Purification and immunoglobulin E epitopes identification of low molecular weight glutenin:an allergen in Chinese wheat

As one of the most important cereals,wheat(Triticum aestivum)has high nutritional value and is widely cultivated in China.However,wheat can cause severe allergic reactions,and a growing number of people are developing allergies to Chinese wheat.Low molecular weight glutenin(LMW-GS),an important allergen in susceptible populations,is responsible for celiac disease and wheat contacts dermatitis.In this study,LMW-GS was highly purified from Chinese wheat(Xiaoyan 6)and further identified and characterized.In addition,8 peptides were predicted efficiently by 5 immunological tools,among which 5 peptides showed significant immunoglobulin E binding abilities.Two specific epitopes were found to be in the non-conserved region of the amino acid sequence of LMW-GS,which was speculated to be the specific epitope of Chinese wheat.This systematic research of LMW-GS may provide new insights into the prevention of wheat allergy and development of hypoallergenic wheat products.

Effect of high-nitrogen fertilizer on gliadin and glutenin subproteomes during kernel development in wheat(Triticum aestivum L.)

Nitrogen(N),a macronutrient essential for plant growth and development,is needed for biosynthesis of protein and starch,which affect grain yield and quality.Application of high-N fertilizer increases plant growth,grain yield,and flour quality.In this study,we performed the first comparative analysis of gliadin and glutenin subproteomes during kernel development in the elite Chinese wheat cultivar Zhongmai 175 under high-N conditions by reversed-phase ultra-performance liquid chromatography and twodimensional difference gel electrophoresis(2D-DIGE).Application of high-N fertilizer led to significant increases in gluten macropolymer content,total gliadin and glutenin content,and the accumulation of individual storage protein components.Of 126 differentially accumulated proteins(DAPs)induced by high-N conditions,24 gliadins,12 high-molecularweight glutenins,and 27 low-molecular-weight glutenins were significantly upregulated.DAPs during five kernel developmental stages displayed multiple patterns of accumulation.In particular,gliadins and glutenins showed respectively five and six accumulation patterns.The accumulation of storage proteins under high-N conditions may lead to improved dough properties and bread quality.

Effect of HMW-GS 5+10 on Quality Parameters in Four Leading Wheat Cultivars

HMW-GS 5 + 10 was introduced to four major wheat cultivars Kehan9, Kefeng3, Longmai20 and Kenda4 through 5 or 6 consecutive backcrosses. No significant difference in protein content and dry gluten content was observed between cultivars containing 5 + 10 and 2 + 12 or 3 + 12 (P>0.1). In cultivars containing HMW-GS 5 + 10, the ratio of wet gluten content to dry gluten content was 2. 9 - 5.0% (P<0. 01) lower than the ratio from the cultivars containing 2 + 12 or 3 + 12, the ratio of Zeleny sedimentation volume to dry gluten content was 4. 5 - 13. 4% (P<0.05) higher. The degree of softening in cultivars having 5 + 10 was 15 -25 FU (P<0. 01) lower, while the maximum resistance was 82 - 193 EU (P<0. 05) higher. Backcrosses with biochemical marker assisted selection in the improvement of quality in wheat cultivars was discussed.

Allelic Variation and Genetic Diversity at Glu-1 Loci in Chinese Wheat (Triticum aestivum L.) Germplasms

