- Email: [email protected]
Molecular characterization and functional properties of two novel x-type HMW-GS from wheat line CNU608 derived from Chinese Spring × Ae. caudata cross
Accepted Manuscript Molecular characterization and functional properties of two novel x-type HMW-GS from wheat line CNU608 derived from Chinese Spring × Ae. caudata cross Chang Wang, Xixi Shen, Ke Wang, Yanlin Liu, Jianwen Zhou, Dr. Yingkao Hu, Friedrich J. Zeller, Sai L.K. Hsam, Dr. Yueming Yan, Prof. PII:
S0733-5210(15)30078-3
DOI:
10.1016/j.jcs.2015.11.002
Reference:
YJCRS 2060
To appear in:
Journal of Cereal Science
Received Date: 6 January 2015 Revised Date:
13 September 2015
Accepted Date: 7 November 2015
Please cite this article as: Wang, C., Shen, X., Wang, K., Liu, Y., Zhou, J., Yingkao Hu, Zeller, F.J., Hsam, S.L.K., Yueming Yan, Molecular characterization and functional properties of two novel x-type HMW-GS from wheat line CNU608 derived from Chinese Spring × Ae. caudata cross, Journal of Cereal Science (2015), doi: 10.1016/j.jcs.2015.11.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Molecular characterization and functional properties of
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two novel x-type HMW-GS from wheat line CNU608
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derived from Chinese Spring × Ae. caudata cross
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Chang Wanga, Xixi Shena, Ke Wanga, Yanlin Liua, Jianwen Zhoua,
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Yingkao Hua*, Friedrich J. Zellerb, Sai L. K. Hsamb, Yueming Yana,c*
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a
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b
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College of Life Science, Capital Normal University, 100048 Beijing, China;
D-85354 Freising, Germany.
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Division of Plant Breeding and Applied Genetics, Technical University of Munich,
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c
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*
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Yueming Yan ([email protected]), Laboratory of Molecular Genetics and
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Proteomics, College of Life Science, Capital Normal University, 100048 Beijing,
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China. Phone and Fax: +86-010-68902777.
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Abbreviations: AS-PCR, allelic-specific polymerase chain reaction. HMW-GS,
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high molecular weight glutenin subunits; LC-MS/MS, liquid chromatography-tandem
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mass
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MALDI-TOF/TOF-MS, matrix-assisted laser desorption ionisation/time-of-flight/
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time-of-flight/mass spectrometry; PBs, protein bodies; SDS-PAGE, sodium dodecyl
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sulphate polyacrylamide gel electrophoresis; TEM, transmission electron microscopy.
Hubei Collaborative Innovation Center for Grain Industry; 434025 Jingzhou, China
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Corresponding authors: Dr. Yingkao Hu ([email protected]) and Prof. Dr.
spectrometry;
LMW-GS,
low
molecular
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weight
glutenin
subunits;
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Abstract Two novel HMW-GS, designed as 1Dx2s and 1Dx2f in the wheat line
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CNU608 derived from hybrids between Chinese Spring (CS) and Ae. caudata were
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identified. Quality analysis showed that the introgression of 1Dx2s and 1Dx2f subunits
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led to significant improvement of dough strength and breadmaking quality. The
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complete encoding sequences of 1Dx2s and 1Dx2f subunits were isolated and cloned.
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1Dx2s and 1Dx2f genes encode 831 and 808 amino acid residues, respectively. Their
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presence and authenticity were confirmed through E. coli expression, Western
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blotting and tandem mass spectrometry identification. The subunit 1Dx2s had an
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octapeptide deletion (ELQELQER) in the N-terminal domain compared to 1Dx2
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while the subunit 1Dx2f was encoded by a chimeric gene originated from 1Dx2 and
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1Cy genes. Particularly, 1Dx2f had an extra cysteine residue from 1Cy subunit at
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position 730 as well as longer repetitive domain and higher glutamine content,
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indicating its potential values for wheat gluten quality improvement. Both 1Dx2s and
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1Dx2f subunits were simultaneously expressed in the derived line, suggesting that one
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x-type gene duplication event at the Glu-D1 locus occurred. A putative origin of both
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1Dx2s and 1Dx2f genes indicated that they could be originated from one octapeptide
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deletion and two unequal cross-over events.
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Keywords: Ae. caudata, gluten quality, HMW-GS, wheat.
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1. Introduction Wheat (Triticum aestivum L.) is one of the three main grain crops in the world
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and serves as human staple food and important protein source. Wheat flour with
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unique physical properties can be used to produce various food products such as
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breads, cakes, and noodles. These unique properties are mainly determined by seed
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storage proteins that consist of polymeric glutenins and monomeric gliadins. Glutenin
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proteins, which give dough strength and elasticity, include high and low molecular
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weight glutenin subunits (HMW-GS and LMW-GS) while gliadins impart dough
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extensibility (Shewry et al., 1992). Although HMW-GS only account for about 10%
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of the total grain proteins, they play a key role in breadmaking quality thanks to their
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ability to form disulphide bonded polymers. (Shewry et al., 1992).
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It is well known that HMW-GS are encoded by three Glu-1 loci located on the
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long arms of chromosomes 1A, 1B and 1D. Each locus contains two closely linked
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genes encoding two types of HMW-GS: the larger x-type subunits with 80–88 kDa
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and the smaller y-type subunits with 67–73 kDa. Glu-1 loci exhibited different allelic
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variations in bread wheat and related species, which are closely associated with the
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end-use quality (Shewry et al., 1992). Particularly, some HMW-GS such as 1Dx5 +
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1Dy10, 1Ax1 and 1By8 have positive effects on gluten quality while 1Dx2 +1Dy12,
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1Bx20 have negative effects on dough strength (Altpeter et al., 1996; Redaelli et al.,
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1997; Shewry et al., 2003; Yan et al., 2009).
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The molecular structures of HMW-GS have three distinct domains, a central
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1992). Differences in subunit size are the result of variation in the central repetitive
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domain, particularly in the number of tripeptides and hexapeptides (Anderson et al.,
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1989; Shewry et al., 1992). So far, considerable work has been performed on the
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structures and functions of HMW-GS and some important features have been found to
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be closely related to superior gluten quality properties, including extra cysteine
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residues (Pirozi et al., 2008), longer repetitive domains, glutamine residues (Tatham et
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al., 1985), and higher expression amount and accumulation rates during grain
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development (Gao et al., 2012; Gupta et al., 1996; Liu et al., 2012).
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Extensive investigations have shown that the variation at Glu-1 loci in bread
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wheat is limited (Gianibelli et al., 2001; Payne and Lawrence 1983). However,
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Aegilops and other related species possess more extensive HMW-GS variation and
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may provide potential candidate genes for wheat quality improvement (Liu et al.,
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2003; Wang et al., 2013; Zhang et al., 2008). The Aegilops genus includes 22 species
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with S, M, C, U, N and T genomes, and has considerable HMW-GS variations (Liu et
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al., 2003), but their functional properties and application potential are still not known.
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This study reveals the potential value of HMW-GS from a wheat-Ae. caudata
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derived line, which resulted in significant improvement of dough strength and
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breadmaking quality. Two novel HMW-GS in the derived line were identified by
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proteomic approach and their molecular characterization were investigated. Both
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subunits are expected to be used as superior gene resources for wheat quality
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improvement.
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2. Materials and Methods
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2.1. Plant materials The materials used in this work included Chinese Spring (CS) and the wheat line
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CNU608. This line was developed by crossing CS with a wheat-Aegilops caudata
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1C(1A) substitution line. The original 1C(1A) substitution line was kindly provided
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by Dr. K. Tsunewaki, Kyoto University, Japan and was derived from a Triticum
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aestivum–Ae. caudata 1C(1D) substitution line P168 in which the substituted
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chromosome 1C of Ae. caudata was cytologically identified (Muramatsu 1959). The
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1C Ae. caudata chromosome was later transferred to CS (Tahir and Tsunewaki 1971).
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Two backcrosses followed by an inbred generation were further made between CS
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and the 1C(1A) substitution line in Freising, Germany. Selection was made for 42
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chromosome plants which were resistant to powdery mildew disease. Tests for
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powdery mildew were according to the method described by Hsam and Zeller (1997).
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Selection for powdery mildew resistance was facilitated by the fact that the CS-Ae.
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caudata 1C (1A) substitution line was resistant to powdery mildew whereas CS was
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susceptible. The presently used CNU608 wheat-Ae. caudata derived line was
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eventually obtained after several inbred generations, grown in Freising, Germany and
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in Beijing, China.
