# Cloning and Characterization of Low-Molecular-Weight Glutenin Subunit Alleles from Chinese Wheat Landraces (Triticum aestivum L.)

Authors: Hongqi Si; Manli Zhao; Xin Zhang; Guoliang Yao; Genlou Sun; Chuanxi Ma
Journal: The Scientific World Journal
 (2014)

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## Abstract

Low-molecular-weight glutenin subunits (LMW-GS) are of great importance in processing quality and participate in the formation of polymers in wheat. In this study, eight new LMW-GS alleles were isolated from Chinese wheat landraces (Triticum aestivum L.) and designated as Glu-A3-1a, Glu-A3-1b, Glu-B3-1a, Glu-B3-1b, Glu-B3-1c, Glu-D3-1a, Glu-D3-1b, and Glu-D3-1c, which were located at the Glu-A3, Glu-B3, and Glu-D3 loci, respectively. Based on the proteins encoded, the number of deduced amino acids of Glu-B3 alleles was approximately 50 more than those of Glu-A3 and Glu-D3 alleles. The first cysteine of Glu-A3 and Glu-D3 alleles was located at the N-terminal domain, while that of Glu-B3 alleles was found in the repetitive domain, which may lead to the different functioning in forming disulfide bonds. All the eight genes were LMW-m types and the new allele of Glu-B3-1a which had nine cysteine residues may be the desirable LMW-GS gene for improving bread-making quality.


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## Body

## 1. Introduction

Wheat dough possesses unique viscoelastic properties determined by the structure and interaction of storage proteins in making bread and noodles [1]. Wheat storage proteins are mainly composed of monomeric gliadins and polymeric glutenins [2, 3], and these glutenins are divided into the low-molecular-weight glutenin subunits (LMW-GS) and the high-molecular-weight glutenin subunits (HMW-GS) according to their electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [4]. The HMW-GS have been recognized as the major determinants of dough and gluten properties [5], but LMW-GS also play an important role in determining wheat dough’s viscoelastic properties [6].The LMW-GS genes are encoded byGlu-A3,Glu-B3, andGlu-D3 loci on the short arms of chromosomes 1A, 1B, and 1D, respectively, in hexaploid wheat [7]. Allelic variation in LMW-GS is generally accepted to have an important effect on wheat processing quality [8].Glu-A3a,Glu-B3d, andGlu-D3a have a better effect on dough strength than other alleles at theGlu-3 loci. The effects ofGlu-A3d,Glu-B3i,Glu-A3d, andGlu-B3d contribute most to dough extensibility and Zeleny sedimentation volume, whileGlu-D3 loci do not have a significant effect on either [9]. According to He et al. [10], noodle quality is decided by the protein subunits ofGlu-A3d andGlu-B3d, andGlu-A3d is significantly more important in this process than other alleles. Si et al. [11] reported thatGlu-B3b,Glu-B3g, andGlu-B3h significantly heightened the SDS sedimentation volume, whileGlu-B3a,Glu-B3c, andGlu-B3j significantly lowered the SDS sedimentation volume. So far, no consistent conclusion has been reached regarding the influence of LMW-GS allelic variation on wheat processing quality, perhaps due to the different materials used, and the interaction between genotypes and the environment.Although the mobility of many LMW-GS subunits is very similar and sometimes overlapped with each other in SDS-PAGE, a lot of researches have recently been conducted on LMW-GS genes by allelic-specific polymerase chain reaction (PCR) in common wheat and related species [12–14], which lead to a better understanding of the function, structure, and diversity of LMW-GS.The landraces possess many useful traits that have been lost in modern cultivars. The phenological, morphological, physiological, and quality traits in landraces are genetically diverse [15]. Although more than 15 LMW-GS genes have been found so far in wheat [16], further studies of the diversity of these LMW-GS genes and cloning of new and rare alleles are interesting and challenging. In this study, we isolated and characterized eight new alleles located atGlu-A3,Glu-B3, andGlu-D3 loci from landraces for which no LMW-GS genes have been previously detected by the corresponding molecular markers [17–19].


## 2. Materials and Methods

### 2.1. Plant Materials

The landraces Jiangdongmen, Daqingmang, Hongjinbaoyin, Dabaimai, Hongmangzi, Hongdougou, and Baimangmai used in this study were kindly provided by the National Gene Bank of China, Institute of Crop Science, CAAS, China. In our previous research, none of the LMW-GS genes were detected in the varieties Daqingmang and Hongjinbaoyin using the PCR markers forGlu-A3 [17] alleles, theGlu-B3 [18] alleles from the varieties Hongdougou, Dabaimai, and Hongmangzi, or theGlu-D3 [19] alleles from the varieties Jiangdongmen, Daqingmang, and Baimangmai. To determine whether the genes at theGlu-A3,Glu-B3, andGlu-D3 loci corresponding to the above varieties are missing or whether it is a novel gene, we used locus-specific primers to amplify the LMW-GS genes specifically for theGlu-A3,Glu-B3, andGlu-D3 loci.


