| 朱 虹 1，曾 俊 1,2，孔祥锦 1,2，彭 宽 1,2，朱孝扬 3，温玲蓉 1，屈红霞1，蒋跃明 1.香蕉 miR164-NAC 调控模块的鉴定与分析[J].广东农业科学,2023,50(9):1-15 |
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香蕉 miR164-NAC 调控模块的鉴定与分析 |
| Identification and Characterization of miR164-NAC Regulatory Modules in Banana |
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| DOI：10.16768/j.issn.1004-874X.2023.09.001 |
| 中文关键词: 香蕉 后熟 低温胁迫响应 miR164-NAC 调控模块 鉴定分析 |
| 英文关键词: banana ripening cold stress response miR164-NAC module identification |
| 基金项目:国家自然科学基金面上项目（31772371）；广东省自然科学基金面上项目（2021A1515011258）；中国科学院华南植物园青年人才团队项目（QNXM-04） |
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| 中文摘要: |
| 【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。 |
| 英文摘要: |
| 【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. |
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