遗 传 学 报 Acta Genetica Sinica, October 2006, 33 (10)：929–936 ISSN 0379-4172

Molecular Characterization of a HMW Glutenin Subunit Al-lele Providing Evidence for Silencing of x-type Gene on Glu-B1

YANG Zu-Jun1,①, LI Guang-Rong2, LIU Chang1, FENG Juan1, ZHOU Jian-Ping1,

REN Zheng-Long1,2 1. School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China;

2. Key Laboratory of Plant Genetics and Breeding, Sichuan Agricultural University, Ya’an 625014, China Abstract: Understanding the molecular structure of high-molecular-weight glutenin subunit (HMW-GS) may provide useful evi-dence for the study on the improvement of quality of cultivated wheat and the evolution of Glu-1 alleles. Sodium dodecyl sul-fate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that the subunits encoded by Glu-B1 were null, named 1Bxm, in a Triticum turgidum var. dicoccoides line PI94640. Primers based on the conserved regions in wheat HMW-GS gene promoter and coding sequences were used to amplify the genomic DNA of line PI94640. The PCR products were sequenced, and the total nu-cleotide sequence of 3 442 bp including upstream sequence of 1 070 bp was obtained. Compared with the reported gene sequences of Glu-1Bx alleles, the promoter region of the Glu-1Bxm showed close resemblance to 1Bx7. The Glu-1Bxm coding region differs from the other Glu-1Bx alleles for a deduced mature protein with only 212 residues, and a stop codon (TAA) at 637 bp downstream from the start codon was present, which was probably responsible for the silencing of x-type subunit genes at the Glu-B1 locus. Phylogenetic tree based on the nucleotide sequence alignment of HMW glutenin subunit genes showed that 1Bxm was the most ancient type of Glu-B1 alleles, suggesting that the evolution rates are different among Glu-1Bx genes. Further study on the contri-bution of the unique silenced Glu-B1 alleles to quality improvement was also discussed. Key words: gene silence; Glu-1Bx; high molecular weight glutenin subunit; Triticum turgidum

Received: 2006-06-29; Accepted: 2006-09-02 This work was supported by the National Natural Science Foundation of China (No. 30170502) and Chunhui Plan of Ministry of Education (No. 2004251008). ① Corresponding author. E-mail: yangzujun@uestc.edu.cn; Tel: +86-28-8320 6556

High-molecular-weight glutenin subunits (HWM-GS) are encoded by three Glu-1 loci, namely, Glu-A1, Glu-B1, and Glu-D1, located on the long arms of group-1 chromosomes of common wheat (Triticum aestivum L.), which can be easily extracted and separated by the conventional SDS-PAGE. The allelic composition of Glu-1 loci was firmly associ-ated with the bread-making quality of wheat flour [1]. By far, a total of 22 Glu-A1, 56 Glu-B1, and 37 Glu-D1 alleles were listed in the catalog of gene symbols, some of which are only identified in species of wheat progenitors, T. turgidum and Aegilops tauschii [2]. Each Glu-1 locus consists of two tightly

linked genes, designated x-type and y-type. In total, six HMW-GS genes are present in cultivated wheat, but a few of them, such as gene 1Ay, and sometimes 1By and 1Ax, are not expressed [3, 4]. However, to date, studies on Glu-1 gene silencing involving 1Bx subunit genes have not been carried out.

Wild emmer wheat, Triticum turgidum L. var. dicoccoides (AABB, 2n=4x=28), is the ancestor of bread wheat and durum wheat, which are distributed along the boundary of the Mediterranean area [5]. Abundant allelic variations of HMW-GS in cultivated emmer wheat, which are believed to be useful genetic resources for quality improvement of bread wheat,

930 遗传学报 Acta Genetica Sinica Vol.33 No.10 2006

were revealed by several studies [5-7]. Several novel Glu-A1 and Glu-B1 alleles separated by SDS-PAGE were never identified in common wheat. However, the molecular identification of the T. turgidum L. var. dicoccum unique HMW glutenin locus was limited.

Vallega and Waines [7] first reported a novel variant, Glu-B1 null, in four T. turgidum L. var. di-coccoides lines. The Glu-B1 null was designated as Glu-B1m based on a standard T. dicoccoides line PI 94640 from Iran by McIntosh et al [2]. To determine the molecular mechanism of this silenced Glu-B1 locus in PI94640, the cloning and sequencing of the x-type of gene Glu-B1 was attempted using PCR-based methods, and the structure and evolution of the Glu-1Bx genes were discussed.

1 Materials and Methods

1. 1 Plant materials

Common wheat lines SY-95-71 and Chinese Spring (CS) were maintained at Triticeae Research Institute, Si-chuan Agricultural University, China. T. turgidum var. dicoccoides lines PI94640 and PI94660 as well as a wheat variety Moulin were provided by Dr. Harold Bockelman, National Plant Germplasm System, USDA-ARS, Aberdeen, Idaho, USA.

