Transformation of common wheat (Triticum aestivum L.) with avenin-like b gene improves dough functional properties
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This document summarizes research on transforming common wheat with an avenin-like b gene to improve dough functional properties. Key findings include: 1) The avenin-like b gene was found to belong to a multigene family and be specifically expressed in wheat seeds. 2) In vitro studies found that addition of purified avenin-like b protein decreased dough mixing time and increased resistance. 3) Transgenic wheat lines were generated with endosperm-specific expression of the avenin-like b gene. Analysis confirmed transgene integration and expression.
Transformation of common wheat (Triticum aestivum L.) with avenin-like b gene improves dough functional properties
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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. HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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? China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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). China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 11. Whether avenin-like b proteins play a role in determining the functional properties of dough? China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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 ) China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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. China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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? China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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. China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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). China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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). China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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. China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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. China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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 China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14
- 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! China-UK HUST-RRes Genetic Engineering & Genomics Joint Laboratory 2012/8/14