### 2.2. Genomic DNA Extraction and PCR Amplification

Genomic DNA was isolated from single dry seeds according to the procedure of SDS-phenol-chloroform with minor modification. Six locus-specific primer sets, Glua3f1/Glua3r2 (R: GTACGCTTTTGTAGCTTGTGC, F: GATGCCAACGCCTAATGGCACAC) [17], 5/7 (R: TCCTGAGAAGTGCATGACATG, F: GTAGGCACCAACTCCGGTGC) [20], and 3/4 (R: TTGTAGAAACTGCCATCCTT, F: GTCACCGCTGCATCGACATA) [20], were used for amplifying LMW-GS genes at theGlu-A3 locus on chromosome 1A and theGlu-B3 andGlu-D3 loci on chromosomes 1B and 1D, respectively. The locus-specific primers were synthesized by Shanghai Sangon Biological Engineering & Technology and Service Ltd. (http://www.sangon.com/). PCR amplification was performed usingEx Taq DNA polymerase (0.5 U, TaKaRa, Shiga, Japan) in 10 μL of 1x PCR buffer (comprising 2 mM MgCl2; TaKaRa) containing 50 ng genomic DNA, 0.1 mM dNTP, and 20 μM of each primer. The PCR conditions were 94°C for 5 min followed by 38 cycles at 94°C for 1 min (or 35 s for primer pair 3/4 at 52°C, 5/7 at 54°C, and Glua3f1/Glua3r2 at 59°C), followed by 72°C for 90 s and a final extension at 72°C for 10 min.


### 2.3. DNA Cloning and Sequencing of LMW-GS Genes

The PCR products were separated using 1.2% agarose gels and the expected fragments were isolated from the gels using an EasyPure Quick Gel Extraction Kit (TransGen, Beijing, China). The isolated PCR products were cloned into the pMD18-T Simple Vector (TaKaRa) and transformed intoE. coli Competent Cells DH5α (TaKaRa). The positive colonies were verified using M13 universal primers, and the selected clones were sequenced by Shanghai Sangon Biological Engineering & Technology and Service Ltd. Each PCR and sequencing analysis was repeated at least three times to avoid technical errors.


### 2.4. Accession Numbers

The LMW-GS gene sequences identified from Jiangdongmen, Daqingmang, Hongjinbaoyin, Dabaimai, Hongmangzi, Hongdougou, and Baimangmai were deposited in GenBank under accession numbers KF020658–KF020665. KF020658 and KF020659 were cloned usingGlu-A3 locus-specific primers from Daqingmang and Hongjinbaoyin, designated asGlu-A3-1aandGlu-A3-1b, respectively. The sequences KF020660–KF020662 were cloned usingGlu-B3 locus-specific primers from Hongdougou, Dabaimai, and Hongmangzi, designated asGlu-B3-1a,Glu-B3-1b, andGlu-B3-1c, respectively, and KF020663–KF020665 were cloned usingGlu-D3 locus-specific primers from Jiangdongmen, Daqingmang, and Baimangmai, designated asGlu-D3-1a,Glu-D3-1b, andGlu-D3-1c, respectively.


### 2.5. LMW-GS Gene Analysis

Sequence analysis and characterization were performed using DNAMAN software (http://www.lynnon.com/), BLAST tools at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi/), and the PlantCare database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The nomenclature of the LMW-GS genes followed the “Catalogue of Gene Symbols for Wheat” at http://wheat.pw.usda.gov/ggpages/wgc/98//.


## 2.1. Plant Materials

The landraces Jiangdongmen, Daqingmang, Hongjinbaoyin, Dabaimai, Hongmangzi, Hongdougou, and Baimangmai used in this study were kindly provided by the National Gene Bank of China, Institute of Crop Science, CAAS, China. In our previous research, none of the LMW-GS genes were detected in the varieties Daqingmang and Hongjinbaoyin using the PCR markers forGlu-A3 [17] alleles, theGlu-B3 [18] alleles from the varieties Hongdougou, Dabaimai, and Hongmangzi, or theGlu-D3 [19] alleles from the varieties Jiangdongmen, Daqingmang, and Baimangmai. To determine whether the genes at theGlu-A3,Glu-B3, andGlu-D3 loci corresponding to the above varieties are missing or whether it is a novel gene, we used locus-specific primers to amplify the LMW-GS genes specifically for theGlu-A3,Glu-B3, andGlu-D3 loci.


