 g of highMrand 1·64 g of lowMrglutenin subunits from 5 g of gluten. The purities of these fractions, determined by RP-HPLC, were 90% and 95%, respectively, and their amino acid compositions were similar to those of high and lowMrsubunits separated by RP-HPLC.  相似文献   

4.
The importance of glutenin in bread-making quality has led to a substantial research effort. Studies on glutenin can be grouped into four categories: studies that determine the statistical relationships between the quantity of fractions and quality, studies of reconstitution and fortification, breeding and genetic modification, and those that assess structure–function relationships during processing. Statistical relationships between glutenin, glutenin fractions and glutenin polypeptides and quality have been established. The SDS or acetic acid unextractable glutenin correlated strongly with quality parameters. For highMrglutenin subunits the relationships with quality are less strong. In some studies it was demonstrated that the presence of some highMrglutenin subunits is correlated with the quantity of unextractable glutenin. Therefore, subunits are probably indirectly linked with bread-making qualityviathe quantity of unextractable glutenin. Recombination and fortification studies are hampered by changes in functionality of proteins after their separation. Recently, small scale tests have been developed in which small amounts of glutenin fractions can be studied. Controlled breeding studies have demonstrated the importance of highMrglutenin subunits 5+10 and, to a lesser extent, 1 or 2* for quality. In most of these studies the quantity of unextractable glutenin is not reported. This hampers adequate conclusions on cause–effect relationships. During dough processing large changes occur in the extractability of glutenin. The significance of these changes for dough properties and bread quality still requires investigation.  相似文献   

5.
The relationship between allelic composition of the low molecular mass glutenin subunits (LMr GS) and dough properties is poorly understood. Differentiating the L MrGS on the basis of their N-terminal sequence type may provide an important alternative in understanding the relationship. Polyclonal and/or monoclonal antibodies were produced using synthetic peptides corresponding to each of the seven N-terminal amino acid sequence types of the B- and C- L MrGS, namely SHIPGLERPS-, METSHIPGL-, METSRVPGL-, METSCIPGL-, METRCIPGL-, NMQVDPSGQVQ- (γ-type) and VRVPVPQLQP- (α-type). Each of the polyclonal antisera recognised both the corresponding peptide and LMr GS. For monoclonal antibodies, the proportion of hybridoma clones that produced antibody which recognised either the peptide or L MrGS varied between 1 and 88%. However, antibodies from only 4% of antibody-secreting stable cell lines recognised both the peptide immunogen and intact L MrGS. Using ELISA, the majority of the antibodies cross-reacted with related synthetic peptides corresponding to more than one N-terminal LMr GS sequence, although several of these bound small groups of L MrGS on immunoblots. Different polyclonal antisera prepared to a given immunogen exhibited similar patterns of subunit recognition on immunoblots. Monoclonal antibodies prepared to the same immunogen exhibited a variety of patterns, although each of the antibodies specific for a particular peptide or combination of peptides on ELISA recognised a similar pattern of L MrGS on immunoblots. For each sequence type, polyclonal or monoclonal antibodies specific for individual N-terminal sequences were identified. These probes may be useful tools to determine whether the type and amount of each N-terminal sequence is correlated with dough properties.  相似文献   

6.
Three hundred and eighty four immobilised overlapping nonapeptides, corresponding to the full amino acid sequences of three high Mr subunits of glutenin from bread wheat (Triticum aestivum) grain, were used to determine the linear epitopes recognised by four monoclonal antibodies. These antibodies were selected on the basis of significant and positive correlations between their binding to wheat flour extracts in a two-site ('sandwich') enzyme immunoassay and rheological measures of dough strength, an important aspect of bread wheat quality. The antibodies did not bind to a single, specific sequence but bound a series of related peptides in each high Mr glutenin subunit examined. The sequences recognised were not identical for the four antibodies, but in each case were in the central repeating domain of the high Mr glutenin subunits, and usually comprised regions that overlapped the degenerate repeat nonamer and hexamer sequences. High Mr glutenin subunits that have been associated with greater dough strength, such as the D-genome allelic products 1Dx5 and 1Dy10, displayed an increased number of the epitope sequences. The location of the epitopes in sequences of overlapping β-turns in the repetitive region supports the hypothesis that dough elasticity arises partly from β-turn-forming secondary structure in the repeat regions of the Mr glutenin subunits. Additional β-turn within high Mr subunits may extend their structure to allow increased interaction between the glutenin subunits and with the other proteins of the gluten complex, thus improving dough strength.  相似文献   