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2.2. SDS-PAGE and MALDI-TOF/TOF-MS
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HMW-GS were extracted from a half kernel (about 20 mg) and analyzed by
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sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) based on
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Yan et al. (2009). For matrix-assisted laser desorption ionisation/time-of-flight/time-of-flight/mass
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spectrometry (MALDI-TOF/TOF-MS) analysis, protein gels were visualized by a
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scanner with optics resolution setting at 300 dpi. Protein bands were excised manually
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and digested by trypsin. Tryptic peptides were analyzed with a MALDI-TOF mass
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spectrometer (SM, Shimadzu Biotech, Kyoto, Japan). All MS and MS/MS spectra
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were searched in the NCBI non-redundant green plant database MASCOT program
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using GPS ExplorerTM software version 2.0 (Applied Biosystems).
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2.3. Gluten property and breadmaking quality testing
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Both CS and CNU608 were planted in 2013 at the Beijing field trial station of
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the Chinese Academy of Agricultural Sciences. Each line included three replicates and
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each plot was 20 m2. Dough rheological properties during mixing were determined by
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Farinograph (Brabender, Duisburg, Germany) following the AACC (2000) and the
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quality parameters included dough development time, stability, degree of softening
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and Farinograph quality number (FQN). The 10-gram Mixograph (National
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Manufacturing) was used to assess the dough functional properties, and carried out in
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three duplicates based on AACC (2000), including gluten content, dry and wet gluten
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content and gluten index.
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Bread baking experiments were carried out to evaluate the bread-making
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qualities of the lines. The baking procedure was the standard rapid-mix-test with 1 kg
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Image analysis of bread was carried out with a C-Cell image analysing software and
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equipment (Calibre Control International Ltd.) referred to Sun et al. (2010). Slice area,
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height (max), breadth, wrapper length, volume of holes, slice brightness, cell contrast
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and average cell elongation were tested and used to estimate the bread properties.
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2.4. Light microscopy and TEM observation
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The developing grains in CS and CNU608 were collected at 7, 10, 13, 15, 19 and
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23 days post anthesis (DPA). The seed fixation, rinse, dehydration and thin sections
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preparation were as reported by Loussert et al. (2008) with some modifications. The
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sections were stained with Coomassie brilliant blue, rinsed extensively with water
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before examination on the Leica DRD microscope in bright field. For transmission
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electron microscopy (TEM) analysis, the sections were fixed in primary antibody
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(HMW-GS Polyclonal antibody) and second antibody (15 nm gold conjugated goat
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anti-rabbit IgG) (Loussert et al. 2008).
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2.5. DNA extraction and PCR amplification
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Genomic DNA was extracted from leaves following the procedure of Wang et al.
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(2013). About 20 mg of crushed seed were placed in a 1.5 ml tube followed by
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maceration in 1 ml of extraction buffer (200 mM Tris-Hcl (pH 7.5), 288 mM NaCl, 25
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mM EDTA and 0.5% SDS). The supernatant was retained and 700 µl phenol:
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chloroform: iso-amyl alcohol (25:24:1) was added and centrifuged for 10 min at
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13000 rpm. The last step was repeated and then isopropanol (700 ml) was added and
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the sample stored at -20°C for 30 min. After centrifugation, the DNA pellet was
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washed two times with 70% ethanol and dissolved in H2O. Two pairs of allelic-specific polymerase chain reaction (AS-PCR) primers were
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designed based on the published HMW-GS gene sequences and used to amplify the
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complete coding region
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(5'-ATGGCTAAGCGGTTAGTC CT-3') and Cx-1R (5'-GCTGCAGAGAGTTCTA
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TC-3'), Cx-2F (5'-ATGGCTAAGCGGTTAGTCCTC T-3') and Cx-2R (5'-CTATCA
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CTGGCTGGCCGACAAT-3'). The PCR amplification included an initial denaturation
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step at 94°C for 5 min followed by 34 cycles at 94°C for 45 s, 62/55°C for 1 min,
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72°C for 150 s, and a final extension step at 72°C for 10 min. The PCR products were
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separated by 1% agrose gel in Tris-acetic acid-EDTA buffer.
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2.6. Molecular cloning and sequencing
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of HMW-GS genes from CNU608, namely Cx-1F
The amplified products with expected sizes were purified from the gel and
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cloned with pGEM-T plasmid vector (Promega). Three recombined DNA clones were
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sequenced by TaKaRa Biotechnology (Dalian) CO., LTD, China, and each clone was
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sequenced three times to reduce the sequencing error.
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2.7. Heterologous expression, Western blotting and LC-MS/MS
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The cloned HMW-GS genes were amplified again by newly designed expression
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primers to remove the signal peptides. The forward primer was pETyF:
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5'-AAACATATGGCTGAAGGTGAGGCCTCTGA-3' with Nde I restriction site while
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the reverse primer was pETyR1: 5'-AGGCTCGAGCTATCACTGGCTGGC-3' and
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site. After amplification and purification, the PCR products of the cloned genes were
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ligated into the expression vector pET-28a (Novagen) and transformed into
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Escherichia coli BL21(DE3) plysS cells. The positive hybrid plasmid was induced by
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IPTG. The extraction and separation of expressed proteins were carried out by
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SDS-PAGE according to Yan et al. (2009). The Western blotting was performed based
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on Yan et al. (2009). The sensitive first antibody (anti-His-tag mouse monoclonal
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antibody) was used to detect the expressed proteins based on the His-tag sequence
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present at the downstream of the cloned gene.
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The peptide sequences of the expressed HMW glutenin subunit were determined
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by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The specific
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HMW-GS band on the SDS-PAGE gel was excised and digested with trypsin. A
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sample of the digested protein (0.5 ml) was subject to MS analysis in a Waters
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SYNAPT High Definition Mass Spectrometry™ (HDMS) mass spectrometer.
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2.8. Phylogenetic analysis
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The multiple alignment was performed with homologous nucleotide sequences
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by using the ClustalW program. The alignment file was converted to MEGA format
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and a phylogenetic tree was constructed with the Molecular Evolutionary Genetics
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Analysis software MEGA 6 by the complete coding regions of HMW-GS.
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3. Results
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3.1. Characterization of the derived line CNU608
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substitution line, the derived line CNU608 was developed. The line CNU608 shows
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significant changes of plant morphological characteristics as well as growth and
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development features, including increasing plant height, spike length and grain sizes
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and colour (Suppl. Fig. S1). It also differs from CS in that it is awned and is powdery
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mildew resistant (data not shown). The many morphological and physiological
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differences between CS and the line CNU608 are not justified, only by the presence of
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the 1C(1A) substitution; it is likely that in the line CNU608 additional portions of the
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genome of Ae. caudata have been introgressed in CS
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3.2. Quality performance of Chinese Spring and the derived line CNU608
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Various gluten and breadmaking parameters were tested to estimate the quality
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performance of CNU608. The results indicated that dough development time, stability
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time, Farinograph quality number, gluten content and gluten index in CNU608 were
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significantly higher than those in CS while the degree of softening of CNU608 was
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lower than that in CS (Table 1). Breadmaking quality showed that in CNU608, the
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loaf volume, bread sensory evaluation score and most of the C-Cell parameters related
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to bread structural features were significantly improved, including bread slice area,
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height, breadth, wrapper length, volume of holes, slice brightness and average cell
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elongation as shown in Table 1 and Fig. 1A.
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3.3. Identification of seed proteins in Chinese Spring and CNU608
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SDS-PAGE analysis showed that, compared to CS, CNU608 still had 1Bx7 and
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novel HMW-GS, which were slightly larger and smaller than 1Dx2 and designated as
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1Dx2s and 1Dx2f , respectively (Fig. 1B CNU608 is not known. The deduced
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molecular mass of 1Dx2s and 1Dx2f subunits were 88244.42 Da and 85285.12 Da,
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respectively. To further confirm the authenticity of the two novel HMW-GS in
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CNU608 and acquire their structural information, 1Dx2s and 1Dx2f subunits from
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SDS-PAGE gel were excised manually, digested with trypsin, and then identified by
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MALDI-TOF/TOF-MS. The results showed that 1Dx2s and 1Dx2f subunits were
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identified as x-type HMW-GS (Suppl. Table S1). Two peptides (QYEQQIVVPPK and
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AQQLAAQLPAMCR)
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GGSFYPGETTPPQQLQQ
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respectively well matched to 1Dx2s and 1Dx2f with protein score C.I. % of 100%.
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3.4. Protein body (PB) changes during grains development
and
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peptides
(ELKACQQVM
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RYYPSTSPQQVSYYPGQASPQRPGR)
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Wheat seed storage proteins are deposited into organelles of protein bodies (PBs)
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after synthesis and folding in the lumen of the endoplasmic reticulum (Loussert et al.,
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2008). Since HMW-GS have major effects on gluten strength and bread-making
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quality, the significant improvement of gluten quality in CNU608 could mainly be
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attributed to the introgression of the two novel subunits above described. In order to
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compare the PB differences between CS and CNU608 during grain development,
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different microscopy techniques were used to observe the PB changes at different
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grain developmental stages. The results from light microscopy observation showed
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of PBs (Fig. 2A). The PBs appeared at 10 DPA in CS and CNU608, grew in size by
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fusion among themselves and finally reached the maximum size at 19 DPA in CS and
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CNU608, respectively. At 19 DPA, microscopic observations showed that PBs did not
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remain as separate particles but coalesced to form large aggregates in CS, whereas in
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CNU608 the PBs continued to enlarge from 15 DPA to 19 DPA. At 23 DPA, no PBs
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could be observed by light microscopy in both lines. Some clear differences of PB
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development between CS and CNU608 were found. More large PB numbers with the
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diameter more than 5 µm at 13, 15 and 19 DPA in CNU608 were observed (Fig. 2A).
