Effect And Molecular Mechanism On Wheat Processing Quality Of HMW-GS Deletion In Ningmai 9

Wheat gluten protein mainly include glutenin and gliadin,and glutenin determined gluten strength and processing quality.Glutenin is composed of high molecular weight glutenin subunits(HMW-GS)and low molecular weight glutenin subunits(LMW-GS).HMW-GS accounts for 10%percent of the total wheat gluten protein,which is the key factor in gluten strength and processing quality.In this study,we developed a set of single HMW-GS deletion lines of soft wheat Ningmai9 to understand the effects of HMW-GS deletion on kernel quality,quantity of gluten protein fractions,solvent retention capacity,dough rheological properties and sugar-snap cookie quality.In addition,cloning five HMW-GS genes of Ningmai9,the gene sequence and the amino acid sequence were analyzed,Study the Glu-D1y(12)deletion of high molecular weight gluten subunit gene coding sequence characteristics and the molecular basis of expression.The main results are as followed:1.Effect of HMW-GS Deletion on Processing Quality of Ningmai9Compared to the wild type,the HMW-GS deletion lines had significant difference in flour protein content,wet gluten content,kernel hardness,solvent retention capacity,dough rheological properties and sugar-snap cookie quality.In the HMW-GS deletion lines,sugar-snap cookie diameter had significant greater difference from the wild type(30.19cm),varying from 30.59cm-31.79cm.The HMW-GS deletion lines had significantly decreased the quality of mixograph peak time,peak value.peak width,TIMEX width and solvent retention capacity from the wild type.Compared between each HMW-GS deletion lines,the content of GMP and HMW-GS of x-type subunit deletion lines is lower than y-type subunit deletion lines,that is to say,the content of HMW-GS of x-type subunit had significantly bigger difference from y-type subunit,Bx7 deletion line is the lowest in the content of GMP%.Compared to the other lines,Glu-D1x(2)?Glu-B1y(8)deletion lines had significantly difference in flour protein content,wet gluten content.In the quality of sugar-snap cookie diameter,Glu-D1x(2)deletion line is the biggest,Glu-B1y(8)?Glu-B1x(7)deletion lines are second biggest,Glu-D1y(12)deletion line is the least.2.The nucleotide sequences and deduced amino acid sequences of HMW-GS gene of Ningmai9Cloning four HMW-GS genes of Ningmai9 on the Glu-B1?Glu-D1 locus,and analyze the gene sequence and amino sequence.Explore the connection between molecular structure and flour quality characters.In this study,the ?-turn sequences are compared in the central region,it is observed that the total number of four repeat sequences in x-type subunit is more than those of y-type subunit,indicating that the x-type subunits of HMW-GS contain more ?-turn structure and have greater influence.on dough quality in comparison with y-type subunits.The total number and percentage of Glutamine are compared,demonstrating that,1Dx2,1 By8 subunit have more than the other types of subunit.It is indicating that repeat sequence in central area of Glu-D1x(2)?Glu-B1y(8)subunit have a better ability to combined with hydrogen bond between each other to form long chains.The number of Glutamine in Glu-Dly(12)subunit is less,but the percentage is higher up to 35%,the percentage of Glutamine in Glu-B1x(7)subunit is lowest.3.The Molecular Basis of Glu-Dly(12)deletion typeThe coding sequences of Glu-Dly(12)silence gene in Glu-Dly(12)deletion line was isolated and sequenced.Sequence alignment indicated that silence of Glu-D1y(12)in mutant 10 was mainly caused by the single base replacement involved in C to T leaded to the triplet code CAG for glutamine(G)changed to stop codon TAG located at 1663 by positions in the coding sequence.In mutant 11?silence of 1Dy12 was mainly caused by the single base replacement involved in G to A leaded to the triplet code TGG for tryptophan(W)changed to stop codon TGA located at 798 by positions in the coding sequence.Premature termination codon play an important role for the silence of HMW-GS gene.QRT-PCR result show that premature termination codon have influence on the mRNA transcription,make it always in a low level,have influence on translation and expression of the protein and then.In this study,single deletion of HMW-GS weakened gluten strength and improved sugar snap cookie processing quality,indicating that HMW-GS knockout can be used to improve soft wheat quality.