Wheat processing quality is greatly influenced by the seed proteins especially the high molecular weight glutenin subunit (HMW-GS) components, the low molecular weight glutenin subunit (LMW-GS) components and gliadin components. Genes encoding the HMW-GS and LMW-GS components were located on the long arms and the short arms of homoeologous group 1 chromosomes, respectively. HMW-GS components in 5 129 accessions of wheat germplasms were analyzed systematically, including 3 459 landraces and 1 670 modern varieties. These accessions were chosen as candidate core collections to represent the genetic diversity of Chinese common wheat ( Triticum aestivum) germplasms documented and conserved in the National Gene Bank. These candidate core collections covered the 10 wheat production regions in China. In the whole country, the dominating alleles at the three loci are Glu-A1b (null), Glu-B1b (7 + 8), and Glu-D1a (2 + 12), respectively. The obvious difference between the land race and the modern variety is the dramatic frequency increase of alleles Glu-A1a (1), Glu-B1c (7 + 9), Glu-B1h (14 + 15), Glu-D1d (5 + 10) and allele cording 5 + 12 subunits in the later ones. In the whole view, there is minor difference on the genetic(allelic)richness between the landrace and the modern variety at Glu-1, which is 28 and 30 respectively. However, the genetic dispersion index (Simpson index) based on allelic variation and frequencies at Glu-A1, Glu-B1 and Glu-D1 suggested that the modern varieties had much higher genetic diversity than the landraces. This revealed that various isolating mechanisms (such as auto-gamous nature, low migration because of undeveloped transposition system) limited the gene flow and exchange between populations of the landraces, which led up to some genotypes localized in very small areas. Modern breeding has strongly promoted gene exchanges and introgression between populations and previous isolated populations. In the three loci, Glu-Bl has the highest genetic diversity , then Glu-D1, while Glu-A1 always keeps the lowest genetic diversity. In the landrace, the three regions with the highest allelic richness are Huanghuai Winter Wheat Region, Northwest Spring Wheat Region and Southwestern Winter Wheat Region. For the bred varieties, the highest allelic richness existed in Southwest Winter Wheat Region, Huanghuai Winter Wheat Region, Low & Middle Branch Winter Wheat Region of Yangtze River. Introduction and utilization of foreign varieties in cross breeding has had great effects on the allelic components and frequency of the three loci, which greatly affected the genetic dispersion index. This has made 'population' of the modern variety quite different from that of the landrace.

Analysis of Dynamic Accumulation of Three Types of Glutenin Subunits and Their Content in Relation to Sedimentation Value in Common Wheat

The biosynthetic time and accumulations of A-, B-, and C-type glutenin subunits in 7 winter wheat cultivars with different quality （strong, medium, weak gluten） were analyzed by SDS-PAGE. The results showed that no glutenin subunit was observed within 8 d after anthesis. Parts or all A-, B-, and C-type subunits appeared around day 12 in different cultivars. Other A-, B-, and C-type subunits appeared gradually. The accumulation of A-, B-, and C-type subunits fluctuated before maturity. The results of analysis of correlation between the ratios of A/T （total content of glutenin subunits）, A/C, AJ （B＋C）, （A＋B）/C, and （A＋B）/T and SDS-sedimentation value suggested that they were more significant. The negative correlation between the ratio of （B＋C）/T and SDS-sedimentation value was more significant, and the correlations between the ratio C/T and the SDS-sedimentation value were significantly negative.

Allelic Variation of Glu-A1,Glu-B1 and Glu-D1 in Chinese Released Wheat Varieties in the Last 50 Years

The high-molecular-weight glutenin subunit (HMW-GS) components from about 85 varieties were fractionated by SDS-PAGE, including 22 corner stone breeding parents, 45 varieties taking important role in Chinese wheat production and 18 varieties with good bread making quality. The 22 corner stone breeding lines carry either null ? or l(a) summits on Glu-Al loci. Five types are detected on Glu-Bl, which are 7 + 8(b), 7 + 9 ?, 14 + 15 (h), 17 + 18 (i), 6 + 8 (d). 7 + 8 and 7 + 9 are the two major types. Six types have been detected on Glu-Dl, which are 2 + 12 (a), 2 + 10 €, 5 + 10 (d), 4 + 10 (j), 4 + 12 ?, 2 + 11 (g). 2 + 12 and 2 + 11 are the two major types. In the corner stone breeding lines, early premium from American carries 5 + 10, St2422/464 from Italy carries the 14 + 15. In addition, two relatively essential varieties, Mara and Alondra carry 5 + 10. In the 45 commercialized varieties, sowed more than 666 000 ha (10 million mu) annually, only Yangmai 5 conveys the 5 + 10 on Glu-Dl, and Xiaoyan 6 and Yumai 7 carry the 14 + 15 on Glu-B1. Four varieties, Funo, Nongda 139, Zhengzhou 683 and Fan 6 convey the 17 + 18 on Glu-B1. The 18 bread wheat varieties recommended by the Ministry of Agriculture in 1992 could be classified into two groups, 5 + 10 and 14 + 15. Zhongzuo 8131 and its selections are the typical genotype of 5 + 10. Genes coding the two subunits are from either Yecora F-70 or IRN68-181. Xiaoyan 6 and its derivative varieties are the typical genotype of 14 + 15. The genes coding 17 + 18 subunits in Chinese varieties were derived from Funo or its selections. These results basically reflect allelic changes on Glu-1 in Chinese wheat varieties in the past 50 years. It is also proven that HMW-GS components may have great diversity between lines from the same cross, which causes great difference on baking quality.