7.
A panel of anti-peptide antibodies specific for each of the different N-terminal sequence types of B- and C-low molecular mass glutenin subunits (L MrGS) were utilised in immunoblotting studies to identify the chromosomal location of genes encoding different sequences and to characterise the allelic variation of the encoding loci. The MET-type sequences were predominantly found among the B- subunits, while the α- and γ- sequences predominated in the C- subunits. The quantitatively major SHIPGLERPS sequence was found in both the B- and C- mobility regions. Using either biotypes in the cultivar, Aroona or genetic lines containing double rye chromosome 1 substitutions and thus expressing only single LMr GS alleles, the sequences were determined for most of the major polypeptides expressed by each LMr GS allele. The L MrGS from different genomes encoded different numbers of each sequence type. Furthermore, different polypeptides within a particular «block» of subunits encoded by a given allele often had differing N-terminal sequences. However, subunits of similar electrophoretic mobilities encoded by different alleles at each locus usually had identical N-terminal sequences, suggesting that they may instead differ in the number of repeats. In Chinese Spring, genes encoding the SHIPGLERPS and METSHIPGL sequence types were predominantly present on chromosomes 1B and 1D, while the related METSRVPGL sequence was only encoded on 1D. In contrast, the METSCIPGL, α- and γ-sequences were encoded on each of chromosomes 1A, 1B and 1D. Several different electrophoretic and immunoblotting approaches using null lines suggested that some of the α-type L MrGS may also be encoded by group 6 chromosomes, particularly 6D. The anti- SHIPGLERPS antibody also recognised chromosome 1B encoded β-, γ- and ω-gliadins, while the anti-METSRVPGL antibody recognised 1D encoded α- and β-gliadins. The absence of sequences within the major gliadin families that are highly homologous to the latter two N-terminal L MrGS sequences may suggest that some monomeric L MrGS could exist within the electrophoretically-resolved gliadins. These antibodies will provide valuable reagents for the study of the roles of particular L MrGS families in the structure and function of the glutenin macropolymer, the role of different LMr GS types in determining the influence of allelic variation of L MrGS composition on dough properties, and potentially in the development of diagnostics for these flour components.  相似文献   

8.
The high molecular weight glutenin subunits (HMW-GS) play a key role in end-use quality of wheat. Their particular primary structure is mostly derived from DNA sequencing, which gives no information on potential post-translational modifications. This paper reveals the primary structure of HMW-GS 1Dx2 by proteomic analysis. For this purpose, HMW-GS were first isolated from wheat flour (cv. Contra). The relative molecular mass (Mr) of subunit 1Dx2 present in the HMW-GS mixture was then very accurately determined with high-performance liquid chromatography–electrospray ionization-mass spectrometry using a quadrupole-time-of-flight mass analyzer (HPLC–ESI-QTOF-MS). The obtained Mr value (87,105) differed from the value derived from its protein sequence in the NCBI database (87,007). The subunit was further purified by preparative reversed-phase HPLC and partially hydrolyzed with chymotrypsin. The resulting 1Dx2 peptides were then analyzed by HPLC–ESI-MS/MS and the MS data were compared to amino acid sequences in protein databases. The discrepancy between the calculated and the measured Mr of 1Dx2 was explained by a missing proline in the 1Dx2 amino acid sequence from the database and not by any post-translational glycosylation.  相似文献   

