东乡野生稻抗旱性相关候选miRNA的验证及调控机制研究

Dongxiang wild rice (Oryza rufipogon Griff.) is a common wild rice located at 28°14’ N latitude in Dongxiang county, Jiangxi province, China, which is considered to be the northernmost region in the world where Oryza rufipogon is found. Our previous study revealed that Dongxiang wild rice can survive under extremely drought condition and carries very precious drought-resistant gene resources. Using transcriptome sequencing and small RNA sequencing technologies, the expression profiles of mRNA and miRNA were analyzed under drought condition, nine conserved and six novel miRNAs were identified as candidate miRNAs related with drought resistance, and all of them were confirmed by Stem-loop RT-PCR. On this basis, this project intends to validate the candidate miRNAs controlling drought resistance in Dongxiang wild rice by high-throughput sequencing technology combined with molecular biology methods, and analyze the expression patterns of miRNAs and their target genes. Parallel analysis of RNA ends (PARE) and rapid amplification of cDNA ends (RACE) approaches will be used to identify the miRNA-target RNA pairs and analyze the splice sites. By over expression and short tandem target mimic (STTM) approaches, we will validate the functions of 2-3 related miRNA, and clarify their molecular mechanisms in controlled drought resistance. This study can enrich current research results in the field of miRNA, and provide theoretical support and genetic resources for drought-resistant rice breeding.

东乡野生稻是目前发现的全球分布最北的普通野生稻。本课题组前期研究表明，东乡野生稻能在极其干旱的生态环境下生存，具有宝贵的抗旱基因资源。借助转录组测序和small RNA测序技术，我们对东乡野生稻干旱胁迫下mRNA和miRNA的表达情况进行了研究，遴选出9个保守和6个新的miRNAs作为调控抗旱性的重要候选miRNA，并使用Stem-loop RT-PCR法对结果进行了检验。本项目在此基础上，拟采用高通量测序和分子生物学技术相结合的方法，验证东乡野生稻抗旱性相关候选miRNA，分析miRNA与靶基因的表达特征；利用降解组测序技术和靶基因裂解实验鉴定靶基因，分析miRNA对靶基因的剪切方式；并通过过量表达和STTM抑制技术，深入研究2-3个miRNAs在干旱胁迫下的功能，探明它们调控抗旱性的分子机制。本项目将丰富现有miRNA领域的研究成果，并为水稻抗旱育种提供理论支撑和基因资源。

随着气候的全球性恶性变化，非生物胁迫因子已给水稻生产造成了严重的损失，其中干旱是范围最广、危害最严重的非生物影响因子之一。东乡野生稻是全球分布最北的野生稻。本课题组前期研究表明，东乡野生稻能在极其干旱的生态环境下生存，是改良水稻抗旱性的重要种质资源。越来越多的研究表明，microRNA（miRNA）在植物逆境应答中起着重要作用，与植物抗逆性密切相关。本项目通过高通量测序技术和分子生物学方法，共筛选鉴定了东乡野生稻抗旱性相关的已知miRNAs 505个，分别代表了215个家族成员，其中17个已知miRNAs表达量在干旱条件下显著下调，16个在干旱条件下显著上调；同时，筛选到138个新miRNAs，其中27个新miRNAs表达量在干旱条件下显著下调，40个在干旱条件下显著上调；此外，通过生物信息学方法对差异表达的miRNAs进行了靶基因预测，结果表明广泛的生物学途径参与了东乡野生稻干旱胁迫响应。最终遴选出9个保守和6个新的miRNAs作为调控抗旱性的重要候选miRNA。通过构建东乡野生稻抗旱性相关候选miRNAs过量表达载体，进行遗传转化，对转基因阳性株系进行了抗逆性分析，发现其中3个miRNAs转基因株系的抗旱能力与对照中花11相比显著提高，并利用转录组和降解组测序技术分析了其调控机制。本项目的顺利实施将为进一步阐明miRNAs特征、功能及种间进化的科学问题及东乡野生稻抗旱优异性状的育种应用奠定基础。