International Gluten Workshop, 11th; Beijing (China); 12-15 Aug 2012

1. Transformation of common wheat (Triticum aestivum L.) with
avenin-like b gene improves dough functional properties
Guangyuan He
Huazhong University of Science &Technology, China
2012/8/14
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2. Contents
 Background
 Cloning and expression analysis of avenin-like b genes
 in vitro and in vivo analysis of the avenin-like b proteins on
the dough functional properties
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3. Background
 Avenin-like proteins (ALPs) are wheat storage proteins of unknown function
 The distinguishing feature of these proteins is the high levels of cysteine
residues
 ALPs are divided into two types, A and B. Type A proteins, corresponding to
the LMW gliadins, contain 14 cysteine residues, while type b proteins, un-
certainty corresponding to, usually contain 18 or 19 cysteine residues
Fig 1 Schematic depiction of the domain structures. Cysteine residues, which are conserved within the
a-type or b-type proteins are shown in yellow, non-conserved cysteine residues in orange. Kan Y.C, et al.
J Cereal Sci.
China-UK Kan Y.C, et al. J Cereal Sci.
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4. Questions?
 Whether avenin-like b genes belong to a multigene family?
 What expression patterns of avenin-like b genes are in
wheat and related species?
 Whether they play a role in determining the functional
properties of dough?
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5. Cloning of avenin-like b genes in wheat and related species
Table 1 The gene accession numbers and the species of the genes derived from
Fig. 2. PCR amplification products of avenin-like genes from
genomic DNA of 23 different Triticeae species. Lanes 1 and 15, DNA
marker; lanes 2–14, 16–25, PCR products of the materials corresponding
to EU096528–EU096540 and EU096541–EU096550 in Table 1,
respectively; lane 26, negative control
The presence and properties of the type b avenin-like proteins in 23 species of the
Triticeae including 18 species of Aegilops, 1 barley and 1 diploid, 1 tetraploid and 2
hexaploid wheat species
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6. Avenin-like b genes of wheat belong to a multigene family
Southern blot analysis showed that two or three
hybridized bands were observed after restriction
digestion of genome DNA with HincII or HhaI,
indicating that avenin-like genes of wheat
belong to a multigene family, which is similar
to other gluten protein genes
Fig.3 Southern blot analysis of genomic copy number of avenin-like gene
Lane 1,negative control; lane 2, wheat genomic DNA digested with Hinc II;
lane 3, wheat genomic DNA digested with Hha I
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7. Multiple alignment of avenin-like b proteins
Fig. 4. Multiple alignment of the deduced amino acid sequences of 23 avenin-like proteins by using the MegAlign program
of DNAStar software package and visually depicted by Genedoc.
The cysteine residues were shaded in gray with red frame for one residue and with green frame for two residues. The derived
proteins were named after their corresponding GenBank accession numbers (Table 1).
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8. Phylogenetic relationships of avenin-like b proteins
 The phylogenetic relationships of the 23
avenin-like proteins were analyzed by
construction of a dendrogram, including
sequences of other members of the prolamin
superfamily
 Avenin-like sequences form a single
cluster which is closest to the avenins of oats
and the sulphur-rich prolamins of wheat
(a-gliadins, g-gliadins, LMW subunits of
glutenin)
Fig. 5. Phylogenetic relationships of the avenin-like
proteins and other members of prolamin superfamily
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9. Expression patterns of avenin-like genes in wheat and related species
A  RT-PCR results showed that
β-actin avenin-like b transcripts were
expressed only in the seeds of
avenin-like wheat and other related
species, and not in other
B 3 5 7 9 11 13 15 18 20 22 24 NC
tissues
β-actin
avenin-like  Expression of avenin-like b
proteins occurred in the wheat
seeds between 3 and 22 DPA,
C reaching a peak between 11
β-actin and 15 DPA
avenin-like
Fig. 6. RT-PCR analysis of the spatio-temporal expression pattern of avenin-like gene.
A: different organs; B: DPA of immaure seeds; C: Seeds of different species
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10. Identification of avenin-like b proteins in wheat and related species
Fig. 8. Western bolt analysis of avenin-like b proteins
in wheat and related species.