## 2.2. Genomic DNA Extraction and PCR Amplification

Genomic DNA was isolated from single dry seeds according to the procedure of SDS-phenol-chloroform with minor modification. Six locus-specific primer sets, Glua3f1/Glua3r2 (R: GTACGCTTTTGTAGCTTGTGC, F: GATGCCAACGCCTAATGGCACAC) [17], 5/7 (R: TCCTGAGAAGTGCATGACATG, F: GTAGGCACCAACTCCGGTGC) [20], and 3/4 (R: TTGTAGAAACTGCCATCCTT, F: GTCACCGCTGCATCGACATA) [20], were used for amplifying LMW-GS genes at theGlu-A3 locus on chromosome 1A and theGlu-B3 andGlu-D3 loci on chromosomes 1B and 1D, respectively. The locus-specific primers were synthesized by Shanghai Sangon Biological Engineering & Technology and Service Ltd. (http://www.sangon.com/). PCR amplification was performed usingEx Taq DNA polymerase (0.5 U, TaKaRa, Shiga, Japan) in 10 μL of 1x PCR buffer (comprising 2 mM MgCl2; TaKaRa) containing 50 ng genomic DNA, 0.1 mM dNTP, and 20 μM of each primer. The PCR conditions were 94°C for 5 min followed by 38 cycles at 94°C for 1 min (or 35 s for primer pair 3/4 at 52°C, 5/7 at 54°C, and Glua3f1/Glua3r2 at 59°C), followed by 72°C for 90 s and a final extension at 72°C for 10 min.


## 2.3. DNA Cloning and Sequencing of LMW-GS Genes

The PCR products were separated using 1.2% agarose gels and the expected fragments were isolated from the gels using an EasyPure Quick Gel Extraction Kit (TransGen, Beijing, China). The isolated PCR products were cloned into the pMD18-T Simple Vector (TaKaRa) and transformed intoE. coli Competent Cells DH5α (TaKaRa). The positive colonies were verified using M13 universal primers, and the selected clones were sequenced by Shanghai Sangon Biological Engineering & Technology and Service Ltd. Each PCR and sequencing analysis was repeated at least three times to avoid technical errors.


## 2.4. Accession Numbers

The LMW-GS gene sequences identified from Jiangdongmen, Daqingmang, Hongjinbaoyin, Dabaimai, Hongmangzi, Hongdougou, and Baimangmai were deposited in GenBank under accession numbers KF020658–KF020665. KF020658 and KF020659 were cloned usingGlu-A3 locus-specific primers from Daqingmang and Hongjinbaoyin, designated asGlu-A3-1aandGlu-A3-1b, respectively. The sequences KF020660–KF020662 were cloned usingGlu-B3 locus-specific primers from Hongdougou, Dabaimai, and Hongmangzi, designated asGlu-B3-1a,Glu-B3-1b, andGlu-B3-1c, respectively, and KF020663–KF020665 were cloned usingGlu-D3 locus-specific primers from Jiangdongmen, Daqingmang, and Baimangmai, designated asGlu-D3-1a,Glu-D3-1b, andGlu-D3-1c, respectively.


## 2.5. LMW-GS Gene Analysis

Sequence analysis and characterization were performed using DNAMAN software (http://www.lynnon.com/), BLAST tools at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi/), and the PlantCare database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The nomenclature of the LMW-GS genes followed the “Catalogue of Gene Symbols for Wheat” at http://wheat.pw.usda.gov/ggpages/wgc/98//.


## 3. Results and Discussion

### 3.1. Basic Characteristics of the LMW-GS Alleles Identified in This Study

Eight novel LMW-GS alleles with no intron were obtained in this study (see Figure S1 in Supplementary Material available online athttp://dx.doi.org/10.1155/2014/371045/). Their sequences were submitted to GenBank with the accession numbers KF020658–KF020665 and designated asGlu-A3-1a,Glu-A3-1b,Glu-B3-1a,Glu-B3-1b,Glu-B3-1c,Glu-D3-1a,Glu-D3-1b, andGlu-D3-1c, following the nomenclature rules at http://wheat.pw.usda.gov/ggpages/wgc/98//.Glu-A3-1a andGlu-A3-1bhad the same size of 1089 bp and could be translated into 298 amino acids. The nucleotide and deduced amino acid sequences of the other six genes were varied with 1361–1603 bp and 306–351 residues (Table 1). The upstream nucleotide sequences of the cloned genes were searched for in the PlantCare database to analyze the characteristics of the promoter sequences. The eight genes all contained CAAT-boxes and TATA-boxes.Glu-B3-1a,Glu-B3-1b,Glu-B3-1c,Glu-D3-1a,Glu-D3-1b, andGlu-D3-1c contained a Prolamin-box in the upstream sequence (Table 1). All eight genes from landraces in our study belonged to the LMW-m type since their first amino acid at the N-terminal was methionine.Table 1
Comparison of nucleotide sequences of the eight cloned genes.
LMW-GS genes
Upstream
CDS
Total length
Deduced amino acids
Putative transcription binding sites in the upstream region
Glu-A3-1a
195 bp
894 bp
1089 bp
298
CAAT-box, TATA-box, ARE, and MRE
Glu-A3-1b
195 bp
894 bp
1089 bp
298
CAAT-box, TATA-box, ARE, and MRE
Glu-B3-1a
317 bp
1053 bp
1370 bp
351
CAAT-box, TATA-box, and Prolamin-box
Glu-B3-1b
317 bp
1044 bp
1361 bp
348
CAAT-box, TATA-box, and Prolamin-box
Glu-B3-1c
317 bp
1050 bp
1367 bp
350
CAAT-box, TATA-box, and Prolamin-box
Glu-D3-1a
593 bp
921 bp
1591 bp
307
CAAT-box, TATA-box, and Prolamin-box
Glu-D3-1b
605 bp
921 bp
1603 bp
307
CAAT-box, TATA-box, and Prolamin-box
Glu-D3-1c
605 bp
912 bp
1594 bp
306
CAAT-box, TATA-box, and Prolamin-box