Composition and Content of High-Molecular-Weight Glutenin Subunits and Their Effects on Wheat Quality

Sedimentation values, flour glutenin macropolymer (GMP) contents, composition and contents of high-molecular-weight (HMW) glutenin subunits (GS) of 233 flour samples were determined. Our data indicated that subunit 1 occurred more frequently at Glu-A1 , subunit pair 7 + 8 at Glu-B1 and 2 + 12 at Glu-D1. The significant relationships between Glu-1 quality score and total HMW glutenin content, sedimentation value and GMP content suggested that the composition of HMW-GS affects wheat quality strongly. Moreover, the total content of HMW-GS was correlated with certain quality parameters more significantly. Relationship between subunit 5 + 10 content and breadmaking quality was better than others, but 2 + 12, 7 + 8, 7 + 9 and 4 + 12 also correlated with certain quality parameters significantly. The contents of total HMW-glutenin, x-type subunits and y-type subunits related with sedimentation value, flour GMP content, and Glu-1 quality score more strongly than that of individual subunit or subunit pair. The flour GMP content, with excellent correlation to sedimentation value, total contents of HMW glutenin, x- and y-type subunits and many other quality parameters, could be an ideal indicator of breadmaking quality at earlier generations for breeding purpose for its simple procedure and small scale.

Study on Relationship Between the Size Distribution of Glutenin Polymeric Protein and Wheat Flour Mixing Properties

Parental varieties Suneca and Cook have contrasting alleles at each of the five glutenin subunit loci (Glu-B1, Glu-D1 Glu-A3, Glu-B3, and Glu-D3), a set of 60 lines homozygous at these loci from the F4 progeny population of Suneca X Cook was chosen to analyze the variation of the size distribution of glutenin polymeric protein (measured by SE-HPLC) and flour mixing properties of these lines and to study relationship between the size distribution of glutenin polymeric protein and wheat flour mixing properties. The results showed that there were very significant differences among the relative size distributions of glutenin polymeric protein (i.e. percentage of unextracTable polymeric protein in the total polymeric protein, or UPP%) and dough development times (i.e. peak time of mixograph, or PTM) of different homozygous lines, respectively. Flour mixograph shape was closely related to UPP% value. The results also indicated that UPP% was very strongly positive correlation with PTM and negative correlation with peak height of mixograph (PHM). Comparing with flour protein content (FP%), UPP% gave greater effect on PTM and PHM, i.e. flour mixing properties, and it can be considered as one of criteria for quality selecting from early generation of breeding program.

Allelic Variation and Genetic Diversity at HMW Glutenin Subunits Loci in Yunnan, Tibetan and Xinjiang Wheat

Allelic variation and genetic diversity at HMW glutenin subunits loci, Glu-A1, Glu-B1and Glu-D1 were investigated in 64 accessions of three unique wheats of western Chinausing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Two HMWglutenin patterns (i.e., null, 7+8, 2+12 and null, 7, 2+12) in 34 Yunnan wheataccessions, 3 HMW glutenin patterns (i.e., null, 7+8, 2+12; null, 6+8, 2+12 andnull, 7+8, 2) in 24 Tibetan accessions and 1 HMW glutenin pattern (null, 7, 2+12) in6 Xinjiang wheat accessions were found. The Tibetan accession TB18 was found to be witha rare subunit 2 encoded by Glu-D1. A total of 4 (i.e., Glu-A1c, Glu-B1a, Glu-B1b andGlu-D1a), 5 (i.e., Glu-A1c, Glu-B1d, Glu-B1b, Glu-D1a and Glu-D1) and 3 alleles (i.e.,Glu-A1c, Glu-B1a and Glu-D1a) at Glu-1 locus were identified among Yunnan, Tibetan andXinjiang unique wheat accessions, respectively. For Yunnan wheat, Tibetan wheat andXinjiang wheat, the Neis mean genetic variation indexes were 0.1574, 0.1366 and 0,respectively, which might indicate the higher genetic diversity at HMW glutenin subunitsloci of Yunnan and Tibetan wheat accessions as compared to that of Xinjiang wheataccessions. Among the three genomes of hexaploid wheats of western China, the highestNeis genetic variation index was appeared in B genome with the mean value of 0.2674,while the indexes for genomes A and D were 0 and 0.0270, respectively. It might bereasonable to indicate that Glu-B1 showed the highest, Glu-D1 the intermediate and Glu-A1 always the lowest genetic diversity.