(A) Proteins extracted from different tissues of wheat.
(B) Proteins extracted from mature endosperms of different
cereals.
Fig. 7. SDS-PAGE of total proteins from E. coli
transformed with the control plasmid pET32a or the
recombinant expression vector pET32a-avel.
 Polyclonal anti-serum was generated by immunizing New Zealand rabbits with the purified
and re-natured avenin-like protein
 Polyclonal antibodies raised against recombinant protein has been used to identify the
corresponding proteins in extracts of seeds
 Although the antibody was not completely specific for the b-type proteins, a reactive band
of the expected mass (about 34 kDa) was observed in all seed protein extracts
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11. Whether avenin-like b proteins play a role in
determining the functional properties of dough?
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12. Heterologous expression and dough mixing studies
of a cysteine-rich avenin-like b protein
Proteins: heterologous expression proteins
The avenin-like b gene sequence in this research was 855 bp long and
encoded a protein with 284 amino acid residues containing 19 cysteine
residues.
Heterologous expression vector : pET-32a-avel
Plant material: wheat cultivar En 1
Positive control: HMW-GS 1Bx14 purified directly from the flour of wheat
cultivar Emai 18
Method: two-gram Mixograph tests (Simple addition and incorporation )
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13. Expression and purification of Avenin-like b protein in large scale
The presence of a His tag on the
recombinant protein allowed it to
be purified in high purity. The
His tag was then removed by
incubating with enterokinase to
eliminate its effect on gluten
mixing properties.
Fig.9. Expression and purification of Avenin-like b protein from E. coli.
(a) SDS-PAGE of total reduced cell proteins from E. coli transformed with control plasmid pET-32a or the
recombinant expression vector pET32a-avel.
(b) SDS-PAGE of Avenin-like protein purified by Bind affinity chromatography.
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14. Two-gram Mixograph tests
Fig.10. A comparison of the functional properties of different glutenin subunit proteins in this study.
(a) Result of simply addition experiment; (b) Result of incorporation experiment.
Table 2. The means of mixing time (MT), peak dough resistance (PR) and resistance at breakdown (RBD) of the dough and
the dough mixed (by addition or incorporation) with 1Bx14 and Avenin-like determined from triplicate mixing experiments
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15. Two-gram Mixograph tests
1. Simple addition:
 Addition of 10 mg 1Bx14 HMW glutenin subunits or 10 mg Avenin-like
protein, the effects were marginal
 Addition of 15 mg Avenin-like protein caused a significantly decreased
mixing time (MT) and peak dough resistance (PR). No statistically
significant differences in resistance at breakdown (RBD) were observed in
the addition experiments
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16. Two-gram Mixograph tests
2. Incorporated:
 When 10 mg 1Bx14 HMW glutenin subunits or Avenin-like b protein were
incorporated into the base flour through reduction and re-oxidation treatment
both of them caused significantly increase in MT and PR and decrease in
RBD values
 While 15 mg Avenin-like b protein was incorporated into 2 g base flour, the
effects on these mixing properties were strengthened remarkably even
compared to that of 10 mg 1Bx14. This suggested that the role of the
Avenin-like b protein in dough quality properties could be enhanced with
increase of the protein quantity
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17. Distribution of incorporated proteins in reconstituted doughs
Fig.11. Example of SE-HPLC separation of total
proteins extracted from dough.
The chromatograms are divided into four parts
containing large polymeric proteins (LPP), smaller
polymeric proteins (SPP), large monomeric proteins
(LMP) and smaller monomeric proteins (SMP).
Table 3. Distribution of added/incorporated proteins in the SE-HPLC regions of total-protein extracts isolated from doughs after
10 min mixing
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18. Distribution of incorporated proteins in reconstituted dough
 An increased proportion of LMP and a lowed ratio of (LPP +
SPP)/(LMP + SMP) were found when Avenin-like b protein was
simply added
 When Avenin-like b protein and HMW-GS 1Bx14 were incorporated
into base flour, increased proportions of LPP and/or SPP and ratio of
(LPP + SPP)/(LMP + SMP) were observed
 This indicated that both the Avenin-like b protein and HMW-GS
1Bx14 were incorporated into the polymeric protein in the
reconstituted dough by disulphide bonds
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19. From the results of in vitro reduction and re-oxidation
experiment, it is demonstrated that Avenin-like b proteins
play an important role in determining functional properties
of dough and provided a preliminary result about the
relationships between avenin-like b proteins and functional
properties of dough
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20. When Avenin-like b protein was over expressed specifically
in the endosperm by transgenic approach, whether it can
lead the improvement of qualities of wheat dough?
