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1.
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   

2.
Putative continuous epitopes, recognised by five panels of monoclonal antibodies (MAb) with differing specificities for gliadins and glutenin subunits, were identified using overlapping nonapeptides. These peptides corresponded to the entire sequence of an α/β-gliadin, a γ-gliadin, an ω-prolamin (homologous to ω-gliadin), a low molecular weight glutenin subunit (L MrGS) and several high molecular weight glutenin subunits (HMr GS). Antibodies that bound to γ- or ω-gliadins, L MrGS or HMr GS bound to the peptides at similar concentrations used normally in direct ELISA, but little binding to the peptides was seen for several antibodies that bound specifically to small groups of α/β-gliadins. Epitopes for these antibodies in α/β-gliadin may be discontinuous (i.e. derived from amino acid residues that are brought together by folding of the polypeptide chain or by juxtaposition of two polypeptide chains), since binding of these antibodies to gliadins was greatly decreased following the reduction of intra-molecular disulphide bonds. While some regions in particular subunits were immunodominant, such as the cysteine–cysteine containing peptide found in the central domain of many prolamins, a diversity of reaction patterns was found. Cross-reaction of antibody with peptides from other prolamin families was often due to binding to a peptide having significant sequence homology, but in some cases no homology was obvious. Some major trends were as follows. Antibodies which bound to most or all H MrGS recognised the central repeat region, while those that were selective for one or two subunits bound to epitopes in the unique N- and/or C-terminal domains. A high proportion of the epitopes recognised by MAb to α-, β-, ω-gliadins and L MrGS contained cysteine; these MAb may be useful in detecting covalent binding sites within or between subunits. Although a number of MAb bound a wide range of gliadins and GS, several of these recognised single (and differing) epitopes in the target proteins. However, comparatively few MAb recognised epitopes from either the N- or C-terminal regions of the target proteins. Several explanations are possible; either these regions are buried in the immunogen and not accessible for antibody production or alternatively the repeat sequences are immunodominant.  相似文献   

3.
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   

4.
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   

5.
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   

6.
A collection of 173 Triticum tauschii accessions was analysed to evaluate the variability of low molecular weight (Mr) glutenin subunits. These proteins were analysed by one-step one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and were divided into B-, C- and D-subunits in accordance with their electrophoretic mobility. Extensive polymorphism, both in the number and electrophoretic mobility, was detected in lowMr glutenin subunits present in T. tauschii. Thirty different patterns for B-subunits and forty-three for C-subunits were identified, some of which were with identical electrophoretic mobility than those observed in hexaploid wheat. Glutenin subunits with the same electrophoretic mobilities of low Mr D-glutenin subunits as well as subunits encoded at the Glu-D4 and Glu-D5 loci, were also detected in accessions of T. tauschii. These results provide new basic knowledge regarding the genetics variability of the low Mr glutenin subunits, as well as their potential to create novel germplasm for the improvement of wheat quality in breeding programs.  相似文献   

7.
In an attempt to detect highMrglutenin subunits specifically by immunochemical means, antisera were produced against synthetic peptides corresponding to three N-terminal sequences and to two repetitive motifs of highMrglutenin subunits. The three N-terminal peptides, NT1, NT2 and NT3, differed by a single substitution at the sixth position and correspond, respectively, to the N-termini off Dx subunits, Ax and Bx subunits and By and Dy subunits. The anti-peptide sera did not cross react with gliadins or with lowMrglutenin subunits, and differed in their ability to recognise highMrglutenin subunits. The antisera to the repetitive motifs recognised all highMrglutenin subunits, whereas the antisera to the N-terminal peptides detected only some of the subunits. The antiserum directed against the N-terminal peptide from Dx subunits detected these subunits specifically, whereas the antiserum directed against the N-terminal peptide corresponding to y type subunits did not react with the homologous subunits although it did react with Dx or Bx subunits. Antisera were also produced against internal sequences present in the N-terminal domain specific for x and for y-type subunits, but these antisera did not react with the cognate proteins. The failure of some anti-peptide sera to recognise the homologous highMrglutenin subunits may be due to differences in conformation between peptides and the corresponding regions in proteins.  相似文献   

8.
A new wheat endosperm protein subunit that was found in accessions belonging to different collections was identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Insoluble in 0·5 M NaCl, 70% ethanol, dimethyl sulphoxide (DMSO) and 50% propan-1-ol, it appeared in the pellet corresponding to the polymeric proteins along with high (HMW) and low molecular weight (LMW) glutenin subunits (GS). In the reduced form, it had an electrophoretic mobility between those two types of glutenin subunits. The apparent Mr of this novel protein was estimated by SDS-PAGE to be 71 000. N-terminal sequence and amino acid analyses indicated a composition similar to the ω-gliadins encoded by genes located on chromosome 1B. This protein can be ascribed to the D-subunits of LMW-GS with at least one cysteine residue that allows it to form part of the polymeric structure of glutenin, as shown by reaction with a fluorogenic reagent specific for sulphydryl groups. Fractions collected after size exclusion high-performance liquid chromatography (SE-HPLC) fractionation and further characterised by SDS-PAGE, confirm that the protein participates in the glutenin polymeric structure. An increase in its concentration was observed in fractions collected within the polymeric peak as elution time increased, implying that a larger amount of this protein is present in small size polymers. The role of this protein in the complex relationship between endosperm proteins and quality parameters is discussed in relation to its likely role as a chain terminator.  相似文献   

