Genome-wide identification and analysis of NAC gene family in walnut (Juglans regia L.)

Abstract:【Objective】Flowering is an important way for plants to propagate progeny, and the normal development of flower organs plays an important role in floral formation. NAC transcription factors are involved in many processes of plant growth and development, and also regulate the development of flower organs. The regulation of floral organ development by NAC has been identified in other plants, but the regulation in the flowering process of walnut is still unclear. Exploring NAC gene family in walnut can provide reference for further study on the mechanism of walnut flower formation.【Methods】Walnut variety Wen 185 was collected in Southern Xinjiang. The female and male flower buds were col- lected at the undifferentiated stage of the female flower bud morphology (FB-1), the initial stage of mor- phological differentiation (FB-2) and the pistil primordium differentiation stage (FB-3), and the tran- scriptome sequencings were performed. The candidate NAC members in walnut were obtained using HMMER software, NAC domains of candidate members were identified by Pfam and SMART, and- NAC transcription factor members in walnut were finally obtained. The physicochemical properties and subcellular localization of JrNAC members were analyzed by online software. Phylogenetic analysis was performed using MEGA6, Bootstrap was set to 1000, based on the neighbor-joining (NJ) method, and the default values. The phylogenetic tree, gene structure and conserved motif of walnut NAC genes  were analyzed by TBtools. Chromosome mapping, gene replication and collinearity analysis were also performed on TBtools. The NAC proteins of walnut and Arabidopsis thaliana were compared using BLASTP. Then, NAC proteins of Arabidopsis thaliana with high homology were selected, and then the online software String was used to analyze the NAC proteins regulatory network of walnut. Finally, heat maps of JrNAC gene expression patterns in the male and female flower buds of walnut at different stages were drawn and analyzed.【Results】Among the 121 JrNAC members identified on 16 chromo- somes in walnut, most of them were hydrophilic proteins with an average molecular weight of 41.138 kDa. The subcellular localization showed that the NAC transcription factors in walnut were localized in the nucleus, chloroplast, cytoplasm, peroxisome, Golgi body and mitochondria, respectively. Among them, 86 JrNAC proteins were localized in the nucleus. The NAC members of walnut were divided into 15 sub-families, the largest one was OSNAC7 subfamily, and only one JrNAC member in the ATAF sub- family, but no member of ANAC001 subfamily was identified. The number of introns and exons of theJrNAC gene ranged from 1 to 7 and from 1 to 8, respectively. Most JrNAC members contained three ex- ons. Ten conserved motifs were identified in walnut. Most JrNAC members contained seven conserved motifs, and some subgroups contained unique motifs . As the motif 10 exist only in the subgroup IV, the motif 8 existed in the subgroup XVI and belonged to the subfamily ONAC003, but this subgroup did not contain motif 4 and 1. Synteny analysis showed that four JrNAC genes in walnut were collinear with the other three plants, and the number of homologous genes between walnut and other two woody plants was higher than that between walnut and Arabidopsis thaliana. Gene duplication showed that a total of 26 JrNAC gene duplication events were identified in walnut genome. Notably, both the Jr- NAC44 and JrNAC108 were identified in both analyses. The gene expression pattern showed that the 7JrNAC genes (JrNAC2, JrNAC38, JrNAC42, JrNAC66, JrNAC25, JrNAC8 and JrNAC115) were highly expressed in both the female and male flower buds, the gene expression level of 6 genes was higher in the initial stage of morphological differentiation of female flower buds than in the other two stages, while only 3 genes (JrNAC2, JrNAC38 and JrNAC42) showed similar gene expression characteristics in the male flower buds. The JrNAC2 belonged to the OSNAC7 subfamily, and its expression was obvious- ly up-regulated in the initial stage of morphological differentiation of female flower buds, which was speculated to be related to flower bud differentiation. The high expressions of the JrNAC8 and Jr- NAC115 at MB-1 stage were speculated to be related to the development of anther or anther sac. In addi- tion, the JrNAC97 was highly expressed in pistil primordium formation stage, and it was also highly ex- pressed in the male flower bud at MB-1 and MB-2 stages, suggesting that the JrNAC97 may played a role in pistil or stamen formation and development process. In the study of the protein interaction net- work, a total of 9 JrNAC proteins (JrNAC47, JrNAC111, JrNAC91, JrNAC120, JrNAC59, JrNAC16, Jr- NAC68, JrNAC2 and JrNAC53) were obtained, which played a key role in the network. Meanwhile, there were interaction relationships between NAC and other transcription factors.【Conclusion】In our study, the NAC family members of walnut were identified, and the NAC genes of walnut were relatively conservative, but it still exerted the function of diversity. The JrNAC2, JrNAC8, JrNAC115 and Jr- NAC97 might be the key genes in the development of walnut flower organs. This study would provide a reference for further study on walnut NAC gene family.