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TEM observation at 15 and 19 DPA through the immunogold labelling using
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anti-HMW-GS further verified that CNU608 had more and larger PBs than CS (Fig.
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2B).
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3.5. Molecular characterization of 1Dx2s and 1Dx2f subunits
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The complete open reading frames (ORFs) encoding 1Dx2s and 1Dx2f subunits
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were isolated and cloned from CNU608 by AS-PCR. Their sequences showed that
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1Dx2s gene consisted of 2499 bp encoding 831 amino acid residues while the 1Dx2f
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comprised 2427 bp encoding 808 amino acid residues. Their nucleotide and deduced
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amino acid sequences were deposited in the GenBank with accession number of
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KF466259 and KF466260, respectively.
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Comparison of the deduced amino acid sequences showed that both 1Dx2s and
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1Dx2f had similar structural characteristics with 1Dx2 subunit (Fig. 3). 1Dx2s
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of 21 amino acid residues, a non-repetitive N-terminal domain of 81 amino acid
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residues, followed by a repetitive domain of 687 amino acid residues and a
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non-repetitive C-terminal domain of 42 amino acid residues. But one octapeptide
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deletion (ELQELQER) occurred in the N-terminal domain of 1Dx2s compared to
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1Dx2 subunit (Fig. 3A). The 1Dx2f subunit, it showed distinct structural feature: its
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first 779 amino acid residues were highly homologous with the corresponding
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sequence of 1Dx2, while the final 120 amino acid residues were highly similar with
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those of 1Cy subunit (GenBank accession number GQ403046, Fig. 3B). 1Dx2f
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subunit had a long repetitive domain, high glutamine content and an extra cysteine
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residue from 1Cy subunit at position 730 near the C-terminal domain. Thus, five
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cysteine residues were present in the chimeric 1Dx2f subunit: three in the N-terminal
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domain, one in the repetitive domain near the C-terminal domain and one in the
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C-terminal domain.
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3.6. Heterologous expression and identification of 1Dx2s and 1Dx2f genes
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To verify that the cloned DNA fragment was the accurate representative of the
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1Dx2s and 1Dx2f genes, the ORF without signal peptide was ligated with the
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expression vector pET-28a and transformed into E. coli BL21 (DE3) pLysS cells. The
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positive hybrid plasmid was induced by IPTG. The expressed proteins were purified
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by 50% (v/v) propanol including 1% DTT (w/v) and separated by SDS-PAGE (Fig.
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4A). The results showed that the expressed proteins had similar mobilities to those of
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expressed proteins were further confirmed by Western blotting. The bacterially
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expressed proteins of 1Dx2s and 1Dx2f genes and the endogenous subunits from
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CNU608 displayed a strong reaction to the polyclonal antibody specific for HMW-GS
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(Fig. 4B).
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To further confirm the authenticities of the cloned sequences, the heterologous
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expressed proteins collected from SDS-PAGE gel were digested by trypsin and
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identified by LC-MS/MS. The results showed that three and five peptides could be
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well matched with 1Dx2s and 1Dx2f, respectively as listed in Suppl. Table S2. The
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coverage rates were 6.1 % for 1Dx2s and 9.1 % for 1Dx2f, confirming the
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correspondence between the expressed proteins and their encoding genes cloned.
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3.7. Phylogenetic analysis
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To investigate the phylogenetic relationships among HMW-GS gene family from
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different genomes, 27 HMW-GS genes were used to construct a phylogenetic tree,
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which included 1Dx2s and 1Dx2f genes obtained in this study and other 25 genes from
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GenBank (http://www.ncbi.nlm.nih.gov/). As showed in Suppl. Fig. S2, the
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phylogenetic tree was apparently clustered into two separate groups, well
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corresponding to x-type and y-type subunit genes, respectively. Three subgroups for
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both x-type and y-type subunit genes were clearly separated by the A, B and D
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genomes. In particular, 1Dx2s, 1Dx2f and 1Dx2 genes were clustered to a small
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subgroup, implying their highly similar structural features and close phylogenetic
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relationship.
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4. Discussion Aegilops caudata (2n=2x=14, CC) is a diploid species whose genome is involved
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in the formation of many polyploid species (Friebe et al., 1992), so it is expected to
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serve as valuable genetic sources for wheat quality improvement. To date, HMW
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glutenin subunits in Aegilops caudata have been identified and characterized, which
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implied that they have potential values in improving the processing properties of
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bread wheat (Liu et al., 2003). In this study, we developed the derived line CNU608
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from crossing between Chinese Spring (CS) and CS-Ae. caudata substitution line
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1C(1A) that possessed two novel HMW-GS genes 1Dx2s and 1Dx2f, which showed
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high similarity and phylogenetic relationships with 1Dx2 (Suppl. Fig. S2). The
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deduced amino acid sequences revealed that both novel subunits were the variants of
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the 1Dx2 subunit. The 1Dx2s subunit had an octapeptide (ELQELQER) deletion in
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the N-terminal while the 1Dx2f was a chimeric subunit originated from combination
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of 1Dx2 and the C-terminal (120 amino acid residues) of 1Cy subunit respectively
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(Fig. 3). Since two x-type HMW-GS are present in the derived line CNU608, two
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gene copies should be located at the Glu-D1 locus. This suggests that a gene
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duplication event occurred during the development process of the derived line.
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It is known that the allelic variations at Glu-1, Glu-3 loci are mainly resulted from
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point
mutations,
unequal
crossing-over,
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illegitimate recombination and homoeologous recombination (Guo et al., 2013; Zhang
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slip-mismatching,
intrachromosomal
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and 1Dx2f genes. As shown in Fig. 5, firstly, an octapeptide (ELQELQER) deletion
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occurred in 1Dx2 subunit of Chinese Spring, which resulted in a new subunit 1Dx2s.
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Subsequently, a duplication event of 1Dx2s gene by unequal crossing-over occurred at
322
Glu-D1 locus, leading to two x-type subunits at Glu-D1. Finally, the chimeric gene
323
1Dx2f was originated from the unequal crossing-over between 1D and 1C
324
chromosomes. Cross overs between the 1D and 1C chromosomes could have already
325
occurred in the original wheat-Ae. caudata addition line (Triticum aestivum strain
326
P168) initially obtained by Dr. H. Kihara, Kyoto University, Japan, in which the
327
1C(1D) substitution line was first identified by Muramatsu (1959). Alternatively, this
328
event could have occurred during the backcrossing process with CS.
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In the past several decades, the molecular mechanisms of HMW-GS allelic
330
variations affecting wheat quality properties have been widely studied. Considerable
331
work showed that the molecular structure of HMW gluten proteins and their amino
332
acid compositions have important effects on dough properties (Hanssini et al., 2005).
333
In general, x-type HMW-GS have longer repetitive domains and higher expression
334
amount compared to y-type subunits. A long repetitive domain can form more β-turns
335
structure conferring elasticity to the protein molecule (Gianibelli et al., 2001; Tatham
336
et al., 1985). In addition, high glutamine content can stabilize the polymeric structure
337
of glutenin through forming more hydrogen bonds (Gilbert et al., 2000), so larger
338
subunits rich in glutamines had a greater positive effect on dough strength than
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340
is the proportion of the consensus hexapeptides and nonapeptides in the repetitive
341
domain. Higher proportion of the consensus hexapeptides and nonapeptides can
342
produce a more regular pattern of repetitive β-turns in the protein, and contribute to
343
better dough elasticity (Flavell et al., 1989). Also, the number and position of cysteine
344
residues have important roles in dough viscoelasticity, which form the inter- and
345
intra-molecular disulfide bonds and also link with LMW-GS and gliadins to form
346
gluten macropolymer (Pirozi et al., 2008). For example, an extra cysteine residue
347
present in 1Dx5 subunit is considered to be responsible for its superior quality
348
property (Anderson et al., 1989). In this study, both novel subunits 1Dx2s and 1Dx2f
349
identified in CNU608 have longer repetitive domains rich in glutamine residues.
