| Wheat was one of the major grain crops in China,and wheat quality was an important indicator of wheat market value.Arabinoxylan(AX)was formed by the oxidative cross-linking of feruloyl groups in the structure of Feruloyl Arabinoxylan(FAX),which affected the processing and nutritional quality of wheat products.Arabinoxylan feruloyl transferase(AFT)catalyzed the synthesis of FAX,and its content was a key factor in determining the structure and properties of wheat AX.Therefore,cloning the key enzyme gene(AFT)affecting FAX content and verifying its gene function was an important way to improve wheat processing quality.In this study,253 wheat varieties(lines)from Yellow and Huai River Valley Winter Wheat Region and Northern Winter Wheat Regions and 124 winter wheat varieties(lines)from Xinjiang were used as test materials to clone the TaBAHD genes of wheat chromosome 3 B and 3 D by homologous cloning and PCR validation,Finally,the functions of TaBAHD-A1,TaBAHD-B1 and TaBAHD-D1 genes were verified by gene knockout,and to develop and validate functional markers based on sequence variability.The main results were as follows:1.The full-length sequences of TaBAHD-B1 and TaBAHD-D1 were 1450 bp and 1469 bp,respectively,and both contained a 1266 bp complete open reading frame(ORF),two exons and one intron,with the splice site structure of the intron conforming to the typical GT-AG type.The TaBAHD-B1 gene of different materials had one SNP site at 1425 bp,and the similarity between the two allelic variants was 98.08%,which could encode 422 amino acid residues with a predicted molecular weight of 45.1 k Da.TaBAHD-D1a and TaBAHD-D1b with 99.73%similarity between the sequences of the allelic variants had four single nucleotide polymorphic sites with a 20 bp 5'untraslated region(UTR)and a 44 bp 3'UTR.The TaBAHD-A1 and TaBAHD-B1 and TaBAHD-D1 genes had ten amino acid nonsynonymous mutations,and the same structural domain proved to be an important component of the catalytic mechanism of acetyltransferase.A dominant complementary pair of markers AFT 97/AFT 85 was developed based on the allelic variant sequence of TaBAHD-B1 gene.AFT 97 amplified a 539 bp fragment in material with TaBAHD-B1a type,which was associated with high FAX content.The difference in FAX content between genotypes in materials from the Yellow and Huai River Valley Winter Wheat Region reached a significant level(P<0.05).Two complementary dominant markers,AFT 19 and AFT 27,were developed based on the19 bp SNP of TaBAHD-D1 gene.There was 908 bp fragment of AFT 27 was amplified in TaBAHD-D1a material,which was associated with high FAX content.Therefore,the developed functional markers were correlated with FAX content and could be effectively used for FAX content genetic improvement.The TaBAHD-A1a/TaBAHD-B1a/TaBAHD-D1a genotype combination was found in the Northern Winter Wheat Rregion,the Yellow and Huaihua Winter Wheat Region and the overall material(P<0.05).Based on the above results,the functional role of TaBAHD-B1 gene on the catalytic synthesis of FAX content was presumed to be small,which was basically consistent with the results of the preliminary linkage analysis and association analysis.2.The expression of TaBAHD-A1,TaBAHD-B1 and TaBAHD-D1 genes in 12 wheat ears seeds with genotypes matching the phenotypic values at 7 d,14 d,21 d,28 d and 35 d after flowering were statistically analyzed by real-time fluorescence quantitative PCR(Quanti-ficational,PCR).The results showed that TaBAHD-A1,TaBAHD-B1 and TaBAHD-D1 genes all showed the highest expression at 21 d,and were more similar to other 7 d,14 d and 21 d genes.The relative expressions of TaBAHD-A1,TaBAHD-B1 and TaBAHD-D1 genes were significantly different from those of other 7 d,14 d,28 d and 35 d periods(p<0.05),which was consistent with the performance of wheat seeds in the dough stage.The clustering heat map also showed the same results,all the materials 12 were divided into two categories,category I for high FAX content varieties and category II for low FAX content varieties.The relative expression of TaBAHD-A1a was about 1.3 times higher than that of TaBAHD-B1a allelic,two times the relative expression of TaBAHD-D1a allelic variant,and five times the relative expression of TaBAHD-B1b allelic variant.The relative expression of the TaBAHD-B1b allele was 5-fold.Nongda 212(TaBAHD-A1a/TaBAHD-B1a/TaBAHD-D1a)and Xiaoyan 54(TaBAHD-A1a/TaBAHD-B1a/TaBAHD-D1b)were the high FAX content materials of the superior allelic variant combination type,which were different from Huaimai 18(TaBAHD-A1b/TaBAHD-B1b/TaBAHD-D1a).Jimai 21(TaBAHD-A1b/TaBAHD-B1b/TaBAHD-D1b),and Shanxi 354(TaBAHD-A1b/TaBAHD-B1b/TaBAHD-D1a)with low FAX content at different developmental periods.The differences in expression were significant(p<0.05)and the relative expression was consistent with the phenotypic characteristics of FAX content in the previous study,indicating that the TaBAHD-A1,TaBAHD-B1b and TaBAHD-D1 genes of chromosome group 3 may be involved in catalyzing the cross-linking reaction of FAX content during the development of synthetic wheat seeds,and there were differences in the degree of involvement of different genotypes in the regulatory role,and validation of TaBAHD gene function at the transcriptional level.3.The p ET-28a(+)-TaBAHD-A1,p ET-28a(+)-TaBAHD-B1,p ET-28a(+)-TaBAHD-D1prokaryotic expression vectors were constructed by double digestion,and the whole proteins were incubated for 6 h at 37?and 215 rpm with IPTG at a final concentration of 1 m M.The results of SDS-PAGE gel electrophoresis were consistent with the predicted results,and the TaBAHD protein was successfully induced.Further optimization of the recombinant protein expression conditions showed that p ET-28a(+)-TaBAHD were all expressed at 37?,while p ET-28a(+)-TaBAHD-A1 and p ET-28a(+)-TaBAHD-D1 and p ET-28a(+)-TaBAHD-B1 were expressed at 1.0 m M and 1.5 m M,respectively,and p ET-28a(+)-TaBAHD-D1 were expressed under optimal conditions at 4 h and 8 h,respectively.4.The intermediate vector pMETa U 6.1 was used as the template for the double g RNA gene editing vector construction.The first sequencing results showed that the mutation types were mainly Bi and He,mainly in the PAM recognition sequence of TaBAHD-222 target upstream 3 bp with 4 bp,5 bp deletion mutation and downstream 2 bp single base substitution.In the PAM recognition sequence of TaBAHD-322target upstream 4 bp with 2 bp deletion mutation and downstream 2 bp single base substitution.The seed FAX content of plants with simultaneous gene editing of three chromosomal TaBAHD genes was 3.57×10-5mol/L,which was lower than that of the recipient Fielder variety(4.07×10-5mol/L).Preliminary analysis verified that the TaBAHD gene may be correlated with endosperm cell wall FAX content. |
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