9.
The sensitivities of flour proteins to precipitation by NaCl at acid pH were investigated by extraction with 0·05macetic acid solution containing varying concentrations of salt and by precipitation of the proteins extractable in acetic acid solution by addition of salt to varying concentrations. Flours of two Canadian hard red spring wheat cultivars (Glenlea and Katepwa) were used because of their different dough strengths. Electrophoresis results showed that as the NaCl concentration was raised, higherMrproteins of gliadins and glutenins were less extractable and were more easily precipitated. This tendency was more evident for the proteins of cv. Glenlea than those of cv. Katepwa, indicating that the former (stronger) is more sensitive to NaCl than the latter. SDS–PAGE results indicated that differences in the molecular size and subunit composition (i.e.relative proportion of high:lowMrglutenin subunits, and relative proportion of x-:y-type highMrglutenin subunits) of glutenin polymer contribute to differences in NaCl sensitivity. The differences appear to be related to the baking strength of the flour.  相似文献   

10.
Polyclonal and monoclonal antibodies (Mabs) were produced against the major type ofN-terminal amino acid sequence of lowMrglutenin subunits. The reactivities of these antibodies were determined using glutenin extracts of several bread wheat cultivars of known allelic composition. Analyses were performed by immunoblotting after one or two-dimensional electrophoresis. One Mab (Mab 6x1) was found to react with lowMrglutenin subunits encoded by chromosomes 1B and 1D but not with subunits controlled by chromosome 1A. Only some of the subunits encoded at theGlu-D3locus were recognised. In contrast, this Mab reacted with all the subunits controlled by theGlu-B3locus. After single dimension SDS–PAGE, we observed significant differences between immunoblot patterns of cultivars expressing different lowMrglutenin subunits from chromosome 1B. Mab6 x1 is a useful reagent for analysing the allelic composition at theGlu-B3locus.  相似文献   

11.
The effect of several additives (1·215 μmol KIO3, 0·892 μmol cysteine, endo-xylanase and 0·5% (w/w) rye-water-extractable arabinoxylans) on changes in the level and glutenin subunit composition of the sodium dodecyl sulphate (SDS)-unextractable protein during breadmaking was investigated. Protein extractability drastically increased during dough mixing and was enhanced both by cysteine and KIO3. The mixing-induced increase in protein extractability was partly reversed during fermentation. Fermenting doughs containing endo-xylanase had a higher level of SDS-unextractable protein than control doughs, while with KIO3the amount of SDS-unextractable protein remained very low. During baking most protein became SDS-unextractable. Bread baked from doughs with added KIO3contained a significantly higher level of SDS-extractable protein. Changes in subunit composition of the SDS-unextractable glutenin polymers, determined with RP-HPLC, coincided with changes in protein extractability during dough processing. Mixing decreased the ratio of high to lowMrglutenin subunits. Simultaneously, the relative proportions of the different highMrglutenin subunits in the unextractable glutenin polymers changed. During fermentation changes in subunit composition of the SDS-unextractable glutenin were opposite to those during mixing.  相似文献   

12.
A highly repetitiveMr58 000 peptide based on residues 102 to 643 of subunit 1Dx5 and forms containing one to four cysteine residues were expressed inE. coliand purified to homogeneity. Incorporation into dough using a 2 g Mixograph showed that most peptides resulted in reduced strength, which was possibly due to dilution or chain termination of glutenin polymers. However, a form containing four cysteines (two each close to the N-terminus and C-terminus) resulted in increased strength, indicating that the repetitive domains of the HMW subunits are sufficient to contribute to dough strength when incorporated into glutenin polymers.  相似文献   