350
Particularly, the 1Dx2f subunit has an extra cysteine residue from 1Cy subunit at
351
position 730 near to the C-terminal domain. The repetitive domain of 1Dx2f subunit
352
contains 25 consensus hexapeptide (PGQGQQ) and 8 consensus nonapeptide
353
(GYYPTSP/LQQ) motifs, and thus has high proportion of the consensus hexapeptide
354
and nonapeptide repetitive motifs. These constructural features could contribute to the
355
superior gluten strength and breadmaking quality of the derived line (Table 1 and
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Fig.1A).
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Our results demonstrated that two x-type subunits 1Dx2s and 1Dx2f were present
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at Glu-D1 locus and both subunits were simultaneously expressed in CNU608 (Fig.
359
1B). This results in an increase of the amount of x-type HMW-GS and improve the
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361
absent, CNU608 also has significantly improved gluten strength and breadmaking
362
quality compared to CS (Table 1). Similarly, HMW-GS overexpression such as
363
1Bx7OE resulted from a retroelement mediated gene duplication event at Glu-B1 locus
364
can result in a significantly higher proportion of HMW-GS, and then produce an
365
positive effect on flour quality (Li et al., 2014). Therefore, the duplication and
366
structural variations of two x-type subunit genes at Glu-D1 locus in the derived line
367
CNU608 can significantly increase the expression amount of x-type HMW-GS and
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improve breadmaking quality.
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5. Conclusions
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Two novel x-type HMW-GS designated as 1Dx2s and 1Dx2f in CNU608 derived
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from hybridation between Chinese Spring and CS-Ae. caudata substitution line 1A
372
(1C) were identified, and their complete encoding sequences were amplified and
373
cloned by AS-PCR. Molecular characterization demonstrated that both subunits
374
showed higher similarity and closer phylogenetic relationships with 1Dx2.
375
Particularly 1Dx2s had a octapeptide (ELQELQER) deletion and 1Dx2f belonged to a
376
chimeric gene originated from the recombination between 1Dx2 and 1Cy subunits and
377
had an extra cysteine residue from 1Cy subunit at position 730 near to the C-terminal
378
domain. Both novel subunit genes were probably originated from one deletion and
379
two unequal cross-over events. Their authenticity were confirmed through
380
MALDI-TOF/TOF-MS, prokaryotic expression, Western blotting and LC-MS/MS.
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382
improvement of gluten strength and breadmaking quality. Their specific molecular
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structural features and high expression proportion of x-type HMW-GS could
384
contribute to the formation of superior breadmaking quality. Particularly, the chimeric
385
1Dx2f gene showed potential values for wheat gluten quality improvement.
386
Acknowledgments
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This research was financially supported by grants from the National Natural
388
Science Foundation of China (31471485), Natural Science Foundation of Beijing City
389
and the Key Developmental Project of Science and Technology from Beijing
390
Municipal Commission of Education (KZ201410028031), Key Project of National
391
Plant Transgenic Genes of China (2014ZX08009-003), and International Science &
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Technology Cooperation Program of China (2013DFG30530).
393
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Wellner, N., D’Ovidio, R., Békés, F., Halford, N.G., 2003. Sequence and
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Tahir, Ch.M., Tsunewaki, K., 1971. Monosomic analysis of fertility-restoring genes in
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Triticum aestivum strain P168. Canadian Journal of Genetics and Cytology 13,
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Zeller, F.J., Hsam, S.L.K., Yan, Y.M., 2013. Molecular mechanisms of HMW
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glutenin subunits from 1Sl genome of Aegilops longissima positively affecting
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wheat breadmaking quality. PLoS ONE 8, e58947.
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Yan, Y.M., Jiang, Y., An, X.L., Pei, Y.H., Li, X.H., Zhang, Y.Z., Wang, A.L., He, Z.H.,
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Xia, X.C., Békés, F., Ma, W.J., 2009. Cloning, expression and functional analysis
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of HMW glutenin subunit 1By8 gene from Italy pasta wheat (Triticum turgidum
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L. ssp. durum). Journal of Cereal Science 50, 398–406.
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Appels, R., Yan, Y.M., 2008. Novel x-type HMW glutenin genes from Aegilops
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tauschii and their implications on the wheat origin and evolution mechanism of
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Glu-D1-1 proteins. Genetics 178, 23–33.
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Figure legends: Fig. 1 A. Bread loaves of CS and CNU608. B. Identification of HMW-GS in CS and CNU608 by SDS-PAGE; The two arrowed bands were selected for further
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MALDI-TOF/TOF- MS identification. Fig. 2 Developmental changes of protein body (PB) during grain development of CS and CNU608. A. Light microscopy observation of PBs in the endosperm cells
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during developing grains at 7, 10, 13,15, 19 and 23 DPA; B. Ultrathin sections of
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wheat endosperm demonstrating the immunolocalization of HMW-GS to PBs at 15 and 19 DPA.
Fig. 3 Comparison of the deduced amino acid sequences of 1Dx2s and 1Dx2f genes with 1Dx2 subunit. A. 1Dx2s and 1Dx2; B. 1Dx2f, 1Cy and 1Dx2 subunits. The
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signal peptide, N-terminal domain, repetitive domain and C-terminal domain are indicated. The positions of cysteine residues are marked by black frames. Deleted and identical residues are indicated by red frames.
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Fig. 4 Heterologous expression and identification of 1Dx2s and 1Dx2f genes in E. coli.
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A. SDS–PAGE analysis;1. 1Dx2f; 2. CNU608; 3. 1Dx2s; 4,pET28a; B. Western blot of the expressed protein of 1Dx2s and 1Dx2f genes in E. coli. 1, Blue Plus Ⅱ; 2, pET28a; 3, 1Dx2f; 4, 1Dx2s; 5, CNU608.
Fig. 5 The putative genetic mechanisms for the origination of 1Dx2s and the chimeric gene 1Dx2f. Green area: signal peptides; yellow area: N-terminal domains; red area: repetitive domains; blue area: C-terminal domains; dotted lines: deletions. The letter “S” indicats cysteine residues.
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Supplementary data Suppl. Table S1 The 1Dx2s and 1Dx2f subunits in CNU608 identified by matrix-assisted laser desorption ionisation/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS)
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Suppl. Table S2 Peptides identification of the 1Dx2s and 1Dx2f subunits in CNU608 by liquid chromatography-tandem mass spectrometry (LC-MS/MS)
Suppl. Fig. S1 Morphological characteristics of plants, spikes and seeds of CS and
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CS-Ae. caudata translocation line 1AL.1CS.
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Suppl. Fig. S2 Phylogenetic tree of 27 HMW-GS genes based on complete coding DNA. The GenBank accession numbers for the published HMW-GS genes were 1Dx5*t (DQ681076), 1Dx5 (X12928), 1Dx2 (X03346), 1Dx2.1t (AF480486),
1Dx1.5t
(AY594355),
1Dx3t
(HM347447),
1Dx4t
1Ax2*
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(DQ307383), 1Dx2.1 (AY517724), 1Dx2.2* (AJ893508), 1Ax1 (X61009), (M22208),
1Bx7
(X13927),
1Bx23
(AY553933),
1Bx20
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(AJ437000), 1Bx13 (EF540764), 1Bx14 (AY367771), 1Bx17 (JC2099), 1Ay (X03042), 1Ay/Tu-e (AY245578), 1By8 (AY245797), 1By9 (X61026),
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1By15 (DQ086215), 1Dy10 (X12929), 1Dy11 (EU528008) and 1Dy12 (X03041).
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Farinograph Development
Stability
quality number time(min)
(min)
softening
(%) (mm)
Gluten index
Loaf mass
Loaf volume
(%)
(g)
(cm3)
content
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Materials
Gluten
Dry basis
Degree of
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Table 1. Comparison of gluten and bread-making quality parameters between CS and CNU608
(g)
2.0±0.10**
3.0±0.10**
103±1.6**
40±0.70**
17.92±0.03*
4.83±0.03*
57.80±0.73**
148.91±0.31
738±1.67**
CS
1.5±0.07
0.8±0.07
215±1.78
18±1.11
15.42±0.01
3.91±0.02
28.35±1.64
147.89±0.01
548±1.67
Wrapper
Volume of
Slice
Cell
Average cell
length/px
holes
brightness
contrast
elongation
576±2.33**
1838±0.67*
14.2±0.83
140.9±0.10**
0.705±0.01
1.53±0.01*
419±5.33
1634±3.00
13.6±0.53
127.7±0.10
0.690±0.01
1.41±0.01
Total bread
Height Slice area/px
Breadth/px
score
(max)/px
49**
247916±478.33**
539±3.67*
CS
28
167704±1232.33
523±1.67
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Significant level: *p < 0.05, **p < 0.01.
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Materials
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Highlights 1. Two new HMW-GS 1Dx2s and 1Dx2f significantly improve breadmaking quality. 2. The chimeric gene 1Dx2f was originated from recombination between 1Dx2 and
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3. A putative origin of 1Dx2s and 1Dx2f genes was proposed
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1Cy genes.
