Effect Of New Mutant 1Ax1 Subunit On The Functional Properties Of Common Wheat With The Addition Of Extra Cysteine Residue

Characteistics of wheat storage proteins determine its end-use qualities mainly.Gluten polymers,formed from the crosslinkage between high molecular weight glutenin subunits(HMW-GSs)and low molecular weight glutenin subunits(LMW-GSs)by inter-chain disulfide bonds,are the major determinants of wheat dough properties.Subunit pairs 1Dx5+1Dy10 were recognised as superior subunit combination in common wheat and contained an extra repetitive-domain cysteine residue in 1Dx5 that was important for understanding the formation of dough viscoelasticity.Untill now,the role of this extra cysteine residue in wheat flour functional properties is still unclear.In this study,one specific serine codon of the 1Ax1 gene corresponding to the extra cysteine residue of 1Dx5 was substituted by a cysteine codon through site-directed mutagenesis.Recombinant vectors p LRPT-Glu-Mut1Ax1 and p LRPT-Glu-WT1Ax1 were constructed and transformed into L88-31(Triticum aestivum L.cv)by particle bombardment,respectively.Transgenic independent lines were confirmed by PCR,Southern blot,RT-PCR and SDS-PAGE analyses.Four homozygous transgenic lines expressing mut1Ax1 and one homozygous transgenic lines expressing wt1Ax1 were produced through successive screening of five generations(T0-T4).Photodensitometric software,Glutomatic 2200,Mixograph,size exclusion-high performance liquid chromatography(SE-HPLC),Malvern laser scattering particle size analyzer,scanning electron microscopy(SEM),and Fourier transform infrared spectroscopy(FTIR)were used to study the impact of exogenous Mut1Ax1/WT1Ax1 on dough mixing properties,GMP particle size distributions,gluten networks and their secondary conformation conversions,and disulfide bond contents.These results would help us understand more about the effect of this extra cysteine on flour mixing properties and gluten physicochemical characteristics,and its influential mechanism.The main results are as follows:(1)Photodensity analysis exhibited that the expression levels of both transgenes mut1Ax1 and wt1Ax1 in transgenic lines were the similar.Gluten index and Mixograph showed that compared with WT1Ax1,subunit Mut1Ax1 has significantly increased gluten content and enhanced dough strength and resistance to extension,indicating that subunit Mut1Ax1 with addition of the extra cysteine residue had a positive effect on the dough strength of wheat flour.(2)SE-HPLC,GMP particle size distribution and SEM analyses showed that subunit Mut1Ax1 has given significant increases on the content of large-sized polymers and average particle sizes of GMP particles in comparison with WT1Ax1,indicating that the former promoted the polymerisation of gluten proteins and further formed gluten networks fully.(3)Regarding all the mutant transgenic lines,the SS contents of gluten powder samples were increased remarkably,which suggested that Mut1Ax1 with extra cysteine participated in forming intermolecular disulfide bond to strengthen the intermolecular interactions among glutenin subunits.Furthermore,the obvious conversions of random coils and ?-turns to ?-sheets indicated that the presence of Mut1Ax1 was more conducive to the orderly folding and polymerisation of gluten proteins.The impact of Mut1Ax1 on dough processing properties should be attributed to the extra repetitive-domain cysteine,which took part in the cross-linking among glutenin proteins through intermolecular disulfide bonds,facilitated subunits crosslinkage,promoted GMP polymerisation,modified gluten network structure for embedding starch and other insoluble particles to fully interact with each others,and thus improved the dough quality.