Improvement Of Wheat HMW-GS In Composition And Structure By Chromosome Engineering And Genetic Engineering Strategies

The composition and structure of high molecular weight glutenin subunits(HMW-GS)in wheat grain have an important effect on flour quality.The improvement or optimization of HMW-GS in composition and structure can beneficent to the breeding of wheat varieties with high bread-processing quality.Firstly in this dissertation,the wheat-Aegilops longisma translocated chromosomes 1S~lL·1BS and 1BL·1S~lS were introduced three commercial varieties by continuous backcrossing associated with molecular marker-assisted selection to develop new translocation lines having better agronomic and yield traits.Secondly,two potentially valuable HMW-GS genes 1S~lx2.3~*and 1S~ly16~*isolated from Ae.longisma were transferred into wheat in different combinations via genetic transformation to develop marker-free transgenic lines.Thirdly,base editing technology was employed to modify the key base pair of 1Bx14 and 1Dx2 of wheat HMW-GS to create new germplasm with increased cysteine number of the two HMW-GS.Finally,the developed translocation lines,transgenic lines,and gene editing lines were analyzed bread-processing quality and/or gene expression profiling.Taking together,the effects of different HMW-GS composition and structure on wheat quality and the possible molecular mechanism related to this point were primarily investigated.The main results achieved are detained presented as follows:1.A total of 15 new 1S~lL·1BS and 1BL·1S~lS homozygous translocation lines showing ideal agronomic traits were developed by backcrossing for five times with recurrent parents Ningchun4,Ningchun50 and Westonia combining marker-assisted selection,among which two 1S~lL·1BS translocation lines and three 1BL·1S~lS translocation lines were bred from each recurrent parent.Fluorescence in situ hybridization test revealed that all the developed translocation lines harbored a pair of chromosomes 1S~lL·1BS or 1BL·1S~lS,in which the translocated chromosomes were not changed in constitution.The 40 K liquid chip analysis indicated that the similarity of all the translocation lines to their corresponding recurrent parents was 91.18～96.37%.Liquid chromatography analysis showed that the content of glutelin in 1BL·1S~lS translocation lines increased significantly in comparison with their recurrent parents The evaluation for bread processing quality recovered that the baking score and quality of 1BL·1S~lS and 1S~lL·1BS translocation lines were obviously improved compared with their recurrent parents.2.The two HMW-GS genes 1S~lx2.3~*and 1S~ly16~*from Ae.longisma controlled by the seed specific promotor p Dx5 promoter were transferred into a few wheat cultivars via Agrobacterium-mediated transformation in single manner each and combination type.Consequently,three 1S~lx2.3~*-1S~ly16~*homozygous marker-free transgenic lines,two 1S~lx2.3~*homozygous transgenic lines,one 1S~ly16~*homozygous marker-free transgenic line,and two 1S~ly16~*homozygous transgenic lines were developed in the genetic background of Fielder identified by molecular markers and SDS-PAGE.Additionally,one homozygous marker-free transgenic line with 1S~lx2.3~*-1S~ly16~*in the genetic background of CB037 and two homozygous transgenic lines with 1S~lx2.3~*-1S~ly16~*in the genetic background of Westonia were also identified.The analysis for bread-processing quality test indicated that the bread volume of 1S~lx2.3~*transgenic lines,1S~ly16~*transgenic lines and 1S~lx2.3~*-1S~ly16~*transgenic lines were increased compared with that of their wild type Fielder.3.Transcriptomic sequencing and quantitative real-time polymerase chain reaction(q PCR)were performed by using the RNAs extracted from the leaves and developing grains 20 days post anthesis of1S~lL·1BS and 1BL·1S~lS translocation lines and their corresponding recurrent parents,and transgenic lines and their wild type for studying gene expression profiles among different materials.Results indicated that the translocation lines showed more differentially expressed genes in both tissues than their recurrent parents;there were more differential expression genes in the immature grains of1S~lx2.3~*-1S~ly16~*and 1S~ly16~*transgenic lines compared to their wild type;after comparing the difference of the identified differentially expressed genes on chromosome distribution,it was found that the differential genes were mainly distributed on the translocated 1B chromosome in the two types of translocation lines,and evenly distributed on each chromosome in the three types of transgenic lines;the expression trends of the genes encoding glutens(most gliadins especially)and their related transcription factors were generally significantly increased in the 1S~ly16~*transgenic lines,and partly increased in the 1S~lx2.3~*and 1S~lx2.3~*-1S~ly16~*transgenic lines.4.Adenine base editors ABE8e-NGG and ABE8e-NG were constructed to edit the 1Bx14 and 1Dx2of wheat HMW-GS genes for increasing the cysteine numbers of the two proteins.The editing plants1Bx14-g223 and 1Dx2-g232 using ABE8e-NGG were obtained with editing efficiencies of 85.00%and52.94%,respectively,including two types of heterozygous and homozygous edited plants at1Bx14-Y70C site,and two types of heterozygous edited plants at 1Dx2-Q72R-Y73C site.In the edited plants the editing sites at the third and sixth positions were away from the PAM terminal.Additionally,three types of marker-free homozygous edited lines of 1Bx14-Y70C,1Dx2-Y73C and1Dx2-Q72R-Y73C were screened in the segregated population identified by PCR analysis.The 1Bx14in the homozygous edited lines of 1Bx14-Y70C and 1Dx2 in the homozygous edited lines of1DX2-Y73C and 1DX2-Q72R-Y73C were identified to have one more cysteine in comparison with the two HMW-GS in the wild type.By SDS sedimentation value identification of three mutants grain flour,it was found that the sedimentation value significantly increased in the edited lines of 1Bx14-Y70C,1DX2-Y73C and 1DX2-Q72R-Y73C.