Transformation of common wheat (Triticum aestivum L.) with avenin-like b gene improves dough...

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Genetic Engineering & Genomics

Joint Laboratory

2012/8/14

Guangyuan He

Huazhong University of Science &Technology, China

Transformation of common wheat (Triticum aestivum L.) with

avenin-like b gene improves dough functional properties

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Joint Laboratory 2012/8/14

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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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

Background

Kan Y.C, et al. J Cereal Sci.

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.

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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?

Questions?

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Cloning of avenin-like b genes in wheat and related species

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

Table 1 The gene accession numbers and the species of the genes derived from

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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Avenin-like b genes of wheat belong to a multigene family

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

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

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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).

Multiple alignment of avenin-like b proteins

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Phylogenetic relationships of avenin-like b proteins

Fig. 5. Phylogenetic relationships of the avenin-like

proteins and other members of prolamin superfamily

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)

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Expression patterns of avenin-like genes in wheat and related species

β-actin

avenin-like

A

3 5 7 9 11 13 15 18 20 22 24 NC β-actin

avenin-like

B

β-actin

avenin-like

C

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

RT-PCR results showed that

avenin-like b transcripts were

expressed only in the seeds of

wheat and other related

species, and not in other

tissues

Expression of avenin-like b

proteins occurred in the wheat

seeds between 3 and 22 DPA,

reaching a peak between 11

and 15 DPA

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Identification of avenin-like b proteins in wheat and related species

Fig. 7. SDS-PAGE of total proteins from E. coli

transformed with the control plasmid pET32a or the

recombinant expression vector pET32a-avel.

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.

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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Whether avenin-like b proteins play a role in

determining the functional properties of dough?

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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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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.

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.

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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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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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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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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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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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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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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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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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Genetic transformation of wheat

Fig.13. Regenrataion of transgenic wheat after particle bomBardment

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

weeks on regeneration medium; I -L. The plantlets in culture bottle；M-Q. The plantlets

cultured in the soil；R. The plantlets in culture bottle

Fig.12. Schematic map of the wheat

transformation vector.

Avenin-like b gene inserted between the

endosperm-specific 1Dx5 promoter and

the CaMV35S terminator

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Fig.14.PCR (A and B) and Southern blotting analysis (C) of the

transgenic plants. Left: PCR amplification results of gus gene (A) and

CaMV35S terminator fragment (B). Lane M: DNA Marker III (A) or Marker I

(B); lane 2: plasmid pLRPT-avel for positive control; lane 3: Water for

negative control; lane 4:DNA of Zhengmai 9023 for negative control; lane 5-

11: DNA of regenerated plants. Right: Southern blotting analysis (C) of the

transgenic plants. Lane 1: Positive control of pLRPT-avel digested with BamHI;

lane 2: genomic DNA of Zhengmai 9023 digested with BamHI and HindIII;

lane: 3-7: genomic DNA of trangenic plants digested with BamHI and HindIII.

Fig.15. SDS-PAGE (A) and Western

blotting analysis (B) of gluten protein

extracted from flours of the transgenic

and non-transformed plants. (A) Lane M:

Protein Marker; lane 1: Zhengmai 9023; lane 2:

M3 line; lane 3: M6 line. Arrow indicates the

position of the transgenic avenin-like b proteins.

(B) Lane 1: Zhengmai 9023; lane 2:M3 line;

lane 3: M6 line.

The transgenic plants were confirmed by PCR, Southern blotting, SDS-PAGE and

Western blotting

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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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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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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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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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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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SE-HPLC analysis

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.

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

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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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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

Conclusions

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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!

Acknowledgements

Transformation of common wheat (Triticum aestivum L.) with avenin-like b gene improves dough...

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