13.
A panel of anti-peptide antibodies specific for each of the different N-terminal sequence types of B- and C-low molecular mass glutenin subunits (L MrGS) were utilised in immunoblotting studies to identify the chromosomal location of genes encoding different sequences and to characterise the allelic variation of the encoding loci. The MET-type sequences were predominantly found among the B- subunits, while the α- and γ- sequences predominated in the C- subunits. The quantitatively major SHIPGLERPS sequence was found in both the B- and C- mobility regions. Using either biotypes in the cultivar, Aroona or genetic lines containing double rye chromosome 1 substitutions and thus expressing only single LMr GS alleles, the sequences were determined for most of the major polypeptides expressed by each LMr GS allele. The L MrGS from different genomes encoded different numbers of each sequence type. Furthermore, different polypeptides within a particular «block» of subunits encoded by a given allele often had differing N-terminal sequences. However, subunits of similar electrophoretic mobilities encoded by different alleles at each locus usually had identical N-terminal sequences, suggesting that they may instead differ in the number of repeats. In Chinese Spring, genes encoding the SHIPGLERPS and METSHIPGL sequence types were predominantly present on chromosomes 1B and 1D, while the related METSRVPGL sequence was only encoded on 1D. In contrast, the METSCIPGL, α- and γ-sequences were encoded on each of chromosomes 1A, 1B and 1D. Several different electrophoretic and immunoblotting approaches using null lines suggested that some of the α-type L MrGS may also be encoded by group 6 chromosomes, particularly 6D. The anti- SHIPGLERPS antibody also recognised chromosome 1B encoded β-, γ- and ω-gliadins, while the anti-METSRVPGL antibody recognised 1D encoded α- and β-gliadins. The absence of sequences within the major gliadin families that are highly homologous to the latter two N-terminal L MrGS sequences may suggest that some monomeric L MrGS could exist within the electrophoretically-resolved gliadins. These antibodies will provide valuable reagents for the study of the roles of particular L MrGS families in the structure and function of the glutenin macropolymer, the role of different LMr GS types in determining the influence of allelic variation of L MrGS composition on dough properties, and potentially in the development of diagnostics for these flour components.  相似文献   

14.
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15.
Genetic transformation via the biolistic method has been used to introduce genes encoding natural and novel high-molecular-weight glutenin subunits (HMW-GS) into wheat. The appearance of new seed proteins of sizes not predicted by the transgene coding sequences was noted in some experiments. In this report, the identities of thirteen of these novel proteins were determined by tandem mass spectrometry (MS/MS). Seven different proteins larger than and two proteins smaller than the native protein were shown to contain peptides from 1Dx5. A novel protein found in some progeny of crosses between a transgenic plant and Great Plains winter wheats was larger than but contained several peptides from 1Dy10. In one line, a protein larger than and a protein smaller than HMW-GS each contained peptides from the N- and C-terminus of 1Dx5 and from the repeat region of 1Dy10. In a sixth transgenic line, the native Bx7 gene was apparently replaced by a gene that encodes a larger version of 1Bx7. The variant proteins accumulate in the polymeric protein fraction, indicating that they can form inter-molecular disulfide bonds. These results show that novel proteins found in some transformants are encoded by altered versions of either the transforming or endogenous HMW-GS genes.  相似文献   

16.
A large collection of accessions of the wild wheat progenitor Triticum tauschii, the donor of the D genome of Triticum aestivum, was evaluated for the variability of high molecular weight (Mr) glutenin subunits by electrophoretic and chromatographic methods. A large range of allelic variation at theGlu-Dt1 locus was found in this collection and some novel subunits were observed in both x- and y-type glutenin subunits, including x- or y-type null forms. A few accessions showed three bands in the high Mrglutenin subunit region. However, only two subunits were observed when monomeric proteins were removed before SDS-PAGE analysis of polymeric proteins. The presence of monomeric proteins in this region is discussed. Characterisation of these subunits was also carried out by reversed phase-high performance liquid chromatography (RP-HPLC). Very different surface hydrophobicities were observed between x- and y-type subunits and in some cases it was possible to identify glutenin subunits with the same apparent molecular weight but different surface hydrophobicity. Differences in elution times that were detected when the same subunit was either reduced or reduced and alkylated were related to the number of cysteine residues present in each glutenin subunit. The newGlu-Dt1 glutenin subunits have the potential to enhance the genetic variability available for improving the quality of bread wheat (T. aestivum).  相似文献   

