Identification, classification and expression analysis of NAC transcriptional factor genes in Malus sieversii during canker disease response

Abstract:【Objective】The ork aimed at identification and analysis of NAC gene family in order to ex-plore the potential canker-resistance gene resources in Malus sieversii, the ancestor of domestic apple.【Methods】The sequences of AtNACs were downloaded from the PlantTFDB v4.0 database to establish Hidden Markov Model (HMM) for identifying the MsNACs from the full-length transcriptome database of M. sieversii. MEME suite.11.2 was used to analyze the conserved motif with the maximum search value (7). Phylogenetic tree of MsNACs and AtNACs was generated by MEGA 6.0 with neighbor-joining (NJ) method. The physicochemical properties, membrane-bound domain and subcellular localization of MsNACs were analyzed by Protparam, TMHMM and WoLF PSORT software, seperately. The qRT-PCR was used to validate the MsNACs gene expression patterns under the infection of canker pathogen (Cytospora mali).【Results】165 MsNACs of M. sieversii were obtained, which differed from each other in molecular weight (MW), theoretical isoelectric point (IP) and hydrophobicity. The maxi-mum MW was 95.86 ku (859 aa) and the minimum was 6.77 ku (58 aa). These MsNACs were divided into 12 subgroups (I-XII) according to the phylogenetic analysis, and AtNACs and MsNACs were both included in each subgroup. However, the proportion of MsNACs and AtNACs in subgroups were dramatically different. The 113 of 165 MsNACs had integral NAC domain and the others had one or more subdomains missed. Conserved motifs analysis showed that NAC domains of MsNACs were composed of 7 conserved motifs. We identified 5 subdomains A (composed of motif 1), B (composed of motif 5), C (composed of motif 4 and 3), D (composed of motif 2 and 7), E (composed of motif 6). In addition, membrane-bound domain analysis showed that 18 of 165 MsNACs had the membrane bound domain, which belonged to subgroup III (1), VII (1), VIII (6), IX (9) and X (1). Subcellular location analysis indicated that most of the MsNACs were located in nucleus while the other MsNACs were located in different organelles such as chloroplast (MsNAC034), endoplasmic reticulum (MsNAC046) and cytoplasm (MsNAC025). Thirty-four MsNACs were significantly differentially expressed under the infection of the C. mali pathogen based on the RNA-seq data, and were predominantly clustered into 3 clades. The clade A MsNACs (6) showed increased expression levels at 1-2 dpi and were decreased at 5 dpi. The the clade B MsNACs (20) were highly expressed at 0 dpi and were down-regulated at 1-5 dpi. On the contrary, clade C MsNACs (8) were up-regulated in the late stage of infection (5 dpi). Furthermore, 12 MsNACs were selected to validate their expression patterns by qRT-PCR, and were consistent with RNA- seq profiles. On the 5th day post infection, MsNAC073 (homologous with AtNTL9,AT4G35580) were decreased by 6- fold, while MsNAC102 (homologous with AtSMB, AT1G79580) were increased by 140-fold compared with the control.【Conclusion】In general, the MsNACs played important roles during the response to canker infection in M. sieversii. The research could lay a founda- tion for further functional studies of the MsNACs, and provide a reference for screening disease-re- sponse NAC genes in other species.