1. 2 SDS-PAGE analysis

Extraction of total protein from wheat en-dosperm and the removal of gliadin was based on the method of Xu et al [7]. The separation of the glutenin was analyzed using 12% SDS-PAGE, and the gels were stained overnight with 12% (w/v) trichloroacetic acid solution containing 0.05% Comassie Brilliant Blue R250 in the absolute ethanol (5% w/v) and de-tained in 10% ethanol.

1. 3 DNA preparation and PCR amplification

Genomic DNA of wheat line SY95-71 was iso-lated from 1–2 g of leaves of single plant about 4–6 weeks old, as described by Yang et al [8]. Based on the published HWM-GS gene sequence, two pairs of primers specific to Glu-1Bx gene were used. Primers P1 (5′-ATTCCGGCAACTTGTGGGT-3′)/P2 (5′-AC-

TACTGCCGCAAAGAGGACCAG-3′) for amplify-ing the upstream of promoter region were from Anderson et al [9], and P3 (5′-GATGGCTAAGCGCC- TGGTCC-3′)/P4 (5′-ATGCAAGCTGCAGAGAGTT- 3′) for amplifying the coding region were from D’ Ovidio et al [10].

PCR was carried out using PTC-100 Thermocy-cler (MJ Research, Inc. Watertown, MA). Each PCR reaction (100 μL) contained 300 ng of template, 0.2 mmol/L of each dNTPs, 1 μmol/L of each of the two primers, 10 μL of 10 × PCR buffer, and 5 U of Ex Taq DNA polymerase (TaKaRa, Japan). The PCR was programmed for 2 min at 95 , followed by 35 cycles ℃

of 94 for 1 min, 62 for 1 min, and 72 for 2 ℃ ℃ ℃

min 30 s. After the amplification, the final extension was kept for 10 min at 72 . The PCR product was ℃

analyzed on 1% agarose gels.

1. 4 Cloning, sequencing, and comparative analyses of the HMW-GS genes

The target PCR products were purified from aga-rose gel using QIAquick Gel Extraction Kit (QIAGEN). The fragment was ligated into a pGEMT plasmid vector (Promega, Madison, Wis.) and was used to transform competent cell of Escherichia coli DH5α strain. The three positive clones were sequenced on an automatic DNA sequencer (TaKaRa Biotech, Japan). The nucleo-tide sequences were assembled, and the sequence alignment among different Glu-1 alleles was carried out using BIOEDIT software, and the phylogenetic tree was constructed using Clustal W program. 2 Results

2. 1 Characterization of 1Bxm by SDS-PAGE and PCR

The composition of HMW glutenin subunits of T. turgidum var. dicoccoides line PI94640 were determined by SDS-PAGE, using wheat lines Chi-nese Spring (N, 7+8, 2+12), SY95-71 (1, 23+18, 5+10) and T. turgidum var. dicoccoides PI94660 (1, 23+18) as control (Fig.1). The results indicated that wheat line PI94640 contained only HMW glutenin subunit 1 of Glu-A1 and the subunits from Glu-B1 were lost.

YANG Zu-Jun et al.: Molecular Characterization of a HMW Glutenin Subunit Allele Providing Evidence for Silencing…… 931

The PCR primers, P1 + P2 and P3 + P4, were used to amplify the upstream sequence and coding sequences of Glu-B1x in PI94640, respectively. The primer P1 + P2 gives a product of 1.1 kb, which in-cludes the promoter region of Glu-1Bx, and primer P3+P4 gives a product of 2.4 kb, which corresponds to the coding region of Glu-1Bx. The results demon-strated that no large insertion/deletion occurred in Glu-B1 gene of PI94640.

2. 2 The nucleotide and the upstream sequence of 1Bxm gene

The PCR product of upstream region and coding region were cloned and sequenced, which resulted in a complete sequence of 3 442 bp containing 1 070 bp of upstream region (Fig. 2). The complete nucleotide have been deposited in the NCBI database with the accession number AY621068.

As shown in the complete sequence of 1Bx23 (Fig. 2), the TATA box located at the position that is 90 bp upstream of the ATG start codon, and a 23 bp conserved enhancer sequence, which is a major regu-latory element for gene expression [11], was also pre-sent at the position -208 to 245 bp. The –300 bp ce-real-box element duplicated in –502 to –465 bp and –449 to –411 bp, is a characteristic of cereal storage protein genes [9]. In addition, at the position of –640–649 bp, a

typical GCN4-like motif (GLM) with sequence GTGAGTCAT, which was confirmed to be regulated by the concentration of nitrogen in yeast, was also found [12, 13].

Compared with the reported upstream sequences of 1Bx7 (X13927) [3], 1Bx17 (JC2099) [14], 1Bx20 (AJ437000) [15], 1Bx14 (AY367771) [16], and 1Bx23 (AY553933) [17], there are only three single-base nu-cleotide substitutions, one base insertion between 1Bxm and 1Bx7, and three single-base nucleotide substitutions, six single-base deletions, and five base insertions between 1Bx23 and 1Bx17. An insertion of 184 bp, flanked by an 8 bp duplication, was only found in 1Bx20 and 1Bx14.

