Whole genomic identification of NAC gene family and expression analysis under flooding stress and salt stress in Salix matsudana

【Objective】 The purpose of the study was to identify members of the NAC gene family in Salix matsudana and analyze their expression differences under flooding stress and salt stress，so as to provide theoretical basis for studying the mechanism of SmNAC family members involved in stress regulation and cultivating S. matsudana varieties with high tolerance to abiotic stress. 【Method】 Using the 4 S. matsudana germplasms（flooding-sensitive S. matsudana variety Yanliu No. 1，the flooding-tolerant S. matsudana variety Suliu 795，the salt-sensitive S. matsudana germplasm Yanjiang， and the salt-tolerant S. matsudana variety 9901）as experimental materials，the NAC gene family members within the whole genome of S. matsudana were identified through genomic data mining，and their protein physicochemical properties，conserved motifs，phylogenetic relationships，gene structures，chromosome localization and collinearity relationships were predicted and analyzed. Expression profiles of SmNAC gene family members under flooding stress and salt stress was conducted；genes that showed obvious response to both flooding stress and salt stress were selected to construct Arabidopsis thaliana overexpression vectors and obtain transgenic A. thaliana. The relative expression levels of these genes in wild-type and transgenic A. thaliana were detected by real-time fluorescence quantitative PCR，and the phenotypic differences between transgenic A. thaliana and wild-type A. thaliana under water flooding stress were analyzed，and the physiological indicators were measured. 【Result】 A total of 290 SmNAC gene family members were identified，named SmNAC001 to SmNAC290；the number of amino acids encoded by SmNAC family genes ranged from 105 to 710，with molecular weights ranging from 12.7 to 77.8 kD and theoretical isoelectric points（pI）ranging from 4.33 to 9.65. Most of the protein members were localized in the nucleus. Based on the phylogenetic evolutionary relationship between SmNAC family proteins and A. thaliana NAC proteins and the results of conserved motifs，the SmNAC family proteins were divided into 16 subfamilies. Most SmNAC family genes contained 3 exons，and SmNAC members within the same subfamily had similar motif composition and arrangement patterns，such as the NAP and OsNAC7 subfamilies. Most SmNAC family gene members promoters contained elements responsive to methyl jasmonate and hypoxia stress. In the SmNAC gene family，244 genes were randomly distributed on 38 chromosomes，among which there were 13 pairs of tandem duplication events. Transcriptome data showed that after flooding stress and salt stress，the expression levels of the ATAF subfamily（SmNAC007，SmNAC008，SmNAC009，SmNAC010，SmNAC011，and SmNAC012 genes）was up-regulated in the S. matsudana genplasms with different resistance. Real-time fluorescence quantitative PCR results showed that the relative expression level of the SmNAC007 gene in transgenic A. thaliana was greatly higher than that in the wild-type by 10 to 15 times；compared to wild-type A. thaliana，the total protein content in transgenic A. thaliana was significantly reduced（P<0.05）. 【Conclusion】 A total of 290 SmNAC gene family members are identified，among which 102 and 151 family members are differentially expressed under flooding stress and salt stress respectively；the SmNAC007 gene is preliminarily confirmed to play a positive regulatory role in the response to flooding stress.