Identification and Characterization of miR164-NAC Regulatory Modules in Banana

Abstract

【Objective】This study aims to understand the roles of MIR164, NAC gene families and miR164-NAC regulatory modules in banana ripening and response to low temperature stress, so as to provide a theoretical basis for banana variety improvement and molecular breeding.【Method】'Brazil banana' was used as test material. Through high-throughput sequencing and bioinformatics analysis using miRBase, NCBI database and Clustal, TBtools, MCScanX and iTOL softwares, miR164 and NAC family members in banana were characterized, including their chromosomal location, structure, physical/ chemical properties, phylogenetic relationships, etc. Multiple miR164-NAC regulatory modules in bananas were identified through degradome sequencing and experimental validation combining transcriptome data. Next, the expression patterns of miR164-NAC regulatory modules during ripening and under cold stress were analyzed by small RNA northern blot and qRT-PCR.【Result】A total of six miR164 family members were identified in banana, of which four were located within the coding genes and two in the intergenic region. Phylogenetic analysis showed that several banana MIR164 precursors with high abundance were clustered together with papaya, suggesting that the origin of banana MIR164 gene family was closer to dicotyledonous plants. The banana genome encodes a total of 222 NAC members, unevenly distributed across all 11 chromosomes. A total of 134 homologous gene pairs were identified in these banana NACs, including 4 tandem repeats and 130 segment-replicating repeats, indicating that the main driving force of banana NAC genes evolution came from segmentreplicating events. Comparative phylogenetic analysis of all NAC proteins in banana, Arabidopsis thaliana and Oryza sativa divided this family into 23 subgroups, and transcriptome data revealed extensive redundancy and expression specificity of banana NAC genes. Physicochemical analysis showed that almost all banana NAC proteins were hydrophilic, and less than 15% were stable proteins. The miR164-NAC176/165 regulatory module in banana was verified, and the accumulation of miR164 in banana was induced by ethylene and gradually increased with fruit ripening, while the expression of MaNAC176/165 negatively regulated by miR164 in this module was gradually decreased during fruit ripening. Under the cold stress, miR164 was also obviously induced, resulting in the downregulation of its targets MaNAC176 and MaNAC165.【Conclusion】This study suggested that MaNAC176 and MaNAC165 may be transcriptional repressors of banana fruit ripening, while miR164 promotes ripening by negatively regulating MANAC176/165. This module may also be a key regulatory pathway of banana chilling injury. This study identified key miR164-NAC candidate modules in banana fruit ripening and cold stress response, which laid a foundation for subsequent gene cloning and functional analysis.

Keywords

• banana
• ripening
• cold stress response
• mir164-nac module
• identification