### 3.2. Nucleotide Comparison Analysis of the LMW-GS Alleles within theGlu-3 Loci

To more accurately compare the differences between LMW-GS genes at theGlu-3 loci, we used BLAST tools to find the sequences most similar to the eight genes obtained in this study. NineGlu-3 genes deposited in GenBank were selected for multiple sequence alignment and included FJ549937, FJ549938, and FJ549946 located at theGlu-A3 locus; EU369729, EU369730, and DQ630441 at theGlu-B3 locus; and DQ357054, DQ357055, and EU189094 at theGlu-D3 locus. Based on the results of these alignments (Table 2), of the eight genes isolated in this study,Glu-A3-1a, andGlu-A3-1b were found to be new alleles at theGlu-A3 locus;Glu-B3-1a,Glu-B3-1b, andGlu-B3-1c were new alleles at theGlu-B3 locus; andGlu-D3-1a,Glu-D3-1b, andGlu-D3-1c were new alleles at theGlu-D3 locus.Table 2
Sequence identities of the eight new LMW-GS alleles to the previously reportedGlu-3 genes.
LMW-GS alleles
Glu-A3 gene accession number
Glu-B3 gene accession number
Glu-D3 gene accession number
FJ549946
FJ549937
FJ549938
DQ630441
EU369729
EU369730
DQ357054
DQ357055
EU189094
Glu-A3-1a
93
92
92
84
84
84
87
87
87
Glu-A3-1b
93
92
91
83
83
83
86
86
87
Glu-B3-1a
85
85
85
95
84
84
83
83
83
Glu-B3-1b
N
85
86
99
85
85
84
84
85
Glu-B3-1c
N
85
86
99
85
85
83
83
84
Glu-D3-1a
87
88
88
88
85
85
95
95
94
Glu-D3-1b
86
87
87
83
82
82
99
99
99
Glu-D3-1c
87
87
87
84
82
82
99
99
99
Note: N means that no highly similar sequences were found using BLAST tools.


