Study On Flour-processing Quality Effects Of Aegilops Longissima 1S~lx2.3* And 1S~ly16* And Wheat 1Dy12 Subunits

The content and composition of high molecular weight glutenin subunits?HMW-GS?and low molecular weight glutenin subunits?LMW-GS?in wheat grains affect flour-processing quality.As an important wild relative specie of wheat,Aegilops longissima contains a few superior quality glutenin subunits,which is a potential genetic resource for wheat processing quality improvement.This study aimed to obtain new wheat-Aegilops longissima translocation lines with good agronomic characters by continuously backcrossing wheat-Aegilops longissima 1S^lL/1BS and 1S^lS/1BL translocation lines with three agronomic wheat varieties including Ningchun4;to obtain homozygous transgenic wheat plants1S^lx2.3*gene by genetic transformation,molecular markers,SDS-PAGE,and fluorescence in situ hybridization?FISH?.In addition,a 1Dy12 silencing mutant was developed by somatic variation and investigated for the gene silence mechanism by SDS-PAGE,PCR,qPCR and sequencing.Finally,all the developed materials were analyzed for the quality-related genes expression,protein body accumulation pattern and flour-processing quality.The achievements achieved in this study will be helpful to understand the quality effects of the related a few HMW-GS and improve wheat processing quality via breeding.The main findings are as follows:1.Wheat-Aegilops longissima translocation chromosomes 1S^lL/1BS and 1S^lS/1BL were transferred to the genetic background of Ningchun 4,Ningchun 50,and Westonia,respectively,by crossing and backcrossing combining molecular marker-assisted selection.We obtained 10 homozygous new translocation lines with good agronomic traits in BC₃F₄ generation.Flour-processing quality analysis showed that the dough and gluten strength in the 1S^lL/1BS translocation lines were lower than those in the non-translocation background materials;the bread volume and sensory scores in 1S^lS/1BL translocation lines were higher than those in the recurrent parents with corresponding genetic background.2.1S^lx2.3*gene was transferred into Westonia via Agrobacterium-mediated transformation.Two homozygous transgenic wheat lines with 1S^lx2.3*gene were obtained by Quickstix strips,molecular markers,SDS-PAGE,and FISH identification.The dynamic expression patterns of the HMW-GS genes including 1S^lx2.3*,1Ax2*,1Bx17,1By18,1Dx2 and 1Dy12,and the quality-related genes including PDI-1,PDI-4,PDI-5,Bip-1,Bip-2,Bip-3,DOF-2,DOF-3,DOF-6,SPA-A,SPA-B and SPA-D in the homozygous line T-W1 during grain filling stage was analyzed by qRT-PCR.It was found that 1S^lx2.3*was normally expressed and the expression of 1Dx2 and 1Dy12 was significantly stimulated in T-W1.The expression of 1Ax2*and 1Bx17 in the most stages of grain development in T-W1 was higher than that in the wild-type,but the expression of 1By18 was significantly lower than that in the wild during the whole grain-filling stage.The expression levels of PDI4-1,PDI5-1,Bip-2,Bip-3,SPA-A and SPA-D were significantly down-regulated during the grain-filling stage in T-W1,while the expression levels of Dof-3,Dof-6 and SPA-B were significantly up-regulated during the grain-filling stage in the transgenic line.The analysis for bread and sponge cake processing quality showed that the grain protein content,dough formation time,dough stabilization time and bread volume and decreased mixing tolerance were significantly increased in transgenic homozygous line T-W1 containing 1S^lx2.3*,which significantly improved bread-processing quality,compared with its wild-type Westonia;the volume and sensory score of sponge cake were slightly increased in T-W1,which indicate the quality of sponge cake was also improved in the T-W1,in comparison with its wild-type.3.The 1Dy12 silencing mutant AS273 was obtained by tissue culture from wheat variety Kenong199?KN199?.It was found that 1Dy12 was expressed in AS273 but significantly lower than that in the wild-type KN199 in the transcriptional level at different grain development stages;however,1Dy12 was not expressed in the translation level.Then,the 1Dy12 in AS273 was amplified,sequenced and aligned.The results showed that there was a base substitution?T/C?in the 1Dy12 in AS273,which produced an early termination codon and truncated translation,which led to 1Dy12 silencing.Furthermore,the results for expression patterns of quality-related genes at different grain development stages by qRT-PCR showed that the transcripts of PDI-4,PDI-5,DOF-3 and SPA-A in AS273 were significantly higher and Bip-1 was slightly higher in AS273 than those in KN199,while the transcript of PDI-1 was slightly decreased.The protein bodies?PBs?morphology differences at different developmental stages in AS273 and KN199 were observed by transmission electron microscope?TEM?.The results demonstrated that the PBs was larger in AS273 than KN199 during the grain developmental period in general,except for those at 7 DPA and 28 DPA;the accumulation rate of PBs in AS273 was faster than that in KN199.Moreover,both AS273 and KN199 formed lamellar structure of PBs at 28 DPA when the formation of the PBs networks is normally completed.The processing-quality of bread,sponge cake,and biscuit in AS273 was significantly decreased such as water absorption,dough formation and stabilization time,and bread volume except mixing tolerance?which was increased in AS273?compared with those in KN199.AS273 showed volume reduction,coarser internal texture and inferior mouth feeling in sponge cake compared to KN199.The biscuits made from AS273 had an increased thickness and coarser internal texture than those from KN199.