S0733-5210(15)30078-3
DOI:
10.1016/j.jcs.2015.11.002
Reference:
YJCRS 2060
To appear in:
Journal of Cereal Science
Received Date: 6 January 2015 Revised Date:
13 September 2015
Accepted Date: 7 November 2015
Please cite this article as: Wang, C., Shen, X., Wang, K., Liu, Y., Zhou, J., Yingkao Hu, Zeller, F.J., Hsam, S.L.K., Yueming Yan, Molecular characterization and functional properties of two novel x-type HMW-GS from wheat line CNU608 derived from Chinese Spring × Ae. caudata cross, Journal of Cereal Science (2015), doi: 10.1016/j.jcs.2015.11.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Molecular characterization and functional properties of
2
two novel x-type HMW-GS from wheat line CNU608
3
derived from Chinese Spring × Ae. caudata cross
4
Chang Wanga, Xixi Shena, Ke Wanga, Yanlin Liua, Jianwen Zhoua,
5
Yingkao Hua*, Friedrich J. Zellerb, Sai L. K. Hsamb, Yueming Yana,c*
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a
8
b
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College of Life Science, Capital Normal University, 100048 Beijing, China;
D-85354 Freising, Germany.
9
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Division of Plant Breeding and Applied Genetics, Technical University of Munich,
10
c
11
*
12
Yueming Yan ([email protected]), Laboratory of Molecular Genetics and
13
Proteomics, College of Life Science, Capital Normal University, 100048 Beijing,
14
China. Phone and Fax: +86-010-68902777.
15
Abbreviations: AS-PCR, allelic-specific polymerase chain reaction. HMW-GS,
16
high molecular weight glutenin subunits; LC-MS/MS, liquid chromatography-tandem
17
mass
18
MALDI-TOF/TOF-MS, matrix-assisted laser desorption ionisation/time-of-flight/
19
time-of-flight/mass spectrometry; PBs, protein bodies; SDS-PAGE, sodium dodecyl
20
sulphate polyacrylamide gel electrophoresis; TEM, transmission electron microscopy.
Hubei Collaborative Innovation Center for Grain Industry; 434025 Jingzhou, China
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Corresponding authors: Dr. Yingkao Hu ([email protected]) and Prof. Dr.
spectrometry;
LMW-GS,
low
molecular
21 1
weight
glutenin
subunits;
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Abstract Two novel HMW-GS, designed as 1Dx2s and 1Dx2f in the wheat line
23
CNU608 derived from hybrids between Chinese Spring (CS) and Ae. caudata were
24
identified. Quality analysis showed that the introgression of 1Dx2s and 1Dx2f subunits
25
led to significant improvement of dough strength and breadmaking quality. The
26
complete encoding sequences of 1Dx2s and 1Dx2f subunits were isolated and cloned.
27
1Dx2s and 1Dx2f genes encode 831 and 808 amino acid residues, respectively. Their
28
presence and authenticity were confirmed through E. coli expression, Western
29
blotting and tandem mass spectrometry identification. The subunit 1Dx2s had an
30
octapeptide deletion (ELQELQER) in the N-terminal domain compared to 1Dx2
31
while the subunit 1Dx2f was encoded by a chimeric gene originated from 1Dx2 and
32
1Cy genes. Particularly, 1Dx2f had an extra cysteine residue from 1Cy subunit at
33
position 730 as well as longer repetitive domain and higher glutamine content,
34
indicating its potential values for wheat gluten quality improvement. Both 1Dx2s and
35
1Dx2f subunits were simultaneously expressed in the derived line, suggesting that one
36
x-type gene duplication event at the Glu-D1 locus occurred. A putative origin of both
37
1Dx2s and 1Dx2f genes indicated that they could be originated from one octapeptide
38
deletion and two unequal cross-over events.
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Keywords: Ae. caudata, gluten quality, HMW-GS, wheat.
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1. Introduction Wheat (Triticum aestivum L.) is one of the three main grain crops in the world
47
and serves as human staple food and important protein source. Wheat flour with
48
unique physical properties can be used to produce various food products such as
49
breads, cakes, and noodles. These unique properties are mainly determined by seed
50
storage proteins that consist of polymeric glutenins and monomeric gliadins. Glutenin
51
proteins, which give dough strength and elasticity, include high and low molecular
52
weight glutenin subunits (HMW-GS and LMW-GS) while gliadins impart dough
53
extensibility (Shewry et al., 1992). Although HMW-GS only account for about 10%
54
of the total grain proteins, they play a key role in breadmaking quality thanks to their
55
ability to form disulphide bonded polymers. (Shewry et al., 1992).
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It is well known that HMW-GS are encoded by three Glu-1 loci located on the
57
long arms of chromosomes 1A, 1B and 1D. Each locus contains two closely linked
58
genes encoding two types of HMW-GS: the larger x-type subunits with 80–88 kDa
59
and the smaller y-type subunits with 67–73 kDa. Glu-1 loci exhibited different allelic
60
variations in bread wheat and related species, which are closely associated with the
61
end-use quality (Shewry et al., 1992). Particularly, some HMW-GS such as 1Dx5 +
62
1Dy10, 1Ax1 and 1By8 have positive effects on gluten quality while 1Dx2 +1Dy12,
63
1Bx20 have negative effects on dough strength (Altpeter et al., 1996; Redaelli et al.,
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1997; Shewry et al., 2003; Yan et al., 2009).
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The molecular structures of HMW-GS have three distinct domains, a central
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1992). Differences in subunit size are the result of variation in the central repetitive
68
domain, particularly in the number of tripeptides and hexapeptides (Anderson et al.,
69
1989; Shewry et al., 1992). So far, considerable work has been performed on the
70
structures and functions of HMW-GS and some important features have been found to
71
be closely related to superior gluten quality properties, including extra cysteine
72
residues (Pirozi et al., 2008), longer repetitive domains, glutamine residues (Tatham et
73
al., 1985), and higher expression amount and accumulation rates during grain
74
development (Gao et al., 2012; Gupta et al., 1996; Liu et al., 2012).
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Extensive investigations have shown that the variation at Glu-1 loci in bread
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wheat is limited (Gianibelli et al., 2001; Payne and Lawrence 1983). However,
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Aegilops and other related species possess more extensive HMW-GS variation and
78
may provide potential candidate genes for wheat quality improvement (Liu et al.,
79
2003; Wang et al., 2013; Zhang et al., 2008). The Aegilops genus includes 22 species
80
with S, M, C, U, N and T genomes, and has considerable HMW-GS variations (Liu et
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al., 2003), but their functional properties and application potential are still not known.
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This study reveals the potential value of HMW-GS from a wheat-Ae. caudata
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derived line, which resulted in significant improvement of dough strength and
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breadmaking quality. Two novel HMW-GS in the derived line were identified by
85
proteomic approach and their molecular characterization were investigated. Both
86
subunits are expected to be used as superior gene resources for wheat quality
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ACCEPTED MANUSCRIPT 87
improvement.
88
2. Materials and Methods
89
2.1. Plant materials The materials used in this work included Chinese Spring (CS) and the wheat line
91
CNU608. This line was developed by crossing CS with a wheat-Aegilops caudata
92
1C(1A) substitution line. The original 1C(1A) substitution line was kindly provided
93
by Dr. K. Tsunewaki, Kyoto University, Japan and was derived from a Triticum
94
aestivum–Ae. caudata 1C(1D) substitution line P168 in which the substituted
95
chromosome 1C of Ae. caudata was cytologically identified (Muramatsu 1959). The
96
1C Ae. caudata chromosome was later transferred to CS (Tahir and Tsunewaki 1971).
97
Two backcrosses followed by an inbred generation were further made between CS
98
and the 1C(1A) substitution line in Freising, Germany. Selection was made for 42
99
chromosome plants which were resistant to powdery mildew disease. Tests for
100
powdery mildew were according to the method described by Hsam and Zeller (1997).
101
Selection for powdery mildew resistance was facilitated by the fact that the CS-Ae.
102
caudata 1C (1A) substitution line was resistant to powdery mildew whereas CS was
103
susceptible. The presently used CNU608 wheat-Ae. caudata derived line was
104
eventually obtained after several inbred generations, grown in Freising, Germany and
105
in Beijing, China.
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2.2. SDS-PAGE and MALDI-TOF/TOF-MS
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HMW-GS were extracted from a half kernel (about 20 mg) and analyzed by
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sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) based on
109
Yan et al. (2009). For matrix-assisted laser desorption ionisation/time-of-flight/time-of-flight/mass
111
spectrometry (MALDI-TOF/TOF-MS) analysis, protein gels were visualized by a
112
scanner with optics resolution setting at 300 dpi. Protein bands were excised manually
113
and digested by trypsin. Tryptic peptides were analyzed with a MALDI-TOF mass
114
spectrometer (SM, Shimadzu Biotech, Kyoto, Japan). All MS and MS/MS spectra
115
were searched in the NCBI non-redundant green plant database MASCOT program
116
using GPS ExplorerTM software version 2.0 (Applied Biosystems).