2. 3 Coding sequence and deduced amino acid sequences of 1Bxm

Based on the prediction of the amino acid sequence deduced from 1Bx allele, it shows that the first 21 resi-dues (63 bp from the initiator codon) function as a sig-nal peptide, which is cleaved during the synthesis of the HMW glutenin subunits. Three cysteine residues are found in the N-terminal domain. Translation of the sequence in the coding region of 1Bxm resulted in the formation of a presumptive mature protein with only 212 residues, which is due to the presence of a typical in-frame premature stop codon at the 637 bp after the translation start site (Fig. 2). The TAA stop codon is an important feature of this silent gene, which probably results from the T to C transition at the first base of glutamine codon (CAA) in other Glu-1Bx allele. If the premature stop codons were ignored, the resulting hypothetic protein would also contain 789 amino acid residues, similar to other Glu-B1x genes that contained repetitive central do-mains and C-terminal domains. Comparison of the deduced protein of 1Bxm with the active product of 1Bx7 revealed that only three amino acid substitu-tions occurred in the 212 residues region (Fig. 3).

2. 4 Phylogenetic tree and the evolution analyses

Sequence comparisons were performed among the Glu-1 with different wheat genomes (A, B and D genomes) to understand the relation and the divergent

Fig. 1 The electrophoretic pattern of HWM-GS in wheat line1: Chinese Spring; 2: SY95-71; 3: PI290911; 4: PI 94660.Arrow shows the region of Glu-B1.

932 遗传学报 Acta Genetica Sinica Vol.33 No.10 2006

-1070 ATTCCGGCAACAACTTGTGGGGTACATCTAGTTACAGTGGAATATTAGTGATGGCGTGAGCAAGCGATAA

-999 GGCCAACGAGAGAAGAAGTGCGTCGTCTATGGAGGCCAGGGAAAGACAATGGACATGCAAAGAGGTAGGGGCAGGGAAGAAACACTTGGA -909 GATCATAGAAGAACATAAGAGGTTAAACATAGGAGGGCATAATGGACAATTAAATCTACATTAATTGAACTCATTTGGGAAGTAAACAAA