### 3.3. Deduced Proteins Comparison Analysis of the LMW-GS Alleles within theGlu-3 Loci

The deduced amino acid sequences of the eight genes comprised four structural regions, including a signal peptide of 20 amino acids, a conserved N-terminal region of 13 amino acids, a diverse repetitive domain, and a C-terminal domain, suggesting that the eight genes conformed to the typical molecular characteristics of LMW-GS (Supplementary Material, Figure S2). We compared the alleles within theGlu-3 loci and found that isoleucine was at the sixth position of the signal peptide in the allele located atGlu-B3 locus, while valine was at theGlu-A3 andGlu-D3 loci (Table 3), which might result from the substitution of ATC for GTC (Supplementary Material, Figure S1). The eleventh position of the N-terminal at theGlu-A3 andGlu-D3 loci was arginine, while lysine filled the same position at theGlu-B3 locus (Table 3), which may be the result of an AGA→AAA transversion (Supplementary Material, Figure S1).Table 3
Comparison of the deduced amino acid sequences for genes isolated in this study and data from GenBank.
Sig.
N-ter.
Rep.
C-ter.
Glu-A3
FJ549937
V
C
R
C
C
C
CC
C
C
FJ549938
V
C
R
C
C
C
CC
C
C
FJ549946
V
C
R
C
C
C
CC
C
C
Glu-A3-1a
V
C
R
C
C
C
CC
C
C
Glu-A3-1b
V
C
R
C
C
C
CC
C
C
Glu-B3
EU369729
I
K
C
C
C
C
CC
C
C
EU369730
I
K
C
C
C
C
CC
C
C
DQ630441
I
K
C
C
C
C
CC
C
C
Glu-B3-1a
I
K
C
C
C
C
CC
C
C
C
Glu-B3-1b
I
K
C
C
C
C
CC
C
C
Glu-B3-1c
I
K
C
C
C
C
CC
C
C
Glu-D3
DQ357054
V
C
R
C
C
C
CC
C
C
DQ357055
V
C
R
C
C
C
CC
C
C
EU189094
V
C
R
C
C
C
CC
C
C
Glu-D3-1a
V
C
R
C
C
C
CC
C
C
Glu-D3-1b
V
C
R
C
C
C
CC
C
C
Glu-D3-1c
V
C
R
C
C
C
CC
C
C
The accession numbers are as follows: FJ549937, FJ549938, and FJ549946 are genes at theGlu-A3 locus; EU369729, EU369730, and DQ630441 are genes at the Glu-B3 locus; and DQ357054, DQ357055, and EU189094 are genes at Glu-D3 locus. I, V, R, K, and C represent isoleucine, valine, arginine, lysine, and cysteine, respectively.Eight cysteine residues were found inGlu-A3-1a,Glu-A3-1b,Glu-B3-1b,Glu-B3-1c,Glu-D3-1a,Glu-D3-1b, andGlu-D3-1c, whileGlu-B3-1a had nine. By comparing theGlu-3 loci alleles, the locations of the first and seventh cysteine were found to be varied. As shown in Figure S2 (Supplementary Material) and Table 3, the first cysteine of theGlu-A3 andGlu-D3 alleles was located at the fifth position of the N-terminal domain, whereas the first cysteine of theGlu-B3 alleles was found at the 46th position of the deduced amino acid sequences in the repetitive domain. The seventh cysteine at theGlu-D3 alleles was located 16 amino acids higher up than that at theGlu-A3 andGlu-B3 alleles. In particular, the extra cysteine of alleleGlu-B3-1a was found at the 321st position of the amino acid sequence.Usually the first and seventh cysteine residues participate in forming the intermolecular disulfide bond, while the remaining residues participate in the formation of intramolecular disulfide bonds [21]. Disulfide bonds have an important influence on determining the properties and structure of wheat gluten proteins [22]. The first cysteine at theGlu-B3 allele was in the repetitive domain, however, the first cysteine atGlu-A3 andGlu-D3 alleles were in the N-terminal. The positions of the seventh cysteine residue at theGlu-D3 alleles were also different from theGlu-A3 andGlu-B3 alleles. The LMW-GS gene, which had nine cysteine residues, resulted in desirable processing quality [20]; therefore, the new allele ofGlu-B3-1a may be the desirable LMW-GS gene for improving bread-making quality because the extra cysteine residue could form more disulfide bonds than other alleles during the development of glutenin macropolymer.Insertions/deletions often appeared within the repetitive domain. The length variation of the LMW-GS genes was mainly due to the numbers of repeat motifs, which ranged from 12 to 25 in the repetitive domain. The repeat motif is shown in Table4. Two long insertions were present in theGlu-B3 sequences, that is, 18 amino acid insertions at positions 44–61 and eight amino acid insertions at positions 81–88. The repetitive region was composed of a representative repeat motif P1-2FP/SQ2–6, of which the number would have an impact on changes in the length of the protein [23]. The length of the diverse repetitive domain ofGlu-B3 alleles was longer than those ofGlu-A3 andGlu-D3 alleles since more insertions occurred in this domain. Masci et al. [24] indicated that a 42 K LMW-GS would have a good processing quality due to its large repeat domain. The repeat domain through intermolecular interactions between large numbers of glutamine side chains, which are both good hydrogen bond donors and acceptors, may prove useful in increasing the viscosity and elasticity of wheat dough [24]. More repeat motifs in the repetitive domain would lead to a better flour quality [25]. Based on Figure S2 (Supplementary Material), the sequences at theGlu-B3 locus had 50 more deduced amino acids than those atGlu-A3 andGlu-D3 loci. Therefore, genes at theGlu-B3 locus are more desirable than those at theGlu-A3 andGlu-D3 loci.Table 4
Repeat motif of repetitive domains in the isolated LMW-GS deduced amino acid sequences.
Glu-A3-1a
Glu-A3-1b
Glu-B3-1a
Glu-B3-1b
Glu-B3-1c
Glu-D3-1a
Glu-D3-1b
Glu-D3-1c
QQQ
QQQ
QQQ
QQQ
QQQ
QQQ
QQQ
QQQ
PLPPQQ
PLPPQQ
PLPPQQQ
PLPPQQQ
PLPPQQQ
PLPPQQ
PLPPQQ
PLPPQQ
SFSQQ
SFSQQ
PPCSQQQQ
PPCSQQQQ
PPCSQQQQ
TFPQQ
TFPQQ
TFPQQ
PPFSQQQQQ
PPFSQQQQQ
PFPQQQQ
PFPQQQQ
PFPQQQQ
PLFSQQQQQQ
PLFSQQQQQQ
PLFSQQQQQQ
PLPQQ
PLPQQ
PIIILQQ
PIIILQQ
SIIILQQ
LFPQQ
LFPQQ
LFPQQ
PSFSQQQ
PSFSQQQ
SPFSQQQQ
SPFSQQQQ
SPFSQQQQ
PSFSQQQ
PSFSQQQ
PSFSQQQ
PPFSQQQ
PPFSQQQ
PVLPQQQ
PVLPQQQ
PVLPQQQ
PPFWQQQ
PPFWQQQ
PPFWQQQ
PILSQQ
PILSQQ
PVIILQQ
PVIILQQ
PVIILQQ
PILPQQ
PPFSQQQ
PPFSQQQ
PPFSQQQQ
PPFSQQQQ
PPFSQQQQQQQQQQQ
PPFSQQQQ
PPFSQQQQ
PPFSQQQQ
PILPQQ
PILPQQ
PVLPQQ
PVLPQQ
PFTQQQ
PVLPQQ
PVLPQQ
LVLPQQ
PPFSQQQQ
PVLPPQQ
SPFSQQ
SPFSQQQQ
PPFSQQ
PPFSQQQQQQQQ
PPFSQQQQQQQQQQ
PVLPPQQ
LVLPQQ
SPFPQQQQ
LVLPPQQQQQQ
LVLPPQQQQQQ
PPISQQQQQQQQQQQ
PPFSQQQQ
PPFSQQQQ
SPFPQQQQQ
PVLPPQQ
LVQQQ
LVQQQ
LVQQQ
PPISQQQQ
PSSQQ
PSSQQ
LVQQQ
SPFPQQQQQ
PPFSQQQQ
PPFPQQ
PPFPQQ
LVQQQ
TPFSQQQQ
FPQQQ
FPQQQ