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21. Transformation of common wheat (Triticum aestivum L.) with
avenin-like b gene improves flour mixing properties
Plant Material: Zhengmai 9023 (Triticum aestivum L. cv Zhengmai9023 )
Wheat expression vector: pLRPT-avel
The avenin-like b gene sequence in this research was 855 bp long and encoded a
protein with 284 amino acid residues containing 18 cysteine residues.
Method: particle bombardment
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22. Genetic transformation of wheat
Fig.12. Schematic map of the wheat Fig.13. Regenrataion of transgenic wheat after particle bomBardment
transformation vector.
A. The scutellum of donor wheat on target plate; B-E. The callus induced from wheat
scutellum; F. The cultures after 1 weeks on regeneration medium; G-H. The cultures after 4
Avenin-like b gene inserted between the
weeks on regeneration medium; I -L. The plantlets in culture bottle；M-Q. The plantlets
endosperm-specific 1Dx5 promoter and cultured in the soil；R. The plantlets in culture bottle
the CaMV35S terminator
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23. The transgenic plants were confirmed by PCR, Southern blotting, SDS-PAGE and
Western blotting
Fig.14.PCR (A and B) and Southern blotting analysis (C) of the
transgenic plants. Left: PCR amplification results of gus gene (A) and Fig.15. SDS-PAGE (A) and Western
CaMV35S terminator fragment (B). Lane M: DNA Marker III (A) or Marker I blotting analysis (B) of gluten protein
(B); lane 2: plasmid pLRPT-avel for positive control; lane 3: Water for extracted from flours of the transgenic
negative control; lane 4:DNA of Zhengmai 9023 for negative control; lane 5- and non-transformed plants. (A) Lane M:
11: DNA of regenerated plants. Right: Southern blotting analysis (C) of the Protein Marker; lane 1: Zhengmai 9023; lane 2:
transgenic plants. Lane 1: Positive control of pLRPT-avel digested with BamHI; M3 line; lane 3: M6 line. Arrow indicates the
lane 2: genomic DNA of Zhengmai 9023 digested with BamHI and HindIII; position of the transgenic avenin-like b proteins.
lane: 3-7: genomic DNA of trangenic plants digested with BamHI and HindIII. (B) Lane 1: Zhengmai 9023; lane 2:M3 line;
lane 3: M6 line.
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24.  The T0 transgenic wheat lines contained relatively simple insertion sites,
resulting in a single band on the blot expect for lane 4 which had no
hybridizing band. The banding patterns in lane 5 and lane 6 were very
similar. The banding patterns in lane 3 and lane 7, however, were different,
confirming that the plants were derived from independent transformation
events and could be therefore considered as independent lines
 Western blotting analysis proved that the levels of avenin-like b proteins
in the M3 and M6 transgenic lines were increased by 3.2- and 3.5-times
respectively, compared to the non-transformed line, calculated by
densitometry method
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25. After analysis for the presence and expression of transgene by PCR, Southern
blotting, SDS-PAGE and Western blotting in two successive generations (T2 and
T3), two transgenic wheat lines (M3 and M6 line) overexpressing avenin-like b
proteins were obtained for functional and biochemical characterization of wheat
flour by mixograph and SE-HPLC
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26. Mixing properties analysis
Fig. 16. Mixograph curves of the dough of two transgenic lines of wheat
(M3 and M6) and non-transformed line (Zhengmai 9023).