-819 ATCCATATTCTGGTGTAAATCAAACTATTTGACGCGGATTTACTAAGATCCTATGTTAATTTTAGACATGACTGGCCAAAGGTTTCAGTT

-729 AGTTCATTTGTCACGGAAAGGTGTTTTCATAAGTCCAAAACTCTACCAACTATTTTGCACGTCATAGCATAGATAGATGTTGTGAGTCAT

-639 TGGATAGATATTGTGAGTCAGCATGGATTTGTGTTGCCTGGAAATCCAACTAAATGACAAGCAACAAAACCTGAAATGGGCTTTAGGAGA

-549 GATGGTTTATCAATTTACATGTTCCATGCAGGCTACCTTCCACTACTCGACATGGTTAGAAGTTTTGAGTGCCGCATATTTGCGGAAGCA

-459 ATGGCACTACTCGACATGGTTAGAAGTTTTGAGTGCCGCATATTTGCGGAAGCAATGGCTAACAGATACATATTCTGCCAAACCCCAAGA

-369 AGGATAATCACTCCTCTTAGATAAAAAGAACAGACCAATGTACAAACATCCACACTTCTGCAAACAATACACCAGAACTAGGATTAAGCC

-279 CATTACGTGGCTTTAGCAGACCGTCCAAAAATCTGTTTTGCAAGCACCAATTGCTCCTTACTTATCCAGCTTCTTTTGTGTTGGCAAACT

-189 GCCCTTTTCCAACCGATTTTGTTCTTCTCACGCTTTCTTCATAGGCTAAACTAACCTCGGCGTGCACACAACCATGTCCTGAACCTTCAC

-99 CTCGTCCCTATAAAAGCCCATCCAACCTTCACAATCTCATCATCACCCACAACACCGAGCACCCCAATCTACAGATCAATTCACTGACAG

-9 TTCACCGAGATGGCTAAGCGCCTGGTCCTCTTTGCGGCAGTAGTCGTCGCCCTCGTGGCTCTCACCGCCGCTGAAGGTGAGGCCTCTGGA 1 M A K R L V L F A A V V V A L V A L T A A E G E A S G Signal 82 CAACTACAATGTGAGCGCGAGCTCGAGGCATGCCAACAGGTGGTGGACCAGCAACTCCGAGACGTTAGCCCCGGGTGCCGCCCCATCACC 28 Q L Q C E R E L E A C Q Q V V D Q Q L R D V S P G C R P I T 172 GTCAGCCCGGGCACGAGACAATACGAGCAGCAACCTGTGGTGCCGTCCAAGGCCGGATCCTTCTACCCCAGCGAGACTACGCCTTCGCAG 58 V S P G T R Q Y E Q Q P V V P S K A G S F Y P S E T T P S Q 262 CAACTCCAACAAATGATATTTTGGGGAATACCTGCACTACTAAGAAGGTATTACCCAAGTGTAACTTCTTCGCAGCAGGGGTCATACTAT 89 Q L Q Q M I F W G I P A L L R R Y Y P S V T S S Q Q G S Y Y N-terminal Repeat 352 CCAGGCCAAGCTTCTCCGCAACAGTCAGGACAAGGACAGCAGCCAGGACAAGGACAGCAACCAGGACAAGGGCAACAAGATCAGCAACCA 119 P G Q A S P Q Q S G Q G Q Q P G Q G Q Q P G Q G Q Q D Q Q P 442 GGACAAAGACAACAAGGATACTATCCAACTTCTCCGCAACAGCCAGGACAAGGGCAACAATTGGGACAAGGGCAACCAGGGTACTACCCA 148 G Q R Q Q G Y Y P T S P Q Q P G Q G Q Q L G Q G Q P G Y Y P 532 ACTTCACAGCAGCCAGGACAAAAGCAGCAGCCAGGACAAGGGCAACAATCAGGACAAGGACAACAAGGGTACTACCCAACTTCCCCGCAA 178 T S Q Q P G Q K Q Q P G Q G Q Q S G Q G Q Q G Y Y P T S P Q 622 CAGTCAGGACAAGGGTAACAACCGGGACAAGGGCAACCAGGGTACTACCCAAGTTCTCCGCAGCAGTCAGGACAATGGCAGCAACCAGGA 208 Q S G Q G * Q P G Q G Q P G Y Y P S S P Q Q S G Q W Q Q P G 712 CAAGGGCAACAGCCAGGACAAGGGCAGCAATCAGGACAAGGGCAACAAGGTCAGCAGCCAGGACAAGGGCAACGACCAGGACAAGGACAA 238 Q G Q Q P G Q G Q Q S G Q G Q Q G Q Q P G Q G Q R P G Q G Q 802 CAAGGGTACTACCCAACTTCTTCGCAACAGCCGGGACAAGGGCAACAATCAGGACAAGGGCAACCAGGGTACTACCCAACTTCTTTGCGG 268 Q G Y Y P T S S Q Q P G Q G Q Q S G Q G Q P G Y Y P T S L R 892 CAGCCAGGACAATGGCAGCAACCAGGACAAGGGCAGCAATCAGGACAAGGGCAACTAGGTCAGCAGCCAGGACAAGGACAACAACCAGGA 398 Q P G Q W Q Q P G Q G Q Q S G Q G Q L G Q Q P G Q G Q Q P G 982 CAAGGACAACAAGGATACTACCCAACTTCTCTGCAACAGCCAGGACAAGGGCAACAACCGGGACAAGGGCAACCAGGGTACTACCCAACT 328 Q G Q Q G Y Y P T S L Q Q P G Q G Q Q P G Q G Q P G Y Y P T 1072 TCGCAGCAGTCGGAACAAGGGCAGCAGCCAGGACAAGGAAAACAACCAGGACAAGGACAACAAGGGTACTACCCAACTTATTCACAACAG 358 S Q Q S E Q G Q Q P G Q G K Q P G Q G Q Q G Y Y P T Y S Q Q 1162 TCAGGACAAGGGCAACAACTGGGACAAGGGCAACCAGGGTACTACCCAACTTCTCCACAGCAGTCAGGACAAGGACAACAATCAAGACAA 378 S G Q G Q Q L G Q G Q P G Y Y P T S P Q Q S G Q G Q Q S R Q 1252 GGACAACAAGGGTATTACCCAACTTCTCCGCAACAGTCAGGACAAGGGCAACAACCGGGACAAGGGCAATCGGGGTACTTCCCAACTTCT 418 G Q Q G Y Y P T S P Q Q S G Q G Q Q P G Q G Q S G Y F P T S 1342 CGGCAGCAGTCAGGACAAGGGCAGCAGCCAGGACAAGGACAACAGTCGGGACAAGGGCAACAAGGTCAGCAACCAGGACAAGGACAACAA 448 R Q Q S G Q G Q Q P G Q G Q Q S G Q G Q Q G Q Q P G Q G Q Q 1432 GCGTACTACCCAACTTCTTCGCAACAGTCAGGACAAAGGCAACAGGCAGGACAATGGCAACGACCGGGACAAGGGCAACCAGGGTACTAC 478 A Y Y P T S S Q Q S G Q R Q Q A G Q W Q R P G Q G Q P G Y Y 1522 CCAACCTCTCCACAGCAGCCAGGACAAGCGCAACAATCAGGACAATGGCAACTAGTGTACTACCCAACTTCTCCGCAACAGCCAGGCCAA 508 P T S P Q Q P G Q A Q Q S G Q W Q L V Y Y P T S P Q Q P G Q 1612 TTGCAACAACCAGCACAAGGGCAACAACCAGCACAAGGGCAACAATCAGCACAAGAGCAACAGCCAGGACAAGCGCAACAATCAGGACAA 538 L Q Q P A Q G Q Q P A Q G Q Q S A Q E Q Q P G Q A Q Q S G Q 1702 TGGCAACTAGTGTACTACCCAACTTCTCCGCAACAGCCAGGACAATTGCAACAACCAGCACAAGGGCAACAAGGGTACTACCCAACTTCT 568 W Q L V Y Y P T S P Q Q P G Q L Q Q P A Q G Q Q G Y Y P T S 1792 CCACAACAGTCAGGACAAGGGCAACAAGGGTACTACCCAACTTCTCCGCAACAGTCAGGACAAGGGCAACAAGGGTACTACCCAACTTCT 698 P Q Q S G Q G Q Q G Y Y P T S P Q Q S G Q G Q Q G Y Y P T S 1882 CCGCAACAGTCAGGACAAGGGCAGCAGCCAGGACAAGGACAACAGCCAAGACAAGGGCAACAAGGGTACTACCCAATTTCTCCGCAGCAG 628 P Q Q S G Q G Q Q P G Q G Q Q P R Q G Q Q G Y Y P I S P Q Q 1972 TCAGGACAAGGGCAACAACCAGGACAAGGGCAACAAGGATACTACCCAACTTCTCCGCAGCAGTCAGGACAAGGGCAACAACCAGGACAT 658 S G Q G Q Q P G Q G Q Q G Y Y P T S P Q Q S G Q G Q Q P G H 2062 GAGCAACAGCCAGGACAATGGCTGCAACCAGGACAAGGGCAACAAGGGTACTATCCAACTTCTTCACAGCAGTCAGGACAAGGGCAGCAA 688 E Q Q P G Q W L Q P G Q G Q Q G Y Y P T S S Q Q S G Q G Q Q 2152 TCAGGACAAGGGCAACAAGGGTACTACCCAACTTCTCTGTGGCAACCAGGACAAGGGCAACAACCAGGACAAGGGCAACAAGGCTACGAC 718 S G Q G Q Q G Y Y P T S L W Q P G Q G Q Q P G Q G Q Q G Y D 2242 AGTCCATACCATGTTAGCGCGGAGTACCAGGCGGCCCGCCTAAAGGTGGCAAAGGCGCAGCAGCTCGCGGCACAGCTGCCGGCAATGTGC 748 S P Y H V S A E Y Q A A R L K V A K A Q Q L A A Q L P A M C 2332 CGGCTGGAGGGCAGCGACGCATTGTCGGCCAGGCAGTGATAG 778 R L E G S D A L S A R Q * *

