Haplotype Analysis And Gene Editing Of Wheat Starch Branching Enzyme TaSBEs

Wheat(Triticum aestivum)is the world’s second largest food crop,with the change of people’s diet structure,wheat has different needs.Mature wheat grains contain certain resistant starch(RS),which has similar function to dietary fiber,but tastes better than the latter.There was a positive correlation between resistant starch and amylose.TaSBEs gene plays the function of starch branching enzyme to produce dextran branching.TaSBEs gene is the key gene of wheat resistant starch content.In this study,TaSBEs genes were identified at the whole genome level of wheat,and the basic characteristics of their family members were analyzed to promote their functional analysis.The relationship between haplotype of TaSBEIIa and amylose and amylopectin content was analyzed in order to provide reference for the improvement of amylose content in wheat.Meanwhile,CRISPR/Cas9 technology was used to double-target edit TaSBEIIa,a major gene for amyloid branching enzymes,in common wheat Fielder.The main research results are as follows:(1)Twelve TaSBEs genes were identified on chromosomes 2A,2B,2D,7A,7B and 7D.The analysis of gene expression patterns in 18 parts of commercial wheat variety Azhurnay during 10 growth stages,including filling stage,showed that TaSBE＿7A/7B/7D was only highly expressed in wheat seedlings and very low expressed in the remaining 9 growth stages.TaSBEIIa＿2A/2B/2D genes was highly expressed in 10 stages,and TaSBEIIb＿2A/2B/2D and TaSBEI＿7A/7B/7D genes were only highly expressed in grouting stage and dough stage.TaSBEs genes had tissue-specific expression and growth-specific expression.(2)In the natural population of wheat,18 polymorphic SNPs loci of TaSBEIIa＿2A gene and 130 polymorphic SNPs loci of TaSBEIIa＿2B gene were screened.All the 18 SNPs of TaSBEIIa＿2A were located in the intron region,which constituted three haplotypes.Among 130 SNPs mutation sites of TaSBEIIa＿2B gene,7 SNPS were antisense c SNPs,and the rest did not cause amino acid changes,constituting 7 haplotypes.The TaSBEIIa＿2D gene was very conserved and no SNP sites were found.(3)Molecular markers were used to identify the natural population haplotype in the laboratory,and the phenotypic difference analysis showed that the amylopectin content and amylopectin content of the three haplotypes of TaSBEIIa＿2A had no significant difference,and the amylopectin content of haplotype 5 and haplotype 1 of TaSBEIIa＿2B gene had significant difference.The amylose content was increased by 2.41% on average,and the haplotype 5 of TaSBEIIa＿2B gene could be regarded as an excellent haplotype with high amylose content.(4)Different mutant types of Fielder wheat were obtained by knocking out the TaSBEIIa gene using CRISPR/Cas9 technology,and most of the edited types were 20-58 bp large fragment deletion.35 of 68 reclaimed Fielder wheat plants were successfully transferred to the edited vector,and the vector conversion efficiency was 51.47%.And 33 wheat Fielders were edited with TaSBEIIa genes(94.29%).(5)For the TaSBEIIa alleles aa Bbdd,aabb Dd,Aabbdd and Aa Bb Dd edited wheat by Fielder of T0 generation,the activity of starch branch enzymes decreased significantly,the epidermis shrank significantly,and the shape of starch grains changed from round wild type to oblate and concave in the middle or even irregular.Alleles Aa BBDd and AABBDd showed no significant decrease in the activity of starch branch enzymes,and the epidermis and starch grain structure were similar to those of the wild type,indicating that TaSBEIIa genes were redundant.