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27. Mixing properties analysis
Table 4. The 10-g Mixograph parameters of the transgenic wheat lines (M3 and M6) and non-transformed line of wheat (Zhengmai 9023)
Flour MT a (min) PR b (AU) g RBD(%) c BWPR (AU) d MRW (AU) e MBW (AU) f
M3 3.46±0.04h 45.67±0.78b 14.44±0.67b 26.44±0.65b 28.98±0.66b 31.73±1.26b
M6 3.56±0.04 46.16±0.67b 13.16±0.44b 24.92±0.48b 25.77±2.27b 35.91±3.5b
Zhengmai 9023 3.42±0.09 40.28±0.14a 16.42±0.76a 17.2±0.43a 18.62±1.81a 21.17±0.21a
LSD0.05 NS i 2.07 1.98 1.82 5.96 7.44
a Mixing time. b Peak resistance. c resistance breakdown. d bandwidth at peak resistance. e bandwidth of midline after
mixing time. f maximum bandwidth during the mixing. g Arbitrary units. h Mean ± standard deviation among three
replications. i Not significant. LSD: least significant difference at P = 0.05.
A number of parameters of the Mixograph curve can be measured, including the mixing time
(MT), peak resistance (PR) (both positively related to strength), resistance breakdown (RBD)
(positively related to stability), the maximum bandwidth during the mixing (MBW), the
bandwidth of midline after mixing time (MRW) and bandwidth at peak resistance (BWPR)
(all positively related to resistance to extension).
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28. Mixing properties analysis
 The increased of avenin-like b proteins in transgenic wheat lines resulted in
a significant increase in dough elasticity and strength measured by PR.
 Based on the RBD, the stability of the transgenic wheat dough was
improved. The RBD of transgenic wheat M3 and M6 lines were decreased
to 14.44 and 13.16, respectively, compared to that of 16.42 in the non-
transformed wheat lines.
 The increased of avenin-like b proteins in transgenic wheat lines resulted in
a significant increase in dough extensibility measured by BWPR, MRW, and
MBW.
 In addition, the MT of transgenic lines M3 and M6 were 0.04 and 0.14 min
higher, respectively, than the non-transformed lines, but this difference was
not statistically significant.
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29. SE-HPLC analysis
Table 5. The molecular size distribution of gluten proteins in flours of the transgenic and non-transformed wheat
lines determinated by SE-HPLC.
a %UPP (polymeric insoluble fraction/total polymeric protein) of flour of the transgenic and non-transformed
parent. b Mean ± standard deviation among three replications. c Not determined. LSD: least significant
difference at P = 0.05
All two transgenic lines had higher values for %F1 and %F1/%F2 while values for (%F3 +
%F4)/%F1 and (%F3 +%F4)/(%F1 +%F2) decreased in the two transgenic lines compared
with the non-transformed lines (Table 2), indicating that two transgenic lines had higher
proportions of polymeric proteins
The %UPP in the two transgenic lines M3 and M6 ,were prominent higher than in the non-
transformed wheat lines.
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30. Base on the above results, increased the avenin-like b protein contents
resulted in significant effect on the molecular weight of glutenins in wheat
grain and increase the proportion of polymeric proteins.
The SE-HPLC analysis demonstrated that the improvement of transgenic
line flour properties were due to increased proportion of large polymeric
proteins
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31. Conclusions
 Avenin-like b proteins are widely existed in Triticeae species, belong to a
multigene family, and specifically expressed in seeds
 Both in vitro and in vivo experiments showed that avenin-like b proteins
improved the dough functional properties obviously
 SE-HPLC analysis indicated that avenin-like b protein was incorporated into
polymeric subunits by intermolecular disulphide bonds
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32. Acknowledgements
Guangxiao Yang, Yuesheng Wang, Kexiu Li, Mingjie Chen, Junli Chang, Peng Chen,
Fengyun Ma, Yin Li, Lingling Yu, Miao Li, Hongwen Wang, Yunyi Liu, Cheng Wang,
Tingting Li, Wei Liu
This work was supported by the National Natural Science Foundation of China
(30871524，31071403), Wuhan Municipal S & T research project (201120922286), 482
International S & T Cooperation Key Projects of MoST (Grant No. 2009DFB30340),
National Genetically Modified New Varieties of Major Projects of China (2011ZX08002-
004, 2011ZX08010-004) and the National Natural Science Foundation of Hubei, China
(2010 CBD 02403)
Thank you for your attention!
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