Characterization Of Starch Synthesis Key Gene TaSSIVb And Mining Of Its Mutation Alleles In Winter Wheat

Wheat starch is the main source of energy and carbohydrate for human,which constitutes approximately 70% of the grain yield.In wheat seed,starch synthesis is a complex biological process which is catalyzed by series of enzymes.SSIV,a key enzyme of starch formation,which affects the starch granules number in Arabidopsis and rice.In wheat,SSIV is coded by three homeologous TaSSIVb genes,but the expression characterization and function of the three genes is still unclear.In this study,expression analysis was carried out in leaf and seed to elucidate the characterization of the three homeologous TaSSIVb genes in wild type.Then the functional mutations were mined through TILLING in TaSSIVb-A and B which further used to develop the double and triple mutants by crossing with TaSSIVb-D mutant.In addition,double mutant(ssIV-D/agp-B1)was evaluated and the effects of TaSSIVb-D mutation on other starch-related genes' expression pattern and the starch contents in seeds were analysed.The results of present study were as following:1.The three homeologous TaSSIVb genes expressed both in leaves and in seeds of wheat,but expression was significantly higher in leaves.Expression of TaSSIVb homoeologous genes in leaves was approximately 10 times higher than seeds,and the expression of TaSSIVb-A gene was significantly lower than TaSSIVb-B and TaSSIVb-D both in leaves and in seeds.In leaves,the expression of TaSSIVb-B was lower than TaSSIVb-D.However,during seed development,the expression of TaSSIVb-B was higher than TaSSIVb-D.Both TaSSIVb-B and TaSSIVb-D genes were highly expressed at 6 and 30 days after flowering,but the expression from 12 to 24 days after flowering was significantly lower.2.Five functional alleles of TaSSIVb-A and TaSSIVb-B genes were mined through TILLING.After that,double and triple mutant lines were developed by pyramiding these functional mutants.By screening the EMS mutated population,forty two point mutations were identified in TaSSIVb-A by using TILLING approach.Mutation density in TaSSIVb-A was 1/276.68 Kb,including ten missense mutations.The mutant lines E228,E325 and E359,which carried mutation G5734 A,G4445A and C5809 T respectively,were predicted to impact on function of SSIV.In TaSSIVb-B,thirty mutations were identified by TILLING and mutation density was 1/393.87 Kb in which including one splice junction and nine missense mutations.Missense mutant lines E1246 and E2-2-321,which carried mutation G3624 A and G6144 A,were predicted to affect on SSIV function.The above mentioned 5 mutation alleles were inherited into next generation.Among them,mutant lines E228,E325 and E1246 were homozygous,while E359 and E2-2-321 were heterozygous.By crossing,the mutant alleles of TaSSIVb in three sub-genomes were pyramided from which 13 double mutant lines and 1 triple mutant line were developed.3.The double mutant of TaSSIVb-D and TaAGP.L-B1 genes affected the expression of other starch synthase genes and starch content during seed development.TaSSIVb-D showed synergistic effects with TaAGP.L-B1 gene on the expression patterns of other starch biosynthesis key genes during seed development.In the seeds of ssIV-D/agp-B1 double mutant,the expression of TaSSIVb-D,TaAGP.L-B1,TaAGPSS,TaSSI and TaSBEII genes showed a significant reduction,but TaGBSSII gene expression was significantly increased compared to WT.Moreover,the starch content in the seeds of ssIV-D/agp-B1 was reduced almost 8.38% while in agp-B1 single mutant the starch content reduced 4.4%.The amylose content in the double mutant was significantly increased approximately 4.47%.Expression characterization of three homeologous TaSSIVb genes were analysed.Double and triple mutants were developed by crossing.Moreover,the effects of TaSSIVb-D mutation on other starch-related genes were elucidated through expression analysis.These mutants can be used for further functional analysis of TaSSIVb gene and for molecular breeding.