9.
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   

10.
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   

11.
An affinity-based purification procedure allowed the resolution of two distinct groups of endoxylanase inhibitors with different molecular structures and endoxylanase specificities from wheat wholemeal. The first group comprises the so-called Triticum aestivum L. Endoxylanase inhibitor (TAXI)-type proteins which are of approx. Mr 40 000 and occur in two different molecular forms. These inhibitors were removed from a concentrated cation exchange chromatography fraction from wheat wholemeal on a Bacillus subtilis endoxylanase affinity column. The second group of structurally different endoxylanase inhibitors, the so-called xylanase inhibiting protein (XIP)-type, of approx.Mr 29 000–32 000, with pI values varying between 8·8 and 9·2, was purified from the unbound fraction from the B. subtilis endoxylanase affinity column by chromatography on an Aspergillus niger endoxylanase affinity column followed by gel permeation chromatography. The XIP-type inhibitors are not active against the B. subtilis endoxylanase and were consequently not retained on the B. subtilis endoxylanase column. Further analysis of the XIP-type proteins by high-resolution cation exchange chromatography, SDS-PAGE and iso-electrofocusing, revealed several forms. They had similar endoxylanase specificities and N-terminal amino acid sequences.  相似文献   

12.
The French wheat cultivar Darius (Da) has very good bread-making quality, even though it possesses the high Mr glutenin subunit combination 2, 7 and 12, which is associated with poor quality, and a null allele at the Gli-D1 locus. Darius was crossed with three cultivars, Corin (Cor), Capitole (Cap) and Courtot (Cou), of poor, medium, and good quality, respectively. The three progenies (Cor × Da, Cap × Da and Da × Cou) were used to investigate the genetic basis of the good quality of Darius. Gliadin and glutenin compositions were analysed by acid polyacrylamide gel electrophoresis (A-PAGE) and SDS-PAGE, respectively, from half F2 grains, and the quality was evaluated using six technological criteria for the corresponding plants. The high Mr glutenin subunit alleles of Darius produced a negative effect on quality. The null allele of Darius, characterised by the absence of the Gli-D1 encoded ω-gliadins, was associated significantly with higher dough tenacity P , and strength W (up to 40% in the Cor × Da progeny). Darius had a higher amount of B zone low Mr glutenin subunits than the three other cultivars. The null allele of Darius reduced the dough extensibility in the two first progenies, and probably increased the ratio of aggregated glutenin to unaggregated gliadins. These results demonstrated that using only one locus breeders can improve particular quality traits.  相似文献   

13.
Using a PCR-based strategy, four new 75K γ-secalin genes were isolated from Secale cereale, Secale vavilovii, Secale sylvestre, and Secale strictum in genus Secale (rye). Based on amino acid sequences, the primary structure of the 75K γ-secalin subunits was demonstrated, which was composed of four main structural regions: (a) a conservative 19 amino acids signal peptide, (b) a steady short N-terminal region of 12 amino acids containing a cysteine residue, (c) a repetitive domain, which began with the conservative tetrapeptides PQ3 and was rich in glutamine and proline. PFPQ1−2(PQQ)1−2 was the core repeat motif in the repetitive region. Besides amino acid substitutions, this region showed variations in length due to the insertion and deletion events. In the repetitive region of EF432549 (Secale strictum), there were two octapeptides (PFPQQPQQ and PVPQQSQQ) insertions. On the contrary, deletion events of two residues (QT) took place in EF432546 (Secale sylvestre). Accounting for the amino acid replacement, an extra cysteine residue appeared in the repetitive region of EF432546, which did not exist in other genes, and (d) a conserved 143 amino acids C-terminal domain including eight cysteine residues. The implications of the results for quality improvement are discussed.  相似文献   

14.
A high Mr glutenin subunit, which has not been described previously, was found in several Swedish wheat (Triticum aestivum L.) breeding lines. The electrophoretic mobility (sodium dodecyl sulphate polyacrylamide gel electrophoresis) of this band was close to the mobility of the subunit that has been referred to as band 21 encoded on chromosome 1B. Reciprocal crosses between wheat materials with and without this band have shown that the synthesis of this subunit is controlled by the locus on chromosome 1A. The new band, called 21*, is thus allelic to bands 1 and 2*. The relevance of the novel-subunit to breadmaking quality was investigated by partial-least-square regression analysis. Using this method, the relationship between the electrophoretic patterns of high Mr glutenin subunits and the specific Zeleny volume was determined. The novel glutenin subunit was found in cultivars with a high specific Zeleny volume. Further investigations are needed before it is possible to determined the influence of the new glutenin subunit on baking quality.  相似文献   