Fig. 2 Nucleotide and deduced amino acids of HMW-GS gene 1Bxm The putative TATA box was underlined. A duplicated –300 bp cereal-box are boxed and a GCN4-like domain was represented bydouble lines.

YANG Zu-Jun et al.: Molecular Characterization of a HMW Glutenin Subunit Allele Providing Evidence for Silencing…… 933

time by the construction of phylogenetic trees. In addition to the nucleotide sequence of Glu-1Bx alleles, other 7 HMW subunits, such as 1Ax1(X61009), 1Ax2*(M22208), 1By9(X61026), 1Dx2(X03346), 1Dx5(X12928), 1Dy10 (X12929), and 1Dy12(X03041), were also included. As shown in Fig. 4, the polygenetic tree was clearly divided into two clades: one composed of Glu-1Bx and y-type alleles; the other composed of 1Ax and 1Dx alleles. The Glu-1Bx branch had two sub-branches: one composed of 1Bx7, 1Bx17, 1Bx23, and 1Bxm; the other composed of 1Bx14 and 1Bx20. It is evident that the subunit 1Bxm may appear before the separation of 1Bx7, 1Bx17, and 1Bx17, and they appear earlier than the split of 1Bx14 and 1Bx20.

3 Discussion

The identification and isolation of storage pro-tein genes including the discovery of novel or mutant genes is of importance to explore the effect of the encoding genes and their combinations. The resulting amounts of individual storage proteins might be re-sponsible for the size distribution of the glutenin macropolymer and the dough properties, which is also important for determining the quality of the end product [15,18].

Variation in the HMW-GS composition among cultivars was found to have different effects on the gluten quality in hexaploid wheat. Therefore, the Glu-1 quality scores system was practically devel-oped [1]. The significant strengthening effect of the Glu-B1 locus both on gluten quality and rheological properties were well known in lines expressing sub-unit 1Bx17+1By18 and 1Bx7+1By8, whereas the subunits 1Bx20 and the 1Bx6+1By8 are associated with poor flour quality [15, 19, 20]. Investigations on the relationship of the HMW-GS and the amount of the gene were usually carried out by constructing the near isogenic lines [21]. Based on the investigation of sev-eral special genotypes (Red River 68, TAA 36), which contain two copies of the gene Glu-1Bx7, studies have shown that sometimes the over-expression of the 1Bx-type subunit may result in significantly higher dough strength [18, 22-23]. Conflict-

Fig. 4 A phylogenetic tree generated from the nucleotidesequence alignment of wheat HMW-GS genes by ClustalW(NJ method) Scale bar 0.01 = 1% difference among nucleotide sequences.

