Cloning And Functional Analysis Of RING-type E3 Ubiquitin Ligase Gene TaSDIR1 In Wheat

Wheat?Triticum aestivum L.?is one of the three major food crops in China,which plays a pivotal role in our food security.With the rapid development of society,the global climate has gradually become warmer,abiotic stresses such as drought,high temperature and salting seriously impact the wheat production of in the world.Therefore,excavating and using abiotic stress-tolerant genes has become an important way to improve wheat productivity under adverse conditions.E3 ubiquitin ligases play pivotal roles in plants signaling pathway.In this study,we get TaSDIR1?SALT-AND DROUGHT-INDUCED REALLY INTERESTING NEW GENE FINGER1?gene from a wheat cultivar Hanxuan 10,and we identified the function of TaSDIR1 gene through tissue expression analysis,analysis of expression patterns under different abiotic stresses and in vitro ubiquitination assay.Explore the relationship between haplotypes and agronomic traits by developing molecular markers,genetic mapping,and association analysis and provide favorable information for wheat breeding.There are the following results:1.The E3 ubiquitin ligase genes TaSDIR1-A and TaSDIR1-D were cloned from wheat genomes A and D.They have the same structure which contains eight exons and seven introns.The amino acid sequence identity was as high as 97.14%.TaSDIR1 contains two putative transmembrane domains and a highly conserved C3H2C3-type RING domain,belongs to the RING finger domain family of E3 ubiquitin ligase.2.TaSDIR1-A and TaSDIR1-D expressed in various tissues of wheat at heading stage.They have the highest expression in flag leaves.Both TaSDIR1-A and TaSDIR1-D responded to NaCl,ABA,PEG and 4? treatments.Among them,the response to PEG was the strongest,and the response to NaCl,ABA,and 4? was relatively weak.3.In vitro ubiquitination assay demonstrated that TaSDIR1 has E3 ubiquitin ligase activity.However,TaSDIR1^H244Y,the RING domain was disrupted,has lost the E3 ubiquitin ligase activity.It is proved that the complete RING domain give TaSDIR1 the E3 ubiquitin ligase activity.4.The different ploidy materials about A,B,and D genome donor species of wheat and a set of nullisomic-tetrasomic lines of Chinese Spring were used for chromosome location of the gene.TaSDIR1-A and TaSDIR1-D were located on chromosomes 4A and 4D,respectively.Furthermore,TaSDIR1-A was mapped on chromosome4A flanked by markers AX-111451343?1.60 cM?and Xwmc89?2.31cM?.5.The full length of TaSDIR1-A was 3.2 kb.One SNP?A/G?and one 2bp InDel?CC/--?were detected in the promoter region by detecting the polymorphism of TaSDIR1-A gene sequence in 32 diversity hexaploid wheat accessions.Based on the linkage between the two single-nucleotide variation,the dCAPS marker SNP-397 was developed to scan the wheat natural population.Two haplotypes Hap-4A-1 and Hap-4A-2 were identified in the population.The results of the association analysis showed that the two haplotypes were significantly associated with 1000-grain weight,plant height,length of peduncle and penultimate node,respectively.Hap-4A-2was an excellent haplotype for improving agronomic traits in wheat.6.The full length of TaSDIR1-D was 4.07 kb.Two SNPs were detected in the full-length TaSDIR1-D sequence by sequencing the gene in 32diversity hexaploid wheat accessions.There was one SNP?T/C?in the promoter region,and another in the fourth exon?G/A?,which led to amino acid Arg?CGC?change into His?CAC?.Based on the linkage between the two single-nucleotide variation,the dCAPS marker SNP-583 was developed to scan the wheat natural population and divided into two haplotypes Hap-4A-1 and Hap-4D-2.The results of the association analysis showed that the two haplotypes were significantly associated with 1000-grain weight,length of penultimate node,and spike length,respectively.Hap-4D-2 was an excellent haplotype for improving agronomic traits in wheat.