香蕉miR164-NAC调控模块的鉴定与分析

Identification and Characterization of miR164-NAC Regulatory Modules in Banana

【目的】了解香蕉MIR164和NAC基因家族以及miR164-NAC调控模块在香蕉后熟和低温胁迫应答过程中扮演的角色,为其在香蕉品种改良和分子育种方面的研究提供理论基础。【方法】以‘巴西蕉’为试材,通过高通量测序结合生物信息学分析,利用miRBase、NCBI数据库以及Clustal、TBtools、MCScanX、iTOL等软件对香蕉miR164和NAC家族成员的染色体定位、结构、理化性质及系统发育关系等进行分析。通过降解组测序验证并结合转录组数据,明确了香蕉中多个miR164-NAC调控模块。分别设置后熟和低温实验,利用小分子RNA杂交和qRT-PCR分析miR164-NAC调控模块在香蕉后熟和冷害两种过程中的表达模式。【结果】在香蕉中共鉴定出6个miR164家族成员,其中4个位于编码基因内部、2个位于基因间区。系统进化分析发现,测序丰度较高的多条香蕉MIR164s基因前体均与番木瓜聚为一类,暗示香蕉MIR164基因家族的起源与双子叶植物更为接近。香蕉基因组共编码222个NACs成员,不均匀分布在所有11条染色体上。在香蕉NACs中共鉴定出134对同源基因,包括4对串联重复基因和130对区段复制重复基因,表明香蕉NACs基因进化的主要动力来源于区段复制事件。针对香蕉、拟南芥和水稻NACs蛋白比较系统发育的分析,将该家族划分为23个亚群,结合转录组数据证实香蕉NACs基因存在广泛的冗余性和表达特异性。理化分析结果表明,几乎所有香蕉NACs蛋白均为亲水性,且仅有不到15%属于稳定蛋白。验证了香蕉中miR164-NAC176/165调控模块,香蕉miR164的积累受乙烯诱导且随果实后熟逐渐增强,而该调控模块中受miR164负调控的MaNAC176/165基因在果实后熟过程中的表达量逐渐降低。冷害条件下,香蕉miR164同样被明显诱导,导致其靶向的MaNAC176和MaNAC165也出现不同程度的下调表达。【结论】MaNAC176和MaNAC165很可能是香蕉后熟的转录抑制子,而miR164通过负调控MaNAC176/165促进香蕉后熟。同时,这一模块也很可能是香蕉发生冷害的一个关键调控通路。确定了香蕉后熟和冷害响应的关键miR164-NAC候选模块,可为后续的克隆及功能分析奠定基础。

【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 segment-replicating 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.