油棕低温响应microRNA的克隆与功能分析

Oil palm (Elaeis guineensis) is an important oil plant in tropical region, while low temperature is one crucial factor limiting the growth region and the yield of oil palm. In previous research, we conducted small RNA sequencing of Nengyuan16 (a low temperature resistance cultivar) under cold stress treatment and identified 67 cold-responsive microRNAs(miRNAs). Based on the previous results we will conduct research in the following three aspects: 1) Temporal and spatial expression analysis of the identified 67 miRNA and screen for miRNA genes related with cold resistence; promoter cloning of these candidate miRNA genes and identification of cis-motifs which related with cold-response processes. 2) Predication of target genes of the miRNAs and analysis the cleavage sites of the target genes, determining the regulatory relationship between miRNAs and target genes. 3) Analysis of two more oil palm cultivars different in cold tolerance and explore the relationship between genetic diversity of target genes and miRNA regulatory efficiency. In this research, we aim to obtain miRNA gene resources related with cold resistance of oilpalm, and reveal the regulatory mechanism of miRNAs to their target genes in cold endurance process and provide theoretical basis for genetic improving cold tolerance of oil palm.

油棕是热带地区重要的油料作物，低温是限制其扩大种植区域和实现高产稳产的重要影响因素之一。前期对耐低温油棕"能源16号"进行低温处理和小RNA测序，已经获得了67个低温诱导或者抑制表达的microRNA（miRNA）。本研究将在此基础上，开展1）对候选miRNA进行时空表达分析与验证，分离miRNA基因启动子区域，寻找低温响应顺式元件，筛选油棕耐低温性相关miRNA；2）预测miRNA靶基因，分析miRNA对靶基因的剪切位点，确定miRNA与靶基因间的调控关系；3）增加两个低温敏感的油棕品种，探讨靶基因遗传多态性与miRNA对靶基因调控效率间的关系。本研究拟获得油棕耐低温性相关的miRNA基因资源，并解析在油棕耐低温过程中miRNA对靶基因的调控机制，为油棕耐低温的遗传改良提供理论支持。

油棕是是热带地区重要的油料作物，低温是限制其扩大种植区域和实现高产稳产的重要影响因素之一。本项目在实施过程中依据油棕冷处理和对照的小RNA数据，找到油棕基因组miRNA基因位点138个，并对miRNA上游1k区域进行上游启动元件和调控元件进行分析，其中有74个miRNA基因有启动元件和一些调控元件，进一步分析这些调控元件，发现与冷响应相关的调控元件有10个，存在于45个miRNA基因的启动子区。根据课题组已有的200份油棕种质资源的SNP和indel信息，对miRNA基因内部和上游启动区进行序列变异的分析，在25个miRNA的基因和启动子区找到64个SNP和12个indel位点。此外，找到靶基因的基因内部的1836个SNP和87个indel变异。通过高通量数据的miRNA表达量分析和文献报道的冷响应miRNA基因，筛选了88个的成熟miRNA开展RT-qPCR表达验证，找到冷处理后显著差异表达miRNA有27个。分析miRNA的靶基因中，通过表达验证找到与miRNA表达互为消长的属于NAC、ARF和ERF转录因子家族的基因。此外，我们分析了CBF介导的冷响应途径上的14个基因表达，发现CBF受冷胁迫上升表达，但其下游COR基因没有检测到表达，而egu-miR159、egu-miR164和egu-novel等miRNA表达模式与CBF一致，且在启动子区含CBF结合的C-repeat/DRE元件，可能受CBF调控。本项目的开展初步探索了油棕中miRNA在冷胁迫下的表达模式，以及对靶基因的降解作用，还有与CBF介导的冷响应途径的关系。