Fig. 3 Comparison of the deduced amino acid sequence of Glu-1Bxm subunit with 1Bx7 HMW-GS genes

934 遗传学报 Acta Genetica Sinica Vol.33 No.10 2006

ing results were also published on the possible dupli-cation of the Glu-1Bx7 gene in the over-strong Canadian variety, Glenlea [24]. Moreover, the over-expressions of the HMW-GS genes by transgenic approach have been re-cently reported on several glutenin loci including 1Bx7, which also supported the dosage effect of glutenin on the dough quality [25, 26]. Therefore, special genotype, which will be essential to study the contribution of novel genes to quality, needs to be generated. The genotype of PI94640 will facilitate further investiga-tion of the Glu-1Bx gene dosage effect for T. tur-gidum. In view of the fact that the effect of the y-type subunit of Glu-B1 was also missed in PI94640, stud-ies are underway to explore the molecular mechanism of the simultaneously silenced y-type gene.

The molecular characterization of the alleles of HMW glutenin subunits has provided insights on the relationship between gene structure and its functional association. The cysteine residues in the N-terminal and the C-terminal domains form intermolecular di-sulfide bonds, thereby resulting in the formation of large polymers, which appeared to influence the breadmaking and pasta qualities by contributing to the viscoelastic properties of the dough [27, 28]. It was evident that the subunit 1Bx20 displays one cysteine residue in the N-terminal domain, which was believed to be responsible for the negative effects on dough strength [15]. Moreover, the molecular evidences showed that high levels of expression may result ei-ther from more effective transcription/more efficient translation [22, 30], or from gene duplication [24]. In case of the silencing mechanisms in a Glu-1 allele of wheat were investigated as the transposon like ele-ment insertion or nucleotide mutation of the coding region or promoter regions [3, 30]. The present finding of a stop codon (TAA) that is 637 bp downstream from the translation start codon supports the hypothe-sis of the incomplete protein, which results the null phenotype of 1Bx in PI94640. A similar stop codon was also identified in the coding region of Glu-1 silenced Ay and 1Ax alleles [3, 31]. Recently, Zhu et al. [32,33] first re-ported the generation of the Glu-1Bx14 knockout mutant using the treatment with MNU as chemical mutagen,

which also showed the presence of in-frame stop codon in 1Bx14 coding sequence of repetitive domain.

The knowledge of nucleotide sequence of Glu-1Bxm and its deduced protein have led to a better understanding of the divergence and evolution of Glu-1 locus. Based on the sequence of promoter re-gion of 1Bxm and other 1Bx alleles, only the 1Bx14 and 1Bx20 have an additional insertion of 180 bp, which was considered a transposition event in the upstream region. However, this insertion has not dis-rupted the gene expression of 1Bx20 and 1Bx14 [9, 16]. Moreover, as shown in Fig. 4, the phylogenetic tree was constructed based on the sequence variation of Glu-1Bx coding region. The result demonstrated that the production of 1Bx7 and 1Bx17 might be more ancient than that of the 1Bx14 and 1Bx20, which is consistent with the analysis by Li et al [16]. In the pre-sent study, the 1Bxm allele was formed before the sepa-ration of 1Bx7 and 1Bx17. It is likely that the formation of 1Bxm perhaps occurred in the period of tetraploidiza-tion. On the one hand, the 1Bxm locus was not observed in cultivated T. turgidum var. durum [34, 35]. On the other hand, the subunit 1Bx17 was only observed in hexaploid wheat [6], which supported that the possible divergence of 1Bx17 from 1Bx7 may occur at the hexaploid wheat stage. The above evidences allow us to conclude that the Glu-B1x alleles had different divergence times and evolved at different rates.

References:

[1] Payne P I, Holt L M, Jackson E A, Law C N. Wheat stor-age proteins: their genetics and potential for manipulation by plant breeding. Philos Trans R Soc Lond Ser B, 1984, 304 : 359-371.

[2] McIntosh R A, Yamazaki Y, Devos K M, Dubcoveky J, Rogers W J, Appels R. Catalogue of gene symbols for wheat. In: Pogna N E, Romanò M, Pogna E A, Galterio G ,eds. Proceedings of the 10th International Wheat Ge-netics Symposium, vol. 4. SIMI, Rome, Italy. 2003.

[3] Halford N G, Forde J, Anderson O D, Green F C, Shewry P R. The nucleotide and deduced amino acid sequences of an HMW glutenin subunit gene from chromosome 1B of bread wheat (Triticum eastivum L.) and comparison with those of genes from chromosomes 1A and 1D. Theor Appl Genet, 1987, 75 : 117-126.

[4] Sun Y M, Yan Y M, Jiang Y, Xiao Y H, Hu Y K, Cai M H, Li

YANG Zu-Jun et al.: Molecular Characterization of a HMW Glutenin Subunit Allele Providing Evidence for Silencing…… 935

Y X, Hsam S L K, Zeller F J. Molecular cloning and com-parative analysis of a y-type inactive HMW glutenin subunit gene from cultivated emmer wheat (Triticum dicoccum L.). Hereditas(Beijing), 2004, 141 : 46-54 (in Chinese with an English abstract ).