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2.3. Gluten property and breadmaking quality testing
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Both CS and CNU608 were planted in 2013 at the Beijing field trial station of
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the Chinese Academy of Agricultural Sciences. Each line included three replicates and
120
each plot was 20 m2. Dough rheological properties during mixing were determined by
121
Farinograph (Brabender, Duisburg, Germany) following the AACC (2000) and the
122
quality parameters included dough development time, stability, degree of softening
123
and Farinograph quality number (FQN). The 10-gram Mixograph (National
124
Manufacturing) was used to assess the dough functional properties, and carried out in
125
three duplicates based on AACC (2000), including gluten content, dry and wet gluten
126
content and gluten index.
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Bread baking experiments were carried out to evaluate the bread-making
128
qualities of the lines. The baking procedure was the standard rapid-mix-test with 1 kg
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Image analysis of bread was carried out with a C-Cell image analysing software and
131
equipment (Calibre Control International Ltd.) referred to Sun et al. (2010). Slice area,
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height (max), breadth, wrapper length, volume of holes, slice brightness, cell contrast
133
and average cell elongation were tested and used to estimate the bread properties.
134
2.4. Light microscopy and TEM observation
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The developing grains in CS and CNU608 were collected at 7, 10, 13, 15, 19 and
136
23 days post anthesis (DPA). The seed fixation, rinse, dehydration and thin sections
137
preparation were as reported by Loussert et al. (2008) with some modifications. The
138
sections were stained with Coomassie brilliant blue, rinsed extensively with water
139
before examination on the Leica DRD microscope in bright field. For transmission
140
electron microscopy (TEM) analysis, the sections were fixed in primary antibody
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(HMW-GS Polyclonal antibody) and second antibody (15 nm gold conjugated goat
142
anti-rabbit IgG) (Loussert et al. 2008).
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2.5. DNA extraction and PCR amplification
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Genomic DNA was extracted from leaves following the procedure of Wang et al.
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(2013). About 20 mg of crushed seed were placed in a 1.5 ml tube followed by
146
maceration in 1 ml of extraction buffer (200 mM Tris-Hcl (pH 7.5), 288 mM NaCl, 25
147
mM EDTA and 0.5% SDS). The supernatant was retained and 700 µl phenol:
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chloroform: iso-amyl alcohol (25:24:1) was added and centrifuged for 10 min at
149
13000 rpm. The last step was repeated and then isopropanol (700 ml) was added and
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the sample stored at -20°C for 30 min. After centrifugation, the DNA pellet was
151
washed two times with 70% ethanol and dissolved in H2O. Two pairs of allelic-specific polymerase chain reaction (AS-PCR) primers were
153
designed based on the published HMW-GS gene sequences and used to amplify the
154
complete coding region
155
(5'-ATGGCTAAGCGGTTAGTC CT-3') and Cx-1R (5'-GCTGCAGAGAGTTCTA
156
TC-3'), Cx-2F (5'-ATGGCTAAGCGGTTAGTCCTC T-3') and Cx-2R (5'-CTATCA
157
CTGGCTGGCCGACAAT-3'). The PCR amplification included an initial denaturation
158
step at 94°C for 5 min followed by 34 cycles at 94°C for 45 s, 62/55°C for 1 min,
159
72°C for 150 s, and a final extension step at 72°C for 10 min. The PCR products were
160
separated by 1% agrose gel in Tris-acetic acid-EDTA buffer.
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2.6. Molecular cloning and sequencing
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The amplified products with expected sizes were purified from the gel and
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cloned with pGEM-T plasmid vector (Promega). Three recombined DNA clones were
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sequenced by TaKaRa Biotechnology (Dalian) CO., LTD, China, and each clone was
165
sequenced three times to reduce the sequencing error.
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2.7. Heterologous expression, Western blotting and LC-MS/MS
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primers to remove the signal peptides. The forward primer was pETyF:
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5'-AAACATATGGCTGAAGGTGAGGCCTCTGA-3' with Nde I restriction site while
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the reverse primer was pETyR1: 5'-AGGCTCGAGCTATCACTGGCTGGC-3' and
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site. After amplification and purification, the PCR products of the cloned genes were
173
ligated into the expression vector pET-28a (Novagen) and transformed into
174
Escherichia coli BL21(DE3) plysS cells. The positive hybrid plasmid was induced by
175
IPTG. The extraction and separation of expressed proteins were carried out by
176
SDS-PAGE according to Yan et al. (2009). The Western blotting was performed based
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on Yan et al. (2009). The sensitive first antibody (anti-His-tag mouse monoclonal
178
antibody) was used to detect the expressed proteins based on the His-tag sequence
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present at the downstream of the cloned gene.
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The peptide sequences of the expressed HMW glutenin subunit were determined
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by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The specific
182
HMW-GS band on the SDS-PAGE gel was excised and digested with trypsin. A
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sample of the digested protein (0.5 ml) was subject to MS analysis in a Waters
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SYNAPT High Definition Mass Spectrometry™ (HDMS) mass spectrometer.
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2.8. Phylogenetic analysis
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The multiple alignment was performed with homologous nucleotide sequences
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by using the ClustalW program. The alignment file was converted to MEGA format
188
and a phylogenetic tree was constructed with the Molecular Evolutionary Genetics
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Analysis software MEGA 6 by the complete coding regions of HMW-GS.
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3. Results
191
3.1. Characterization of the derived line CNU608
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substitution line, the derived line CNU608 was developed. The line CNU608 shows
194
significant changes of plant morphological characteristics as well as growth and
195
development features, including increasing plant height, spike length and grain sizes
196
and colour (Suppl. Fig. S1). It also differs from CS in that it is awned and is powdery
197
mildew resistant (data not shown). The many morphological and physiological
198
differences between CS and the line CNU608 are not justified, only by the presence of
199
the 1C(1A) substitution; it is likely that in the line CNU608 additional portions of the
200
genome of Ae. caudata have been introgressed in CS
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3.2. Quality performance of Chinese Spring and the derived line CNU608
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Various gluten and breadmaking parameters were tested to estimate the quality
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performance of CNU608. The results indicated that dough development time, stability
204
time, Farinograph quality number, gluten content and gluten index in CNU608 were
205
significantly higher than those in CS while the degree of softening of CNU608 was
206
lower than that in CS (Table 1). Breadmaking quality showed that in CNU608, the
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loaf volume, bread sensory evaluation score and most of the C-Cell parameters related
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to bread structural features were significantly improved, including bread slice area,
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height, breadth, wrapper length, volume of holes, slice brightness and average cell
210
elongation as shown in Table 1 and Fig. 1A.
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3.3. Identification of seed proteins in Chinese Spring and CNU608
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SDS-PAGE analysis showed that, compared to CS, CNU608 still had 1Bx7 and
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ACCEPTED MANUSCRIPT 1Dy12 subunits, but the 1B-encoded 1By8 subunit and 1Dx2 were replaced by two
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novel HMW-GS, which were slightly larger and smaller than 1Dx2 and designated as
215
1Dx2s and 1Dx2f , respectively (Fig. 1B CNU608 is not known. The deduced
216
molecular mass of 1Dx2s and 1Dx2f subunits were 88244.42 Da and 85285.12 Da,
217
respectively. To further confirm the authenticity of the two novel HMW-GS in
218
CNU608 and acquire their structural information, 1Dx2s and 1Dx2f subunits from
219
SDS-PAGE gel were excised manually, digested with trypsin, and then identified by
220
MALDI-TOF/TOF-MS. The results showed that 1Dx2s and 1Dx2f subunits were
221
identified as x-type HMW-GS (Suppl. Table S1). Two peptides (QYEQQIVVPPK and
222
AQQLAAQLPAMCR)
223
GGSFYPGETTPPQQLQQ
224
respectively well matched to 1Dx2s and 1Dx2f with protein score C.I. % of 100%.
225
3.4. Protein body (PB) changes during grains development
and
three
peptides
(ELKACQQVM
DQQLR,
RYYPSTSPQQVSYYPGQASPQRPGR)
were
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Wheat seed storage proteins are deposited into organelles of protein bodies (PBs)
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after synthesis and folding in the lumen of the endoplasmic reticulum (Loussert et al.,
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2008). Since HMW-GS have major effects on gluten strength and bread-making
229
quality, the significant improvement of gluten quality in CNU608 could mainly be
230
attributed to the introgression of the two novel subunits above described. In order to
231
compare the PB differences between CS and CNU608 during grain development,
232
different microscopy techniques were used to observe the PB changes at different
233
grain developmental stages. The results from light microscopy observation showed
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235
of PBs (Fig. 2A). The PBs appeared at 10 DPA in CS and CNU608, grew in size by
236
fusion among themselves and finally reached the maximum size at 19 DPA in CS and
237
CNU608, respectively. At 19 DPA, microscopic observations showed that PBs did not
238
remain as separate particles but coalesced to form large aggregates in CS, whereas in
239
CNU608 the PBs continued to enlarge from 15 DPA to 19 DPA. At 23 DPA, no PBs
240
could be observed by light microscopy in both lines. Some clear differences of PB
241
development between CS and CNU608 were found. More large PB numbers with the
242
diameter more than 5 µm at 13, 15 and 19 DPA in CNU608 were observed (Fig. 2A).