High-Molecular-Weight Glutenin Subunit Composition of Chinese Wheat Germplasm

The objective of the present study was to characterize the high molecular glutenin subunits （HMW-GS） composition and the presence of 1B/1R translocation in newly developed wheat （Triticum aestivum L.） germplasm, which have one or more traits that are useful in wheat improvement. Sodium dodecyl sulphate polyacrylamide-gel electrophoresis （SDS-PAGE） and acid polyacrylamide-gel electrophoresis （A-PAGE） were used to detect HMW-GS composition and the presence of 1B/1R wheat-rye （Secale cereale L.） chromosome translocation in the wheat germplasm. Bread-making quality scores of these lines were determined. A high level of variations in HMW-GS encoded by Glu-1 locus was observed. Sixteen major HMW-GS, with 30 combinations, were detected. The percentage of cultivars with more than two desirable subunits was 38.7%. Thirteen cultivars had bread-making quality scores of 10 in combination with one or two desirable agronomical traits, such as high-yield potential, dwarfing stem, resistance to diseases, and/or tolerance to abiotic stress. Sixty-eight （36.6%） cultivars possessed 1B/1R translocation. The newly developed germplasm with HMW-GS for good quality can be promising resources for improving bread-making quality of wheat.

A genetic evidence of chromosomal fragment from bridge parent existing in substitution lines between two common wheat varieties

Locating of important agronomic genes onto chromosome is helpful for efficient development of new wheat varieties. Wheat chromosome substitution lines between two varieties have been widely used for locating genes because of their distinctive advantages in genetic analysis, compared with the aneuploid genetic materials. Apart from the substituted chromosome, the other chromosomes between the substitution lines and their recipient parent should be identical, which eases the gene locating practice. In this study, a set of chromosome substitution lines with cv. Wichita （WI） as the recipient parent and cv. Cheyenne （CNN） as the donor parent were studied for the composition of high molecular weight glutenin subunits （HMW-GS） as well as a range of agronomic important traits. Results revealed that the substitution lines of WI（CNN5D）, WI（CNN6A） and WI（CNN7B） had higher plant heights than the two parents of WI and CNN, and WI（CNN3D） had later maturity than the parents. By sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS） analysis, a substitution line WI（CNN5B） was found to contain different HMW-GS patterns from its two parents, in which 1 By9 was replaced by 1 By8 on chromosome 1BL. Simple sequence repeat （SSR） analysis confirmed that the variation on 1BL in WI（CNN5B） was originated from Chinese Spring （CS）. It is concluded that chromosomal fragments from bridge material and donor parent were quite often retained in intracultivaral chromosome substitution lines except the substituting chromosomes.

Swelling Index of Glutenin (SIG) Related to Protein Quality, DoughCharacters and Noodle Quality

The swelling index of glutenin (SIG) and the protein fraction of 25 Chinese wheat varieties were studied with new protein fractions extracting method. The protein fractions compose of monomeric protein, soluble glutenin and insoluble glutenin. The relations between other protein index, dough character, and fresh noodle quality were also discussed. The SIG results at different time is positively and highly significantly related to the insoluble glutenin content (r= 0.808 -0.867, P< 0.01). The SIG result can reflect the insoluble glutenin content. The protein content, gluten index, farinograph stability time, extensibility length and extensigram energy were positively and significantly correlated with SIG5 and SIG20 (r= 0.516 - 0.734, P<0.05, 0.01).SIG proved to be applicable in Chinese wheat dough evaluation. Fresh noodle making quality parameters were evaluated by fresh noodle length, thickness, maximum resistance to extension, extension area and extension distance, while cooked noodle texture was determined by cutting firmness, compression recovery, surface firmness and TPA by using a texture analyzer of TA.XT2i. The noodle cooking quality was significantly correlated with SIG value. The surface firmness and cutting firmness were more desirable for predicating the quality difference than TPA test and compression. Cooking loss and water absorption were negatively related to SIG value and insoluble content (r = -0.556 - - 0.787, P < 0.05, 0.01). The results showed that SIG test was also suitable in evaluating noodle making and cooking quality in very small sample size, which was very important in wheat breeding programs. Therefore, SIG test, as a small scale test, is suitable to evaluate dough rheological properties for Chinese wheat varieties, and will be helpful in cereal research and wheat breeding program, especially, in early generations.