[5] Ciaffi M, Lafiandra D, Porceddu E, Benedettelli S. Stor-age protein variation in wild emmer wheat (Triticum tur-gidum ssp. dicoccoides) from Jordan and Turkey. I. Elec-trophoretic characterization of genotypes. Theor Appl Genet, 1993, 86 : 474-480.

[6] Vallega V, Waines J G. High molecular weight glutenin subunit variation in Triticum turgidum var. dicoccum. Theor Appl Genet, 1987, 74 : 706-710.

[7] Xu S S, Khan K, Klindworth D L, Faris J D. Chromoso-mal location of genes for novel glutenin subunits and gliadins in wild emmer wheat (Triticum turgidum L. var. dicoccoides). Theor Appl Genet, 2004, 108 : 1221-1228.

[8] Yang Z J, Li G R, Feng J, Jiang H R, Ren Z L. Morlecu-lar cytogentic characterization and disease resistance ob-servation of wheat-Dasypyrum brebiaristaum partial am-phiploid and its derivatives. Hereditas(Beijing), 2005, 142 : 80-85(in Chinese with an English abstract ).

[9] Anderson O D, Abraham-Pierce F A, Tam A. Conserva-tion in wheat high-molecular-weight glutenin gene pro-moter sequences: comparisons among loci and among al-leles of the Glu-B1-1 locus. Theor Appl Genet, 1998, 96 : 568-576.

[10] D’Ovidio R, Masci S, Porceddu E. Development of a set of oligonucleotide primers specific for genes at the Glu-1 complex loci of wheat. Theor Appl Genet, 1995, 91 : 189-194.

[11] Shewry P R, Halford N G. Cereal seed storage proteins: structures, properties and role in grain utilization. J Experimental Biology, 2002, 53 : 947-958.

[12] Hammond-Kosack M C U, Holdsworth M J, Bevan M W. In vivo footprinting of a low molecular weight glutenin gene (LMWG-1D1) in wheat endosperm. EMBO J, 1993, 12 : 545-554.

[13] Lamacchia C, Shewry P R, Fonzo N D, Forsyth J L, Har-ris N, Lazzeri P A, Napier J A, Halford N G, Barcelo P. Endosperm-specific activity of a storage protein gene promoter in transgenic wheat seed. J Experimental Bot-any, 2001, 52 : 243-250.

[14] Reddy P, Appels R. Analysis of a genomic DNA segment carrying the wheat high-molecular-weight (HMW) glu-tenin Bx17 subunit and its use as an RFLP marker. Theor Appl Genet, 1993, 85 : 616-624.

[15] Shewry P R, Gilbert S M, Savage W J, Tatham A S, Wan Y F, Belton P S, Wellner N, D’ Ovidio R, Bekes F, Hal-

ford N G. Sequence and properties of HMW subunit 1Bx20 from pasta wheat (Triticum durum), which is as-sociated with poor end use properties. Theor Appl Genet, 2003, 106 : 744-750.

[16] Li W, Wan Y, Liu Z, Liu K, Liu X, Li Z, Zhang X, Dong Y, Wang D. Molecular characterization of HMW glutenin subunit allele 1Bx14: further insights into the evolution of Glu-B1-1 alleles in wheat and related species. Theor Appl Genet, 2004, 109 : 1093-1104.

[17] Yang Z J, Li G R, Shu H L, Chang Z J, Hou W R. Mo-lecular characterization of a x-type coding and promoter sequences of wheat high molecular weight gene Glu-B1p in wheat line SY95-71. International Wheat Quality Conference. Beijing, 2004, 47-48.

[18] Juhasz A, Larroque O R, Tamas L, Hsam S L, Zeller F J, Bekes F, Bedo Z. Bankuti 1201- an old Hungarian wheat variety with special storage protein composition. Theor Appl Genet, 2003, 107 : 697-704.

[19] Lawrence G J, Macritchie F, Wrigley C W. Dough and baking quality of wheat lines deficient in glutenin sub-units controlled by the Glu-A1, Glu-B1 and Glu-D1 loci. J Cereal Sci, 1988, 7 : 109-112.

[20] Schwarz G, Felsenstein F G, Wenzel G. Development and validation of a PCR-based marker assay for negative se-lection of the HMW glutenin allele Glu-B1-1d (Bx-6) in wheat. Theor Appl Genet, 2004, 109 : 1064-1069.

[21] Payne P I, Nightingale M A, Krattiger A F, Holt L M.The relationship between HMW glutenin subunit composition and the bread-making quality of British-grown wheat va-rieties. J Sci Food Agric, 1987, 40 : 51-65.

[22] Lukow O M, Forsyth S A, Payne P I. Over-production of HMW glutenin subunits coded on chromosome 1B in common wheat, Triticum aestivum L. J Genet Breed, 1992, 46 : 187-192.

[23] D’Ovidio R, Masci S, Porceddu E, Kasarda D D. Dupli-cation of the Bx7 high-molecular-weight glutenin subunit gene in bread wheat (Triticum aestivum L.) cultivar ‘Red River 68’. Plant Breed, 1997, 116 : 525-531.

