NAC (NAM, ATAF, and CUC) is a family of transcription factors unique to terrestrial plants, including 18 subfamilies, of which ATAF subfamily members are mainly involved in the response processes of biotic and abiotic stresses, such as salicylic acid (SA), methyl jasmonate acid (MeJA), abscisic acid (ABA), pathogenic bacteria, mechanical damage, low temperature, and sodium chloride (NaCl). The data were from the genomic database of Saccharum spontaneum and the cDNA library of a sugarcane cultivar ROC22. Firstly, the ATAF subfamily members in Saccharum were identified and analyzed for their protein multiple sequence alignment, phylogenetic tree construction, and promoter region cis-acting element prediction using comparative genomics methods and various bioinformatics methods. Secondly, one homologous gene of the ATAF subfamily SsNAC2, ScNAC2, was cloned from a prevalent sugarcane cultivar ROC22 in China. The qRT-PCR was used to detect the tissue-specific expression pattern and the relative expression levels of ScNAC2 gene under different exogenous stresses. Finally, the subcellular localization and the transactivation analysis of ScNAC2 protein were performed. The results showed that six members of the ATAF subfamily were identified with the open read reading frames between 889 bp and 1017 bp, relative molecular weights between 32.067 and 35.819 kD, the theoretical isoelectric points from 5.09 to 8.92, and the proteins of all members were predicted to localize on the nucleus. In addition, the Ka/Ks ratios of six gene pairs were all less than 1, indicating that purification selection played an important role during evolution. The amino acid sequence alignment indicated that all members of the ATAF subfamily contained the NAM conserved domains, consisting of I, II, III, IV, and V subdomains. Phylogenetic analysis revealed that the members from sugarcane, sorghum, maize, and rice, that belonged to Gramineae, were clustered together, indicating that they had a close evolutionary relationship. Forty members of the ATAF subfamily from Arabidopsis, rice, maize, and sorghum were divided into two groups (Group A and Group B), in which the subfamily members of maize had obvious gene expansion. Furthermore, the promoter regions of ATAF subfamily members all contained cis-acting elements that responded to stresses such as low temperature, drought, and hormones, and we thus speculated that they were involved in the response processes of a variety of biotic and abiotic stresses. Furthermore, the full-length cDNA sequence of the ScNAC2 gene (GenBank accession number: OL982539) was cloned from the sugarcane cultivar ROC22, with an open reading frame of 891 bp and encoding 296 amino acid residues. The similarity of amino acid sequence between ScNAC2 and SsNAC2 proteins both from ATAF subfamily Group B was 97.99%. The qRT-PCR showed that the ScNAC2 gene was constitutively expressed in different tissues of sugarcane, and its expression level in sugarcane leaves and stem epidermis was higher than that in stem piths, buds, and roots. Besides, the relative expression level of ScNAC2 gene was significantly down-regulated under SA and MeJA stresses, however, it showed an expression pattern from low to high and varied to significant levels under the stress of ABA, 4℃, and NaCl. Subcellular localization revealed that the ScNAC2-GFP fusion protein was localized in the cell nucleus of Nicotiana benthamiana leaves. Furthermore, the transactivation experiment showed that ScNAC2 protein did not have the transcriptional self-activation activity. The above results established the foundation for identifying the biological functions of sugarcane NAC-ATAF subfamily members in response to biotic and abiotic stresses and provided potential genetic resources for sugarcane resistance molecular breeding.
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