## 3.1. Basic Characteristics of the LMW-GS Alleles Identified in This Study

Eight novel LMW-GS alleles with no intron were obtained in this study (see Figure S1 in Supplementary Material available online athttp://dx.doi.org/10.1155/2014/371045/). Their sequences were submitted to GenBank with the accession numbers KF020658–KF020665 and designated asGlu-A3-1a,Glu-A3-1b,Glu-B3-1a,Glu-B3-1b,Glu-B3-1c,Glu-D3-1a,Glu-D3-1b, andGlu-D3-1c, following the nomenclature rules at http://wheat.pw.usda.gov/ggpages/wgc/98//.Glu-A3-1a andGlu-A3-1bhad the same size of 1089 bp and could be translated into 298 amino acids. The nucleotide and deduced amino acid sequences of the other six genes were varied with 1361–1603 bp and 306–351 residues (Table 1). The upstream nucleotide sequences of the cloned genes were searched for in the PlantCare database to analyze the characteristics of the promoter sequences. The eight genes all contained CAAT-boxes and TATA-boxes.Glu-B3-1a,Glu-B3-1b,Glu-B3-1c,Glu-D3-1a,Glu-D3-1b, andGlu-D3-1c contained a Prolamin-box in the upstream sequence (Table 1). All eight genes from landraces in our study belonged to the LMW-m type since their first amino acid at the N-terminal was methionine.Table 1
Comparison of nucleotide sequences of the eight cloned genes.
LMW-GS genes
Upstream
CDS
Total length
Deduced amino acids
Putative transcription binding sites in the upstream region
Glu-A3-1a
195 bp
894 bp
1089 bp
298
CAAT-box, TATA-box, ARE, and MRE
Glu-A3-1b
195 bp
894 bp
1089 bp
298
CAAT-box, TATA-box, ARE, and MRE
Glu-B3-1a
317 bp
1053 bp
1370 bp
351
CAAT-box, TATA-box, and Prolamin-box
Glu-B3-1b
317 bp
1044 bp
1361 bp
348
CAAT-box, TATA-box, and Prolamin-box
Glu-B3-1c
317 bp
1050 bp
1367 bp
350
CAAT-box, TATA-box, and Prolamin-box
Glu-D3-1a
593 bp
921 bp
1591 bp
307
CAAT-box, TATA-box, and Prolamin-box
Glu-D3-1b
605 bp
921 bp
1603 bp
307
CAAT-box, TATA-box, and Prolamin-box
Glu-D3-1c
605 bp
912 bp
1594 bp
306
CAAT-box, TATA-box, and Prolamin-box


## 3.2. Nucleotide Comparison Analysis of the LMW-GS Alleles within theGlu-3 Loci

To more accurately compare the differences between LMW-GS genes at theGlu-3 loci, we used BLAST tools to find the sequences most similar to the eight genes obtained in this study. NineGlu-3 genes deposited in GenBank were selected for multiple sequence alignment and included FJ549937, FJ549938, and FJ549946 located at theGlu-A3 locus; EU369729, EU369730, and DQ630441 at theGlu-B3 locus; and DQ357054, DQ357055, and EU189094 at theGlu-D3 locus. Based on the results of these alignments (Table 2), of the eight genes isolated in this study,Glu-A3-1a, andGlu-A3-1b were found to be new alleles at theGlu-A3 locus;Glu-B3-1a,Glu-B3-1b, andGlu-B3-1c were new alleles at theGlu-B3 locus; andGlu-D3-1a,Glu-D3-1b, andGlu-D3-1c were new alleles at theGlu-D3 locus.Table 2
Sequence identities of the eight new LMW-GS alleles to the previously reportedGlu-3 genes.
LMW-GS alleles
Glu-A3 gene accession number
Glu-B3 gene accession number
Glu-D3 gene accession number
FJ549946
FJ549937
FJ549938
DQ630441
EU369729
EU369730
DQ357054
DQ357055
EU189094
Glu-A3-1a
93
92
92
84
84
84
87
87
87
Glu-A3-1b
93
92
91
83
83
83
86
86
87
Glu-B3-1a
85
85
85
95
84
84
83
83
83
Glu-B3-1b
N
85
86
99
85
85
84
84
85
Glu-B3-1c
N
85
86
99
85
85
83
83
84
Glu-D3-1a
87
88
88
88
85
85
95
95
94
Glu-D3-1b
86
87
87
83
82
82
99
99
99
Glu-D3-1c
87
87
87
84
82
82
99
99
99
Note: N means that no highly similar sequences were found using BLAST tools.