[24] Butow B J, Ma W, Gale K R, Cornish G B, Rampling L, Larroque O, Morell M K, Bekes F. Molecular discrimina-tion of Bx7 alleles demonstrates that a highly expressed high-molecular-weight glutenin allele has a major impact on wheat flour dough strength. Theor Appl Genet, 2003, 107 : 1524-1532.

[25] Blechl A E, Anderson O D. Expression of a novel high-molecular-weight glutenin subunit gene in trans-genic wheat. Nat Biotechnol, 1996, 14 : 875-879.

[26] Alvarez M, Gomez L M, Carrillo L M, Vallejos R H. Analysis of dough functionality of flours from transgenic

936 遗传学报 Acta Genetica Sinica Vol.33 No.10 2006

what. Mol Breed, 2001, 8, 103-108. [27] Wrigley C W. Giant proteins with flour power. Nature,

1996, 381 : 738-739. [28] Gianibelli M C, Larroque O R, MacRitchie F, Wrigley C

W. Biochemical, genetic and molecular characterization of wheat glutenin and its component subunits. Cereal Chemistry, 2001, 78 : 635-646.

[29] Rampitsch C, Jordan M C, Cloutier S. A matrix attach-ment region is located upstream from the high-molecular- weight-glutenin gene Bx7 in wheat (Triticum aestivum L.). Genome, 2000, 43 : 483-486.

[30] Harberd N P, Flavell R B, Thompson R D. Identification of a transposon like insertion in a Glu-1 allele of wheat. Mol Gen Genet, 1987, 209 : 326-332.

[31] De Bustos A, Rubio P, Jouve N. Molecular characteriza-tion of the inactive allele of the gene Glu-A1 and the de-velopment of a set of AS-PCR markers for HMW gluten-ins of wheat. Theor Appl Genet, 2000, 100, 189-194.

[32] Zhu Y F, Li YW, Chen Y, Li H, Liang H, Yue S J, Zhang A M, Zhang X Q, Wang D W, Jia X. Generation and characterization of a high molecular weight glutenin 1Bx14-deficient mutant in common wheat. Plant Breed, 2005, 124 : 421-427.

[33] Deng Z Y, Zhao H X, Fan S H, Gi W Q, Guo A G, Xue X Z. Purification and biochemical characterization of high-molecular-weight glutenin subunit 14 and 15. Acta Genetica Sinica, 2001, 28(1) : 46-51(in Chinese with an English abstract).

[34] Branlard G, Autran J C, Monneveux P. High molecular weight glutenin subunits in durum wheat (Triticum du-rum). Theor Appl Genet, 1989, 78 : 353-358.

[35] Cherdouh A, Khelif D, Carrillo J M, Nieto-Taladriz M T. The high and low molecular weight glutenin subunit polymorphism of Algerian durum wheat landraces and old cultivars. Plant Breeding, 2005, 124 : 338-342.

小麦高分子量谷蛋白亚基 Glu-B1 位点沉默基因的克隆与序列

分析

杨足君 1，李光蓉 1，刘 畅 1，冯 娟 1，周建平 1，任正隆 1,2

1. 电子科技大学生命科学与技术学院，成都 610054；

2. 四川农业大学植物遗传育种重点实验室，雅安 525014

摘 要：二粒小麦(Triticum turgidum L. var. dicoccoides)具有极其丰富的遗传多样性，是栽培小麦品种改良的巨大基因库。在

高分子量谷蛋白基因的组成上，它具有许多栽培小麦不存在的变异类型，在 Glu-B1 位点上的变异更大。我们利用种子贮

藏蛋白的 SDS-PAGE 方法从原产于伊朗的二粒小麦材料 PI94640 中观察到缺失 Glu-B1 区的高分子量谷蛋白亚基。利用

Glu-1Bx 基因保守序列设计 PCR 引物，对该材料的总 DNA 扩增，获得了 x 型亚基编码基因(Glu-1Bxm)的全序列，其全长

为 3 442 bp 含 1 070 bp 的启动子区。序列比较发现，Glu-1Bxm 在启动子区序列与 Glu-1Bx7 的最为相似。而在基因编码区，

我们发现 Glu-1Bxm 仅编码 212 个氨基酸，由于开放阅读框中起始密码子后第 637 位核苷酸发生了点突变，即编码谷酰胺

的 CAA 突变为终止密码 TAA，可能直接导致了该高分子量谷蛋白亚基的失活，这是我们在小麦 Glu-B1 位点基因沉默分子

证据的首次报道。将 Glu-1Bxm 全序列与 Glu-B1 位点其他等位基因进行了系统树分析，发现 Glu-1Bxm 是较为古老的类型。

本文还对该特异高分子量谷蛋白亚基变异类型对品质遗传改良研究的意义进行了讨论。

关键词：Glu-1Bx；高分子量谷蛋白基因；基因沉默；小麦

作者简介：杨足君（1971-），男，博士，研究方向：植物分子细胞遗传与育种。E-mail: yangzujun@uestc.edu.cn