玉米高温胁迫响应相关miRNA及其靶基因的鉴定与功能研究

MicroRNAs (miRNAs) play essential roles in plant growth, development and stress responses. Maize is one of the most important economic crops in China, and it is of great significance to improve maize yield, quality and agronomic traits under stress environments. It has been reported that miRNAs play roles in multiple abiotic stresses, such as high salinity, drought, and nitrogen or phosphate deficiency in maize. However, the roles of miRNAs in high temperature stress haven’t been well studied. High temperature leads to kernel abortion and seriously limits the maize yield, so it is urgent to study the mechanisms of high temperature tolerance in maize. This project aims to characterize and study the function of high temperature induced miRNAs and their target genes in maize. Small RNA-seq, mRNA-seq, degradome-seq and ribosome protected fragments-seq will be constructed, and the gene overexpression and gene silencing strategies will be taken as well to identify the high temperature induced miRNAs and their targets. The categories, expression profiles, functional mechanisms and the downstream regulatory network of these miRNAs and their targets will be analyzed as well. This study will extend our knowledge in the aspect of understanding the regulatory mechanism of maize miRNAs in response to high temperature stress, and accomplishment of this project will provide theoretical basis and novel strategies for trait improvement of maize under adversity stresses.

MiRNA在植物的生长发育和逆境胁迫响应过程中发挥着重要的调控作用。玉米作为国家重要的经济作物，提高和改良其在逆境胁迫下的产量品质和农艺性状意义重大。目前有关玉米miRNA在多种非生物胁迫，如高盐、干旱、低氮、低磷等中的作用已有相关报道，但高温胁迫响应相关miRNA的研究还很少。高温胁迫造成玉米籽粒败育，严重限制玉米产量，玉米耐受高温胁迫的机理亟待研究。本项目将对玉米高温胁迫下参与响应的miRNA及其靶基因进行鉴定和功能研究，通过构建小RNA文库、mRNA文库、降解组文库和核糖体保护序列文库，以及基因过表达和基因沉默等分子和遗传学手段，鉴定获得高温胁迫相关miRNA及其靶基因，并对其属性、表达模式、具体作用机制，以及胁迫下玉米miRNA下游基因的表达调控网络进行深入分析。本研究的开展将有助于理解和阐释玉米miRNA响应高温胁迫的调控机制，为玉米在逆境胁迫下的性状改良提供理论依据和新途径。

高温胁迫影响玉米的光合作用，导致玉米幼苗形态异常、籽粒败育，严重限制玉米的品质和产量。研究玉米的高温耐受机理对于提高玉米的胁迫耐受性和指导农业生产具有重要意义。本项目对重要经济作物玉米高温胁迫下参与响应的miRNA及其靶基因进行了系统的鉴定及功能研究。通过建立小RNA文库、转录组文库和降解组文库，建立了玉米营养生长和生殖生长时期不同组织在短时与长时高温胁迫下全基因组转录水平时空特异的表达图谱，鉴定获得一系列参与热胁迫响应的miRNA及其随从链miRNA*，包括一些未曾在其他物种中发现的新成员，同时也对这些高温胁迫响应miRNA的靶基因进行了预测、表达分析和功能注释，并对其下游基因的表达调控网络进行深入分析。另外，还发现大量24nt的siRNA在玉米的雄穗和根部显著降低，同时产生这些siRNA转座子附近基因的表达也呈现相同的下降趋势，表明这些转座子与其周围基因的表达具有一定的关联性；此外还发现一个来自于质体的19nt小RNA在高温胁迫下发生大量富集，该19nt小RNA是来自于一个三角状五肽重复（pentatricopeptide repeat，PPR）蛋白对叶绿体基因psbB的保护“印记”，表明高温胁迫下PPR蛋白可能通过调控19nt小RNA的丰度抵御或缓解高温胁迫带来的不利影响。项目同时也选取高温胁迫响应中的关键miRNA进行功能研究，通过构建串联短片段核酸靶标模拟技术（short tandem target mimic,STTM）抑制表达和CRISPR/CAS9基因编辑的方法，获得了miRNA表达发生变化的转基因植株，并对转化植株胁迫相关性状进行表型鉴定和分析。该研究对玉米高温胁迫响应全基因组转录水平进行了详细分析，揭示了一系列高温胁迫响应关键调控因子，为玉米耐热性的遗传改良提供靶点，为通过分子设计育种改良玉米耐热性提供了理论支持。