## 3.3. Deduced Proteins Comparison Analysis of the LMW-GS Alleles within theGlu-3 Loci

The deduced amino acid sequences of the eight genes comprised four structural regions, including a signal peptide of 20 amino acids, a conserved N-terminal region of 13 amino acids, a diverse repetitive domain, and a C-terminal domain, suggesting that the eight genes conformed to the typical molecular characteristics of LMW-GS (Supplementary Material, Figure S2). We compared the alleles within theGlu-3 loci and found that isoleucine was at the sixth position of the signal peptide in the allele located atGlu-B3 locus, while valine was at theGlu-A3 andGlu-D3 loci (Table 3), which might result from the substitution of ATC for GTC (Supplementary Material, Figure S1). The eleventh position of the N-terminal at theGlu-A3 andGlu-D3 loci was arginine, while lysine filled the same position at theGlu-B3 locus (Table 3), which may be the result of an AGA→AAA transversion (Supplementary Material, Figure S1).Table 3
Comparison of the deduced amino acid sequences for genes isolated in this study and data from GenBank.
Sig.
N-ter.
Rep.
C-ter.
Glu-A3
FJ549937
V
C
R
C
C
C
CC
C
C
FJ549938
V
C
R
C
C
C
CC
C
C
FJ549946
V
C
R
C
C
C
CC
C
C
Glu-A3-1a
V
C
R
C
C
C
CC
C
C
Glu-A3-1b
V
C
R
C
C
C
CC
C
C
Glu-B3
EU369729
I
K
C
C
C
C
CC
C
C
EU369730
I
K
C
C
C
C
CC
C
C
DQ630441
I
K
C
C
C
C
CC
C
C
Glu-B3-1a
I
K
C
C
C
C
CC
C
C
C
Glu-B3-1b
I
K
C
C
C
C
CC
C
C
Glu-B3-1c
I
K
C
C
C
C
CC
C
C
Glu-D3
DQ357054
V
C
R
C
C
C
CC
C
C
DQ357055
V
C
R
C
C
C
CC
C
C
EU189094
V
C
R
C
C
C
CC
C
C
Glu-D3-1a
V
C
R
C
C
C
CC
C
C
Glu-D3-1b
V
C
R
C
C
C
CC
C
C
Glu-D3-1c
V
C
R
C
C
C
CC
C
C
The accession numbers are as follows: FJ549937, FJ549938, and FJ549946 are genes at theGlu-A3 locus; EU369729, EU369730, and DQ630441 are genes at the Glu-B3 locus; and DQ357054, DQ357055, and EU189094 are genes at Glu-D3 locus. I, V, R, K, and C represent isoleucine, valine, arginine, lysine, and cysteine, respectively.Eight cysteine residues were found inGlu-A3-1a,Glu-A3-1b,Glu-B3-1b,Glu-B3-1c,Glu-D3-1a,Glu-D3-1b, andGlu-D3-1c, whileGlu-B3-1a had nine. By comparing theGlu-3 loci alleles, the locations of the first and seventh cysteine were found to be varied. As shown in Figure S2 (Supplementary Material) and Table 3, the first cysteine of theGlu-A3 andGlu-D3 alleles was located at the fifth position of the N-terminal domain, whereas the first cysteine of theGlu-B3 alleles was found at the 46th position of the deduced amino acid sequences in the repetitive domain. The seventh cysteine at theGlu-D3 alleles was located 16 amino acids higher up than that at theGlu-A3 andGlu-B3 alleles. In particular, the extra cysteine of alleleGlu-B3-1a was found at the 321st position of the amino acid sequence.Usually the first and seventh cysteine residues participate in forming the intermolecular disulfide bond, while the remaining residues participate in the formation of intramolecular disulfide bonds [21]. Disulfide bonds have an important influence on determining the properties and structure of wheat gluten proteins [22]. The first cysteine at theGlu-B3 allele was in the repetitive domain, however, the first cysteine atGlu-A3 andGlu-D3 alleles were in the N-terminal. The positions of the seventh cysteine residue at theGlu-D3 alleles were also different from theGlu-A3 andGlu-B3 