Development of a wheat material with improved bread-making quality by overexpressing HMW-GS 1S^(l)x2.3^(*)from Aegilops longissima

Wheat bread-making quality can be improved by use of high-molecular-weight glutenin subunits(HMW-GSs)from wild relatives.Aegilops longissima is a close relative of wheat that contains a number of HMW-GS-encoding genes including 1S^(l)x2.3^(*).In this study,transgenic wheat lines overexpressing 1S^(l)x2.3^(*)were obtained by Agrobacterium-mediated transformation and used to investigate the genetic contribution of 1S^(l)x2.3^(*)to wheat flour-processing quality.The 1S^(l)x2.3^(*)transgene was stably inherited and expressed over generations.Expression of 1S^(l)x2.3^(*)increased the relative expression of 1Dx2 and 1Dy12 and reduced that of 1By18 during grain development.In general,integration of 1S^(l)x2.3^(*)stimulated the accumulation of endogenous HMW-GSs and low-molecular-weight glutenin subunits in wheat kernels,greatly increasing the glutenin:gliadin ratio and resulting in faster formation of protein bodies in the endosperm during grain development.A wheat material with improved flour-making quality was developed in which 1S^(l)x2.3^(*)improved wheat bread-making quality.

Smart Colorimetric Corn Starch Films Combined with Anthocyanin-Loaded Glutenin-Carboxymethyl Chitosan Nanocomplexes for Freshness Monitoring of Chilled Pork

In this study,intelligent,pH-responsive colorimetric films were prepared by encapsulating anthocyanins in nanocomplexes prepared from glutenin and carboxymethyl chitosan.These nanocomplexes were added to a corn starch matrix and used in the freshness monitoring of chilled pork.The effects of anthocyanin-loaded nanocomplexes on the physical,structural,and functional characteristics of the films were investigated.The addition of anthocyanin-loaded nanocomplexes increased the tensile strength,elongation at break,hydrophobicity,and light transmittance of the films while decreasing their water vapor permeability.This is because new hydrogen bonds are formed between the film components,resulting in a more homogeneous and dense structure.The colorimetric film has a significant color response to pH changes.These films were used in experiments on the freshness of chilled pork,in which the pH changes with changing freshness states.The results show that the colorimetric film can monitor changes in the freshness of chilled pork in real time,where orange,pink,and green represent the fresh,secondary fresh,and putrefied states of pork,respectively.Therefore,the intelligent colorimetric film developed in this study has good application potential in the food industry.

Effects of HMW-GS Dx2 absence on the protein aggregation characteristics and thermal stability of wheat flour during maturation

Background:Wheat flour maturation affects the aggregation and structural stability of proteins.The number of high-molecular-weight glutenin subunits(HMW-GSs)differs in various wheat varieties.The effects of Dx2 absence on the protein aggregation characteristics and thermal sta-bility of flour were investigated during 120 d of maturation using near-isogenic lines(NILs).Results:The absence of Dx2 delayed and decreased the protein aggregation of flours during maturation,i.e.the maturation-induced increases were later and smaller for glutenin,glutenin macropolymer(GMP),glutenin/gliadin ratio,β-sheets,andβ-sheet/a-helix ratio in HMW-D1a without Dx2 than in HMW-D1p with Dx2;these differences were ascribed to the weaker interactions between the sulfhydryl(-SH)groups,disulfide bonds(-S-S-),and hydrophobicity in the flours without Dx2.Flour maturation caused the dough microstructures to be more compact and denser,thereby increasing the flour thermal stability as observed by a higher denaturation peak temperature(Tp),enthalpy of thermal transition(△H),and degradation temperature(Td),These changes led to better dough properties such as dough development time,dough stability time,and protein weakening,but the optimal stage in HMW-D1a without Dx2was reached later.Conclusion:These findings deepen the understanding of how HMW-GS Dx2 modifies protein structures during flour maturation.

Wheat gluten protein and its impacts on wheat processing quality

Before the advent of the wheat genomic era, a wide range of studies were conducted to understand the chemistry and functions of the wheat storage proteins,which are the major determinants of wheat flour the suitability of wheat flour for various end products, such as bread, noodles and cakes.Wheat grain protein is divided into gluten and non-gluten fractions and the wheat processing quality mainly depends on the gluten fractions.Gluten provides the unique extensibility and elasticity of dough that are essential for various wheat end products.Disulfide bonds are formed between cysteine residues,which is the chemical bases for the physical properties of dough.Based on the SDS-extractability, grain protein is divided into SDS-unextractable polymeric protein(UPP)and SDS-extractable polymeric protein.The percentage of UPP is positively related to the formation of disulfide bonds in the dough matrix.In the wheat genomic era, new glutenins with long repetitive central domains that contain a high number of consensus hexapeptide and nonapeptide motifs as well as high content of cysteine and glutamine residues should be targeted.