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TEM observation at 15 and 19 DPA through the immunogold labelling using
244
anti-HMW-GS further verified that CNU608 had more and larger PBs than CS (Fig.
245
2B).
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3.5. Molecular characterization of 1Dx2s and 1Dx2f subunits
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The complete open reading frames (ORFs) encoding 1Dx2s and 1Dx2f subunits
248
were isolated and cloned from CNU608 by AS-PCR. Their sequences showed that
249
1Dx2s gene consisted of 2499 bp encoding 831 amino acid residues while the 1Dx2f
250
comprised 2427 bp encoding 808 amino acid residues. Their nucleotide and deduced
251
amino acid sequences were deposited in the GenBank with accession number of
252
KF466259 and KF466260, respectively.
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Comparison of the deduced amino acid sequences showed that both 1Dx2s and
254
1Dx2f had similar structural characteristics with 1Dx2 subunit (Fig. 3). 1Dx2s
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256
of 21 amino acid residues, a non-repetitive N-terminal domain of 81 amino acid
257
residues, followed by a repetitive domain of 687 amino acid residues and a
258
non-repetitive C-terminal domain of 42 amino acid residues. But one octapeptide
259
deletion (ELQELQER) occurred in the N-terminal domain of 1Dx2s compared to
260
1Dx2 subunit (Fig. 3A). The 1Dx2f subunit, it showed distinct structural feature: its
261
first 779 amino acid residues were highly homologous with the corresponding
262
sequence of 1Dx2, while the final 120 amino acid residues were highly similar with
263
those of 1Cy subunit (GenBank accession number GQ403046, Fig. 3B). 1Dx2f
264
subunit had a long repetitive domain, high glutamine content and an extra cysteine
265
residue from 1Cy subunit at position 730 near the C-terminal domain. Thus, five
266
cysteine residues were present in the chimeric 1Dx2f subunit: three in the N-terminal
267
domain, one in the repetitive domain near the C-terminal domain and one in the
268
C-terminal domain.
269
3.6. Heterologous expression and identification of 1Dx2s and 1Dx2f genes
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To verify that the cloned DNA fragment was the accurate representative of the
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1Dx2s and 1Dx2f genes, the ORF without signal peptide was ligated with the
272
expression vector pET-28a and transformed into E. coli BL21 (DE3) pLysS cells. The
273
positive hybrid plasmid was induced by IPTG. The expressed proteins were purified
274
by 50% (v/v) propanol including 1% DTT (w/v) and separated by SDS-PAGE (Fig.
275
4A). The results showed that the expressed proteins had similar mobilities to those of
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277
expressed proteins were further confirmed by Western blotting. The bacterially
278
expressed proteins of 1Dx2s and 1Dx2f genes and the endogenous subunits from
279
CNU608 displayed a strong reaction to the polyclonal antibody specific for HMW-GS
280
(Fig. 4B).
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To further confirm the authenticities of the cloned sequences, the heterologous
282
expressed proteins collected from SDS-PAGE gel were digested by trypsin and
283
identified by LC-MS/MS. The results showed that three and five peptides could be
284
well matched with 1Dx2s and 1Dx2f, respectively as listed in Suppl. Table S2. The
285
coverage rates were 6.1 % for 1Dx2s and 9.1 % for 1Dx2f, confirming the
286
correspondence between the expressed proteins and their encoding genes cloned.
287
3.7. Phylogenetic analysis
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To investigate the phylogenetic relationships among HMW-GS gene family from
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different genomes, 27 HMW-GS genes were used to construct a phylogenetic tree,
290
which included 1Dx2s and 1Dx2f genes obtained in this study and other 25 genes from
291
GenBank (http://www.ncbi.nlm.nih.gov/). As showed in Suppl. Fig. S2, the
292
phylogenetic tree was apparently clustered into two separate groups, well
293
corresponding to x-type and y-type subunit genes, respectively. Three subgroups for
294
both x-type and y-type subunit genes were clearly separated by the A, B and D
295
genomes. In particular, 1Dx2s, 1Dx2f and 1Dx2 genes were clustered to a small
296
subgroup, implying their highly similar structural features and close phylogenetic
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relationship.
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4. Discussion Aegilops caudata (2n=2x=14, CC) is a diploid species whose genome is involved
300
in the formation of many polyploid species (Friebe et al., 1992), so it is expected to
301
serve as valuable genetic sources for wheat quality improvement. To date, HMW
302
glutenin subunits in Aegilops caudata have been identified and characterized, which
303
implied that they have potential values in improving the processing properties of
304
bread wheat (Liu et al., 2003). In this study, we developed the derived line CNU608
305
from crossing between Chinese Spring (CS) and CS-Ae. caudata substitution line
306
1C(1A) that possessed two novel HMW-GS genes 1Dx2s and 1Dx2f, which showed
307
high similarity and phylogenetic relationships with 1Dx2 (Suppl. Fig. S2). The
308
deduced amino acid sequences revealed that both novel subunits were the variants of
309
the 1Dx2 subunit. The 1Dx2s subunit had an octapeptide (ELQELQER) deletion in
310
the N-terminal while the 1Dx2f was a chimeric subunit originated from combination
311
of 1Dx2 and the C-terminal (120 amino acid residues) of 1Cy subunit respectively
312
(Fig. 3). Since two x-type HMW-GS are present in the derived line CNU608, two
313
gene copies should be located at the Glu-D1 locus. This suggests that a gene
314
duplication event occurred during the development process of the derived line.
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It is known that the allelic variations at Glu-1, Glu-3 loci are mainly resulted from
316
point
mutations,
unequal
crossing-over,
317
illegitimate recombination and homoeologous recombination (Guo et al., 2013; Zhang
15
slip-mismatching,
intrachromosomal
ACCEPTED MANUSCRIPT et al., 2008). Here we proposed a putative genetic mechanism for the origin of 1Dx2s
319
and 1Dx2f genes. As shown in Fig. 5, firstly, an octapeptide (ELQELQER) deletion
320
occurred in 1Dx2 subunit of Chinese Spring, which resulted in a new subunit 1Dx2s.
321
Subsequently, a duplication event of 1Dx2s gene by unequal crossing-over occurred at
322
Glu-D1 locus, leading to two x-type subunits at Glu-D1. Finally, the chimeric gene
323
1Dx2f was originated from the unequal crossing-over between 1D and 1C
324
chromosomes. Cross overs between the 1D and 1C chromosomes could have already
325
occurred in the original wheat-Ae. caudata addition line (Triticum aestivum strain
326
P168) initially obtained by Dr. H. Kihara, Kyoto University, Japan, in which the
327
1C(1D) substitution line was first identified by Muramatsu (1959). Alternatively, this
328
event could have occurred during the backcrossing process with CS.
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In the past several decades, the molecular mechanisms of HMW-GS allelic
330
variations affecting wheat quality properties have been widely studied. Considerable
331
work showed that the molecular structure of HMW gluten proteins and their amino
332
acid compositions have important effects on dough properties (Hanssini et al., 2005).
333
In general, x-type HMW-GS have longer repetitive domains and higher expression
334
amount compared to y-type subunits. A long repetitive domain can form more β-turns
335
structure conferring elasticity to the protein molecule (Gianibelli et al., 2001; Tatham
336
et al., 1985). In addition, high glutamine content can stabilize the polymeric structure
337
of glutenin through forming more hydrogen bonds (Gilbert et al., 2000), so larger
338
subunits rich in glutamines had a greater positive effect on dough strength than
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ACCEPTED MANUSCRIPT smaller ones (Békés et al., 1995). Another important factor related to dough elasticity
340
is the proportion of the consensus hexapeptides and nonapeptides in the repetitive
341
domain. Higher proportion of the consensus hexapeptides and nonapeptides can
342
produce a more regular pattern of repetitive β-turns in the protein, and contribute to
343
better dough elasticity (Flavell et al., 1989). Also, the number and position of cysteine
344
residues have important roles in dough viscoelasticity, which form the inter- and
345
intra-molecular disulfide bonds and also link with LMW-GS and gliadins to form
346
gluten macropolymer (Pirozi et al., 2008). For example, an extra cysteine residue
347
present in 1Dx5 subunit is considered to be responsible for its superior quality
348
property (Anderson et al., 1989). In this study, both novel subunits 1Dx2s and 1Dx2f
349
identified in CNU608 have longer repetitive domains rich in glutamine residues.
