火龙果抗旱相关miRNAs的挖掘及其调控机制

Dragonfruit (Hylocereus undatus) had been marked as the firstly supported fruit industry during the “13th five-year-plan” scheme of Guizhou Province. Our previous work had cloned numerous genes which involve in many metabolism pathways and implicate in the drought tolerance in this fruit. In other species, these genes are the target genes of miRNAs, and the corresponding metabolism pathways were regulated by many miRNAs. Also, small RNA library was constructed using the in vitro plant subjected to drought stress，then Illumina deep sequencing technology was used for genome-wide identification of miRNAs in response to drought stress. To further uncover the drought-tolerance-related miRNAs, as well as to elucidate their regulation mechanism, consequently, huge number of miRNAs and their target genes, which possibly involve in drought tolerance, will be unraveled by degradome and bioinformatics analysis. Based on the expression profiles of miRNAs and their target genes in response to drought stress, the drought-tolerance-related miRNAs and their regulation network may be uncovered in combination with the important drought-tolerance-related genes and their metabolism pathways as obtained from our previous work. Furthermore, function of the candidate miRNAs will be further identified by genetic transformation. The ongoing work will facilitate the better understanding of molecular mechanism underling the high tolerance to drought stress of the elite dragonfruit genotype.

火龙果（Hylocereus undatus）是贵州“十三五”期间第一支持的精品水果产业。前期工作发掘的多个抗旱相关基因是miRNAs的靶基因，相关代谢途径也受很多miRNAs调控；通过干旱诱导的小分子RNAs文库深度测序，大规模地挖掘了miRNAs；结合miRNAs降解组测序，筛选出了可能的抗旱相关miRNAs。本项目拟在此基础上，进一步通过生物信息学方法，分析miRNAs的靶基因作用位点，结合获得的抗旱相关基因，挖掘抗旱相关候选miRNAs；分析干旱胁迫下miRNAs与靶基因的时空表达，特别是结合重要抗旱相关基因及其代谢途径进行分析，揭示miRNAs-靶基因的调控关系；通过遗传转化，对筛选出的miRNAs进行功能鉴定。最终确认火龙果抗旱相关miRNAs，并揭示其对干旱胁迫的调控机制。项目的实施，对深入解析火龙果抗旱的分子机理具有重要理论意义，也将为深刻理解植物抗旱的分子机制提供新信息。

火龙果是贵州省内主栽果树之一，具有较好的经济价值。本项目探讨了miRNAs调控火龙果抗旱的分子机制，主要结果如下：结合生物信息学和分子生物学方法，注释到36个miRNAs基因，其中33个来自21个已知miRNAs家族，3个为火龙果特有miRNAs家族，保守分析发现多数已知家族起源较早，个别家族具有系列特异性，与其他植物相同，火龙果miRNAs首位碱基主要为U，长度集中在21 nt；定量结果显示共30个miRNAs的表达发生了显著变化，其中miR156a-5p、miR159-3p、miR160-3p、miR162-3p、miR164a-5p、miR168-5p、miR171a-3p、miR393-5p、miR396b-5p、miR398-3p等14个miRNAs表达显著上调，而miR166c-3p、miR171b-3p、miR319b.1-3p、miR408a-3p、miR482-3p、miR535a-5p、miRN01-5p、miRN05-3p、miRN10-5p等15个显著下调表达，miR7972-3p表现为先上调然后下调；联合使用psRNATarget、TargetFinder和GSTAr三种算法，对火龙果miRNAs在Unigene上的结合位点进行预测，分别预测到337、1,064和50,119个miRNA结合位点，其中293个结合位点同时被三种算法所预测，降解组数据显示，32个miRNAs对48个被注释Unigenes上的50结合位点进行了显著性剪切（P≤0.05），其中11个剪切事件被RLM-5-RACE所证实，从编码的蛋白类型来看，这些靶基因多为转录因子（45.83%）。靶向关系保守性进行分析显示，多数靶向关系在植物界具有极高的保守性；克隆了miR396b-5p的靶基因HpGRF6的全长，定量结果显示，miR396和GRF在包括干旱、高温、低温、高盐等多种逆境下均显著差异表达，说明miR396-GRF靶向关系作用于火龙果对多种非生物逆境的应答；构建了首个植物isomiR数据库及植物逆境响应isomiR数据库。研究结果为全面认识火龙果抗旱的分子机理提供了新依据。项目发表相关论文10篇，其中SCI收录7篇；受理发明专利1件；资助研究生6名，其中博士1名，硕士5名；有3人职称得到提升。