alleles. The LMW-GS gene, which had nine cysteine residues, resulted in desirable processing quality [20]; therefore, the new allele ofGlu-B3-1a may be the desirable LMW-GS gene for improving bread-making quality because the extra cysteine residue could form more disulfide bonds than other alleles during the development of glutenin macropolymer.Insertions/deletions often appeared within the repetitive domain. The length variation of the LMW-GS genes was mainly due to the numbers of repeat motifs, which ranged from 12 to 25 in the repetitive domain. The repeat motif is shown in Table4. Two long insertions were present in theGlu-B3 sequences, that is, 18 amino acid insertions at positions 44–61 and eight amino acid insertions at positions 81–88. The repetitive region was composed of a representative repeat motif P1-2FP/SQ2–6, of which the number would have an impact on changes in the length of the protein [23]. The length of the diverse repetitive domain ofGlu-B3 alleles was longer than those ofGlu-A3 andGlu-D3 alleles since more insertions occurred in this domain. Masci et al. [24] indicated that a 42 K LMW-GS would have a good processing quality due to its large repeat domain. The repeat domain through intermolecular interactions between large numbers of glutamine side chains, which are both good hydrogen bond donors and acceptors, may prove useful in increasing the viscosity and elasticity of wheat dough [24]. More repeat motifs in the repetitive domain would lead to a better flour quality [25]. Based on Figure S2 (Supplementary Material), the sequences at theGlu-B3 locus had 50 more deduced amino acids than those atGlu-A3 andGlu-D3 loci. Therefore, genes at theGlu-B3 locus are more desirable than those at theGlu-A3 andGlu-D3 loci.Table 4
Repeat motif of repetitive domains in the isolated LMW-GS deduced amino acid sequences.
Glu-A3-1a
Glu-A3-1b
Glu-B3-1a
Glu-B3-1b
Glu-B3-1c
Glu-D3-1a
Glu-D3-1b
Glu-D3-1c
QQQ
QQQ
QQQ
QQQ
QQQ
QQQ
QQQ
QQQ
PLPPQQ
PLPPQQ
PLPPQQQ
PLPPQQQ
PLPPQQQ
PLPPQQ
PLPPQQ
PLPPQQ
SFSQQ
SFSQQ
PPCSQQQQ
PPCSQQQQ
PPCSQQQQ
TFPQQ
TFPQQ
TFPQQ
PPFSQQQQQ
PPFSQQQQQ
PFPQQQQ
PFPQQQQ
PFPQQQQ
PLFSQQQQQQ
PLFSQQQQQQ
PLFSQQQQQQ
PLPQQ
PLPQQ
PIIILQQ
PIIILQQ
SIIILQQ
LFPQQ
LFPQQ
LFPQQ
PSFSQQQ
PSFSQQQ
SPFSQQQQ
SPFSQQQQ
SPFSQQQQ
PSFSQQQ
PSFSQQQ
PSFSQQQ
PPFSQQQ
PPFSQQQ
PVLPQQQ
PVLPQQQ
PVLPQQQ
PPFWQQQ
PPFWQQQ
PPFWQQQ
PILSQQ
PILSQQ
PVIILQQ
PVIILQQ
PVIILQQ
PILPQQ
PPFSQQQ
PPFSQQQ
PPFSQQQQ
PPFSQQQQ
PPFSQQQQQQQQQQQ
PPFSQQQQ
PPFSQQQQ
PPFSQQQQ
PILPQQ
PILPQQ
PVLPQQ
PVLPQQ
PFTQQQ
PVLPQQ
PVLPQQ
LVLPQQ
PPFSQQQQ
PVLPPQQ
SPFSQQ
SPFSQQQQ
PPFSQQ
PPFSQQQQQQQQ
PPFSQQQQQQQQQQ
PVLPPQQ
LVLPQQ
SPFPQQQQ
LVLPPQQQQQQ
LVLPPQQQQQQ
PPISQQQQQQQQQQQ
PPFSQQQQ
PPFSQQQQ
SPFPQQQQQ
PVLPPQQ
LVQQQ
LVQQQ
LVQQQ
PPISQQQQ
PSSQQ
PSSQQ
LVQQQ
SPFPQQQQQ
PPFSQQQQ
PPFPQQ
PPFPQQ
LVQQQ
TPFSQQQQ
FPQQQ
FPQQQ


## 4. Conclusion

This study showed that, of the eight newGlu-3 alleles added to the LMW-GS gene family,Glu-B3-1a had nine cysteine residues and the others had eight. The first cysteine ofGlu-A3 andGlu-D3 alleles was found in the N-terminal domain, while it was located at the repetitive domain ofGlu-B3alleles. The LMW-GS allele ofGlu-B3-1a can be used as candidate gene for improving bread-making quality because it can form more disulfide bonds.



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*Source: 371045-2014-04-10.xml*