350
Particularly, the 1Dx2f subunit has an extra cysteine residue from 1Cy subunit at
351
position 730 near to the C-terminal domain. The repetitive domain of 1Dx2f subunit
352
contains 25 consensus hexapeptide (PGQGQQ) and 8 consensus nonapeptide
353
(GYYPTSP/LQQ) motifs, and thus has high proportion of the consensus hexapeptide
354
and nonapeptide repetitive motifs. These constructural features could contribute to the
355
superior gluten strength and breadmaking quality of the derived line (Table 1 and
356
Fig.1A).
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Our results demonstrated that two x-type subunits 1Dx2s and 1Dx2f were present
358
at Glu-D1 locus and both subunits were simultaneously expressed in CNU608 (Fig.
359
1B). This results in an increase of the amount of x-type HMW-GS and improve the
17
ACCEPTED MANUSCRIPT formation of better gluten quality. Therefore, although 1By8 subunit from CS is
361
absent, CNU608 also has significantly improved gluten strength and breadmaking
362
quality compared to CS (Table 1). Similarly, HMW-GS overexpression such as
363
1Bx7OE resulted from a retroelement mediated gene duplication event at Glu-B1 locus
364
can result in a significantly higher proportion of HMW-GS, and then produce an
365
positive effect on flour quality (Li et al., 2014). Therefore, the duplication and
366
structural variations of two x-type subunit genes at Glu-D1 locus in the derived line
367
CNU608 can significantly increase the expression amount of x-type HMW-GS and
368
improve breadmaking quality.
369
5. Conclusions
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Two novel x-type HMW-GS designated as 1Dx2s and 1Dx2f in CNU608 derived
371
from hybridation between Chinese Spring and CS-Ae. caudata substitution line 1A
372
(1C) were identified, and their complete encoding sequences were amplified and
373
cloned by AS-PCR. Molecular characterization demonstrated that both subunits
374
showed higher similarity and closer phylogenetic relationships with 1Dx2.
375
Particularly 1Dx2s had a octapeptide (ELQELQER) deletion and 1Dx2f belonged to a
376
chimeric gene originated from the recombination between 1Dx2 and 1Cy subunits and
377
had an extra cysteine residue from 1Cy subunit at position 730 near to the C-terminal
378
domain. Both novel subunit genes were probably originated from one deletion and
379
two unequal cross-over events. Their authenticity were confirmed through
380
MALDI-TOF/TOF-MS, prokaryotic expression, Western blotting and LC-MS/MS.
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ACCEPTED MANUSCRIPT The introgression of both subunit genes in CNU608 resulted in significant
382
improvement of gluten strength and breadmaking quality. Their specific molecular
383
structural features and high expression proportion of x-type HMW-GS could
384
contribute to the formation of superior breadmaking quality. Particularly, the chimeric
385
1Dx2f gene showed potential values for wheat gluten quality improvement.
386
Acknowledgments
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This research was financially supported by grants from the National Natural
388
Science Foundation of China (31471485), Natural Science Foundation of Beijing City
389
and the Key Developmental Project of Science and Technology from Beijing
390
Municipal Commission of Education (KZ201410028031), Key Project of National
391
Plant Transgenic Genes of China (2014ZX08009-003), and International Science &
392
Technology Cooperation Program of China (2013DFG30530).
393
References
394
AACC, 2000. Approved Methods of the American Association of the Cereal Chemists,
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the high-molecular-weight glutenin subunit 1Ax1 gene into wheat. Nature Biotechnology 14, 1155–1159.
Anderson,
O.D.,
Greene, J.M.,
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Yip,
1989.
R.E.,
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N.G.,
of
the
P.R.,
400
Malpica-Romero,
401
high-molecular-weight glutenin genes from the D-genome of a hexaploid bread
19
sequences
Shewry,
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wheat, Triticum aestivum L. cv Cheyenne. Nucleic Acids Research 17, 461-462. Békés, F., Gras, P.W., Gupta, R.B., 1995. The effects of purified cereal polypeptides
404
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Figure legends: Fig. 1 A. Bread loaves of CS and CNU608. B. Identification of HMW-GS in CS and CNU608 by SDS-PAGE; The two arrowed bands were selected for further
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MALDI-TOF/TOF- MS identification. Fig. 2 Developmental changes of protein body (PB) during grain development of CS and CNU608. A. Light microscopy observation of PBs in the endosperm cells
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during developing grains at 7, 10, 13,15, 19 and 23 DPA; B. Ultrathin sections of
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wheat endosperm demonstrating the immunolocalization of HMW-GS to PBs at 15 and 19 DPA.
Fig. 3 Comparison of the deduced amino acid sequences of 1Dx2s and 1Dx2f genes with 1Dx2 subunit. A. 1Dx2s and 1Dx2; B. 1Dx2f, 1Cy and 1Dx2 subunits. The
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signal peptide, N-terminal domain, repetitive domain and C-terminal domain are indicated. The positions of cysteine residues are marked by black frames. Deleted and identical residues are indicated by red frames.
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Fig. 4 Heterologous expression and identification of 1Dx2s and 1Dx2f genes in E. coli.
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A. SDS–PAGE analysis;1. 1Dx2f; 2. CNU608; 3. 1Dx2s; 4,pET28a; B. Western blot of the expressed protein of 1Dx2s and 1Dx2f genes in E. coli. 1, Blue Plus Ⅱ; 2, pET28a; 3, 1Dx2f; 4, 1Dx2s; 5, CNU608.
Fig. 5 The putative genetic mechanisms for the origination of 1Dx2s and the chimeric gene 1Dx2f. Green area: signal peptides; yellow area: N-terminal domains; red area: repetitive domains; blue area: C-terminal domains; dotted lines: deletions. The letter “S” indicats cysteine residues.
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Supplementary data Suppl. Table S1 The 1Dx2s and 1Dx2f subunits in CNU608 identified by matrix-assisted laser desorption ionisation/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS)
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Suppl. Table S2 Peptides identification of the 1Dx2s and 1Dx2f subunits in CNU608 by liquid chromatography-tandem mass spectrometry (LC-MS/MS)
Suppl. Fig. S1 Morphological characteristics of plants, spikes and seeds of CS and
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CS-Ae. caudata translocation line 1AL.1CS.
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Suppl. Fig. S2 Phylogenetic tree of 27 HMW-GS genes based on complete coding DNA. The GenBank accession numbers for the published HMW-GS genes were 1Dx5*t (DQ681076), 1Dx5 (X12928), 1Dx2 (X03346), 1Dx2.1t (AF480486),
1Dx1.5t
(AY594355),
1Dx3t
(HM347447),
1Dx4t
1Ax2*
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(DQ307383), 1Dx2.1 (AY517724), 1Dx2.2* (AJ893508), 1Ax1 (X61009), (M22208),
1Bx7
(X13927),
1Bx23
(AY553933),
1Bx20
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(AJ437000), 1Bx13 (EF540764), 1Bx14 (AY367771), 1Bx17 (JC2099), 1Ay (X03042), 1Ay/Tu-e (AY245578), 1By8 (AY245797), 1By9 (X61026),
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1By15 (DQ086215), 1Dy10 (X12929), 1Dy11 (EU528008) and 1Dy12 (X03041).
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Farinograph Development
Stability
quality number time(min)
(min)
softening
(%) (mm)
Gluten index
Loaf mass
Loaf volume
(%)
(g)
(cm3)
content
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Gluten
Dry basis
Degree of
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Table 1. Comparison of gluten and bread-making quality parameters between CS and CNU608
(g)
2.0±0.10**
3.0±0.10**
103±1.6**
40±0.70**
17.92±0.03*
4.83±0.03*
57.80±0.73**
148.91±0.31
738±1.67**
CS
1.5±0.07
0.8±0.07
215±1.78
18±1.11
15.42±0.01
3.91±0.02
28.35±1.64
147.89±0.01
548±1.67
Wrapper
Volume of
Slice
Cell
Average cell
length/px
holes
brightness
contrast
elongation
576±2.33**
1838±0.67*
14.2±0.83
140.9±0.10**
0.705±0.01
1.53±0.01*
419±5.33
1634±3.00
13.6±0.53
127.7±0.10
0.690±0.01
1.41±0.01
Total bread
Height Slice area/px
Breadth/px
score
(max)/px
49**
247916±478.33**
539±3.67*
CS
28
167704±1232.33
523±1.67
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Significant level: *p < 0.05, **p < 0.01.
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Highlights 1. Two new HMW-GS 1Dx2s and 1Dx2f significantly improve breadmaking quality. 2. The chimeric gene 1Dx2f was originated from recombination between 1Dx2 and
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3. A putative origin of 1Dx2s and 1Dx2f genes was proposed
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1Cy genes.