Identification of a novel HMW glutenin subunit and comparison of its amino acid sequence with those of homologous subunits

Aegilops tauschii is the donor of the D genome of common wheat (Triticum aestivum). Genetic variation of HMW glutenin subunits encoded by the Glu-1Dt locus of Ae. tauschii has been found to be higher than that specified by the Glu-1D locus in common wheat. In the present note, we report the identification of a novel HMW glutenin subunit, Dy13t, from Ae. tauschii. The newly identified subunit pos-sessed an electrophoretic mobility that was faster than that of the Dy12 subunit of common wheat. The complete ORF of encoding the Dy13t subunit contained 624 codons (excluding the stop codons). The amino acid sequence deduced from the Dy13t gene ORF was the shortest among those of the previ-ously reported subunits derived by the D genome. A further comparison of Dy13t amino acid sequence with those of the subunits characterized from the A, B, D, R genomes of Triticeae showed that the smaller size of the Dy13t subunit was associated with a reduction in the size of its repetitive domain.

HMW glutenin subunits in multiploid Aegilops species: composition analysis and molecular cloning of coding sequences

The Aegilops genus contains species closely related to wheat. In common with wheat, Aegilops species accumulate high molecular weight (HMW) glutenin subunits in their endospermic tissue. In this study, we investigated the composition of HMW glutenin subunits in four multiploid Aegilops species using SDS-PAGE analysis. Furthermore, by working with Ae. ventricosa, we established an efficient ge-nomic PCR condition for simultaneous amplification of DNA sequences coding for either x- or y-type HMW glutenin sub-units from polyploid Aegilops species. Using the genomic PCR condition, we amplified and subsequently cloned two DNA fragments that may code for HMW glutenin subunits in Ae. ventricosa. Based on an analysis of the deduced amino acid sequences, we concluded that the two cloned sequences encode one x- and one y-type of HMW glutenin subunit, respectively.

Isolation and Molecular Characterization of High Molecular Weight Glutenin Subunit Genes 1Bx13 and 1By16 from Hexaploid Wheat

The high molecular weight glutenin subunit （HMW-GS） pair 1Bx13＋1By16 are recognized to positively correlate with bread-making quality; however, their molecular data remain unknown. In order to reveal the mechanism by which 1By16 and 1Bx13 creates high quality, their open reading frames （ORFs） were amplified from common wheat Atlas66 and Jimai 20 using primers that were designed based on published sequences of HMW glutenin genes. The ORF of 1By16 was 2220bp, deduced into 738 amino acid residues with seven cysteines including 59 hexapeptides and 22 nanopeptides motifs. The ORF of 1Bx13 was 2385bp, deduced into 795 amino acid residues with four cysteines including 68 hexapeptides, 25 nanopeptides and six tripepUdes motifs. We found that 1By16 was the largest y-type HMW glutenin gene described to date in common wheat. The 1By16 had 36 amino acid residues inserted in the central repetitive domain compared with 1By15. Expression in bacteria and western-blot tests confirmed that the sequence cloned was the ORF of HMW-GS 1By16, and that 1Bx13 was one of the largest 1Bx genes that have been described so far in common wheat, exhibiting a hexapeptide （PGQGQQ） insertion in the end of central repetitive domain compared with 1Bx7. A phylogenetic tree based on the deduced full-length amino acid sequence alignment of the published HMW-GS genes showed that the 1By16 was clustered with Glu-1B-2, and that the 1Bx13 was clustered with Glu-1B-1 alleles.

Molecular Characterization of Two Silenced y-type Genes for Glu-B1 in Triticum aestivum ssp.yunnanese and ssp.tibetanum

The high molecular weight glutenin subunits （HMW-GSs） are a major class of common wheat storage proteins. The breadmaking quality of common wheat flour is influenced by the composition of HMW-GSs. In the present study, two unexpressed 1 By genes from Triticum aesitvum L.ssp.yunnanese AS332 and T. aesitvum ssp.tibetanurn AS908 were respectively cloned and characterized. The results indicated that both of the silenced 1By genes in AS332 and AS908 were 1Byg. In contrast to previously reported mechanisms for silenced genes lAx and lay, which was due to the insertion of transposon elements or the presence of premature stop codon via base substitution of C→T transition in trinucleotides CAA or CAG, the silence of 1By9 genes was caused by premature stop codons via the deletion of base A in trinucleotide CA.A, which lead to frameshift mutation and indirectly produced several premature stop codons （TAG） downstream of the coding sequence.