17.
There is a need to develop more sensitive and reliable tests to help breeders select wheat lines of appropriate quality. Gluten thermostability, measured by the viscoelasticity of heated gluten, was assessed for its usefulness in evaluating quality of wheats in breeding programs. Two sets of wheat samples were used: Set I consisting of 20 cultivars and/or breeders' lines (BL), with diverse dough strengths and allelic variations of high Mr glutenin subunits coded at the Glu-A1, Glu-B1 and Glu-D1 loci (N=20) and Set II consisting of 16 near isogenic BL of F7 generation that had been in a quality selection program for three years. Thermostability of the isolated wet gluten was determined by measuring its viscoelastic properties, and was related to noodle texture, flour protein content, protein composition, dough physical properties and other quality predicting tests.Viscoelasticity of heat-treated gluten, isolated with 2% NaCl solution, significantly correlated with most of the tests used to measure dough and/or gluten strength and Chinese white salted noodle texture. The rate of thermal denaturation of proteins depends on Mr and packing density. High ratios of monomeric proteins such as gliadins and low Mr glutenin subunits to high Mr glutenin subunits increase the thermostability of the gluten. The measurement of viscoelasticity of heat-denatured gluten can be a useful test to determine gluten quality. Our study showed that gluten viscoelasticity and most of the tests related to dough and/or gluten strength are independent of allelic variations of the high molecular weight glutenin subunits. This test has been developed for predicting white salted noodle quality.  相似文献   

18.
Electrophoretic and reversed phase high performance liquid chromatographic (RP–HPLC) analyses were performed on gluten proteins extracted from flours milled from two different Swedish bread wheat lines; these lines have been reported to possess a novel highMrglutenin subunit controlled by a gene at theGlu-A1locus, referred to as 21*. Although RP–HPLC indicated that subunit 21* has a surface hydrophobocity similar to that of the commonly occurring allelic subunits 1 or 2*, it differs from them in isoelectric point, being more basic when analysed by two dimensional gel electrophoresis (IEF/SDS–PAGE). RP–HPLC separations of highMrglutenin subunits showed the presence of an additional peak, the behaviour of which was similar to that of y-type subunits encoded by genes at theGlu-A1ylocus and present only in wild wheatsT. urartu(AA) orT. dicoccoides(AABB). Based on chromatographic results and on the tight linkage observed with subunit 21*, it is suggested that the additional component (indicated as 21*y), present in the breeding lines analysed, corresponds to the y-type subunit encoded at theGlu-A1locus. Genes encoding the subunits 21* and 21*y were also analysed by polymerase chain reaction (PCR). Contrary to what was observed for the polypeptide itself, the gene corresponding to subunit 21* was similar in size to that encoding subunit 2* and shorter than that corresponding to subunit 1. Moreover, the amplification product corresponding to the active 21*y gene was shorter than that of the allelic inactive gene present in the bread wheat cultivar Cheyenne. As reported for other highMrglutenin subunits, gene size differences observed were due to a different length of the repetitive region. Because cultivated polyploid wheats have been shown to have only the x-type subunit at theGlu-A1locus, it is speculated that the new combination, with both x- and y-type subunits expressed, might have been introgressed during breeding processes from the wild wheat progenitorsT. urartuorT. dicoccoides, which have genotypes expressing both types of subunits.  相似文献   

19.
Durum wheat genotypes with some novel high Mr (high molecular weight, HMW) and low Mr (low molecular weight, LMW) glutenin subunits were grown in Sicily for two years of testing in order to compare their rheological and baking properties with respect to commercial durum wheat cultivars. Good bread making quality, as measured by Alveograph W and P/L, Farinograph and Mixograph parameters, and loaf volume was observed in genotypes combining high Mr subunits 2+, 1 or 11 encoded at the Glu-A1 locus with the so-called LMW-2 subunit group encoded at the Glu-B3 locus. The cultivar Avanzi, which carries high Mr subunit 2+ and LMW-2-like subunits, and the cultivars Dritto and Keops, which contain novel high and low Mr subunits, gave higher loaf volumes than control cultivars. The LMW-2 group subunits were found to be the main factor in determining dough strength (Alveograph W). The increase in the amount of high Mr subunits in genotypes with one expressed Glu-A1 gene may account for their improved rheological and baking properties.  相似文献   

20.
为进一步明确小麦高分子量麦谷蛋白亚基(HMW-GS)与小麦品质性状的关系,以黄淮麦区的127份小麦品种(系)为材料,利用SDS-PAGE技术、近红外谷物分析仪、粉质仪和拉伸仪等对其进行HMW-GS鉴定和品质检测.结果表明,参试材料在Glu-A1、Glu-B1和Glu-D1 3个位点上分别检测到2(x1、x-null)、...  相似文献   

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