Identification Of Eutrema Salsugineum NAC Gene Family And Analysis Of Response Of EsNAC044 Gene To Salt Stress

E.salsugineum is a cruciferous annual herb naturally distributed in extremely salinized soil.It is closely related to Arabidopsis thaliana.In recent years,it has become a rare salt for plant salt tolerance mechanism research and salt tolerance gene screening.Health model plants.Mining stress-resistance genes from E.salsugineum and analyzing its specific functions and roles in stress response will help us understand the high salt adaptation mechanism of halophytes.NAC transcription factors are involved in many important physiological processes of plants,such as plant growth and development,cell apoptosis,abiotic stress tolerance,formation of secondary cell walls,and endoplasmic reticulum stress.Under abiotic stress conditions,plants can combine NAC transcription factors with cis-acting elements to regulate the expression of downstream genes to improve plant tolerance to drought,salt stress and low temperature stress.At present,there are few studies on the NAC transcription factor of E.salsugineum.In this study,the E.salsugineum genome database was used to systematically analyze the ESNAC gene family combined with bioinformatics,and the expression pattern of the EsNAC gene in response to salt stress was analyzed based on the transcriptome data.On this basis,the EsNAC044 gene was selected,and physiological characteristics analysis,subcellular localization,transcription activation activity and salt tolerance preliminary analysis were carried out,which laid a certain theoretical foundation for the study of EsNAC family genes.The main findings are as follows:(1)120 NAC family members were identified in E.salsugineum,divided into 15 subfamilies,and renamed EsNAC001-120 according to the order of their positions on the chromosome.The NAC genes of the same subfamily have similar gene structure and conserved motifs,which strongly supports the reliability of our subfamily classification.The analysis of cis-acting elements shows that the NAC family genes of E.salsugineum play an important role in the growth and development process,response to adversity stress,and regulation of hormone signaling pathways.The collinearity analysis of gene family members confirmed that E.salsugineum is closely related to Arabidopsis,and repeated events played an important role in the evolution and expansion of the EsNAC gene family.(2)Using 300 mM NaCl to treat E.salsugineum,transcriptome data analysis showed that the expression levels of 55 NAC family genes in E.salsugineum are affected by salt stress.The results of real-time fluorescent quantitative PCR verified the reliability of the transcriptome analysis data.The expression levels of different EsNAC genes are inconsistent under salt stress,and may play different functions in response to salt stress.(3)The EsNAC044 gene was cloned from E.salsugineum.Bioinformatics analysis showed that its ORF sequence was 939 bp in length,encoding 311 amino acids,and its 15-139 amino acids contained a typical NAM domain.EsNAC044 is highly homologous to Arabidopsis ANAC019,ANA C055 and ANAC072.EsNAC044 contains A-E subdomain at the N-terminus,and the nuclear localization signal is located in subdomain D.The EsNAC044 protein is a nuclear-localized hydrophilic protein and does not have a transmembrane domain and signal peptide.The protein structure prediction results indicate that EsNAC044 may be a stress-related protein.Real-time fluorescence quantitative analysis showed that EsNAC044 gene is expressed in E.salsugineum roots,stems,leaves,flowers,and siliques,with the highest expression in flowers and the lowest expression in leaves.(4)The results of subcellular localization analysis show that EsNAC044 is located in the nucleus and has transcription activation activity,and the transcription activation domain is located at the C-terminus of the protein.(5)Using yeast in vitro expression system to analyze the salt tolerance of Saccharomyces cerevisiae strains transfected with EsNAC044 gene,the results showed that EsNAC044 gene may improve the tolerance of yeast strains to salt stress.(6)The EsNAC044 gene was constructed on the pGWB551 plant expression vector,and the recombinant plasmid was transformed into wild-type Arabidopsis through Agrobacterium-mediated transformation to obtain Arabidopsis plants overexpressing the EsNAC044 gene.Under 150 mmol/LNaCl,the roots of Arabidopsis overexpressing EsNAC044 gene were longer than those of wild-type.The Arabidopsis homologous gene ANAC055 mutant plant(SALK₀14331C)showed a clear NaCl inhibition phenotype compared with the wild type.