15.
16.
The high and low Mr glutenin subunit compositions (controlled by the Glu-1 loci and the Glu-B3 locus, respectively) and the bread-making quality characteristics of 26 durum wheat (Triticum turgidum) genotypes were determined. The relationships between quality parameters and Glu-B1 and Glu-B3 controlled glutenin subunit composition were also investigated. The Glu-A1-controlled null allele was present in all the genotypes. High Mr subunits 20, 6 + 8 and 7 + 8 occurred in similar proportions in the cultivars analysed. The Glu-B3 low Mr allelic variants, LMW-1 and LMW-2, were both represented, with LMW-1 being present in lower proportion. Flour protein, SDS-sedimentation volume, dough strength (Alveograph W value), dough mixing time and bread loaf volume varied among the genotypes. Most samples had high Alveograph tenacity/extensibility (P/G) ratios, typical of tenacious gluten character. SDS-sedimentation volume, dough strength, dough mixing time and bread loaf volume were all interrelated. An association with flour protein content was observed only for mixing time, while the Alveograph tenacity/extensibility ratio was not correlated with the other parameters. Comparisons within the Glu-B1 and Glu-B3 loci indicated that the high Mr subunit 7 + 8 and the low Mr subunit LMW-2 had significantly greater beneficial effects on gluten strength and bread-making quality than the high Mr subunits 6 + 8 or 20 and the low Mr subunit LMW-1, respectively. High Mr subunit 6 + 8 had greater beneficial effects on quality than subunit 20.  相似文献   

17.
The seed proteins of 110 commercially-released Indian wheat cultivars were fractionated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to determine their high Mr glutenin subunit compositions. Amongst the cultivars, three alleles were indentified for the Glu-A1 locus (subunits 1, 2* and the null phenotype), eight alleles for the Glu-B1 locus (7, 7 + 8, 7 + 9, 6 + 8, 20, 13 + 16, 17 + 18 and a new allele) and two for the Glu-D1 locus (2 + 12 and 5 + 10). Nine of the cultivars were heterogeneous and possessed two or more 'biotypes' with respect to high Mr subunits. The cultivars were also analysed for the presence of the 1BL/1RS wheat-rye translocation by SDS-PAGE of unreduced prolamins and hybridisation of DNA dot blots with a rye-telomere-specific repetitive DNA probe, pAW-161. Both methods revealed that the majority of newly-released Indian wheat cultivars carry this translocation, thus confirming the agronomic superiority of these lines. While most of the normal wheat cultivars possessed high Mr subunits 2 + 12, 14 of the 18 translocation cultivars had the allelic subunits 5 + 10, even though no selection was made for these subunits during the breeding process. This suggests that the subunits 5 + 10 may play a compensating role for the loss of dough strength associated with the 1BL/1RS translocation.  相似文献   

18.
A group of low Mr wheat proteins with characteristic extractability behavior was isolated using two different isolation procedures. The proteins were extractable with water, salt solution and 70% (v/v) ethanol. After water extraction of flour and separation of gluten, a substantial proportion of these proteins was still extractable from gluten using 70% (v/v) ethanol. Based in their amino acid compositions, Mrs and IEF patterns, the isolated proteins resemble closely most of the alpha -amylase/protease inhibitors described in the literature. This was confirmed by enzyme inhibition studies in which it was shown that they inhibited mammalian, but not wheat, bacterial and fungal alpha-amylases. All proteases tested were inhibited by the low Mr proteins. Their Mrs and their high cysteine contents (6·5-8·1 mol%) indicated that the proteins contain four to five disulphide bonds. Free thiol groups were not detected in the proteins. Upon reduction, the Mr increased from 7-8000 to 14-19000. Furthermore, the disulphide bonds were highly reactive as determined by their reaction with the thiol-specific label monobromobimane. This suggests that the low Mr wheat proteins may play a role in thiol group/disulphide bond exchange in wheat proteins.  相似文献   

19.
The structural features of highMrglutenin subunits of wheat were compared with those of analogous proteins from rye. Subunits of two rye cultivars (Danko and Halo) and of the wheat cultivar Rektor were isolated from defatted flours by extraction with 50% (v/v) aqueous propan-1-ol under reducing conditions at 60°C followed by precipitation using a 60% concentration of propan-1-ol. The yields of dialysed and freeze-dried subunits were 0·33% and 0·32% (w/w of flour), respectively (rye cultivars), and 0·91% (Rektor). SDS–PAGE revealed that the rye cultivars contained at least five subunits with mobilities corresponding to the x-type subunits of wheat. Separation by RP–HPLC indicated that the rye cultivars did not differ in the qualitative composition of subunits, but in their quantitative proportions. The surface hydrophobicities of the rye subunits were significantly lower than those of wheat subunits. The amino acid compositions of single rye subunits were characterised by high contents of Glx, Gly and Pro, and they were closely related to those of wheat subunits, except that the Glx content was generally lower and the Cys content higher. Notable differences between rye and wheat subunits were found in their contributions to gluten strength. Whereas wheat subunits, reoxidised with potassium bromate and mixed with a standard wheat flour, caused a significant increase in gluten strength, reoxidised rye subunits had the opposite effect.  相似文献   

20.
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