菜用大豆低温胁迫应答相关microRNA的鉴定及其调控机制研究

Low temperature stress in the early spring is one of the major environmental factors that limit vegetable soybean production. Plant microRNAs (miRNAs), emerging as a novel and powerful regulator of gene expression at the post-transcriptional level , have been shown to play critical role in plant stresses response. However, the regulatory roles of plant miRNAs in response to cold stress remain largely unknown. Based on the miRNA sequencing data of two small RNA libraries constructed form vegetable soybean with or without cold stress treatment we did previously, in this project, we are going to combine with bioinformatics and molecular biology technology to identify miRNAs in vegetable soybean in response to cold stress. Meanwhile, the miRNA targets will be identified through target gene prediction and degradome sequencing and further validated by RLM-5’RACE and ETPamir (epitope-tagged protein-based amiRNA). Our results will unravel the cold stress induced miRNAs and their targets in the whole genome-wide. Their expression timing and intense in different tissues of several vegetable soybean cultivars with vary low temperature tolerance under cold stress will be also measured. Furthermore, the biological function of some conserved vegetable soybean miRNAs in cold stress tolerance will be investigated by over-expression the miRNAs in Arabidopsis. The aim of our project is to reveal the miRNA-mediated regulatory networks involved in the vegetable soybean plants response to cold stress and also supply theoretical basis for breeding cold resistant varieties of vegetable soybean and other crops.

早春季节的低温寒潮是制约菜用大豆生产的主要环境因子之一。microRNA（miRNA）的发现为我们从转录后水平深入认识基因表达调控提供了新的视角和强有力的工具。miRNA在植物抗逆反应中发挥着重要作用。但国内外对miRNA介导的植物低温抗性及应答机制尚未明确。本项目拟在对低温胁迫菜用大豆miRNA组测序的基础上，鉴定低温胁迫响应miRNA；并通过生物信息学预测及降解组测序鉴定其靶基因，利用RLM-5’RACE和ETPamir对靶基因进行验证；通过实时荧光定量分析miRNA及靶基因在不同低温耐性材料中的时空表达特性；进一步构建部分保守miRNA的过表达载体转化拟南芥验证相关miRNA的功能。本项目试图阐明菜用大豆低温胁迫应答相关miRNA对靶基因的作用方式，探索miRNA在低温胁迫下的表达变化规律及功能，最终揭示miRNA介导的低温胁迫应答机制及调控网络，为菜用大豆等耐低温育种提供理论基础。

miRNA在植物逆境应答中发挥重要作用，本项目研究了miRNA介导的菜用大豆低温胁迫应答机制及调控网络。通过小RNA测序，在菜用大豆低温胁迫和对照材料中共鉴定到434个miRNA，归属于133个miRNA家族，并鉴定到3个新的miRNA。对miRNA表达量进行分析，共发现归属于32个miRNA家族的55个miRNA在低温胁迫处理后显著性差异表达。通过生物信息学方法结合降解组测序，对低温胁迫诱导表达miRNA的靶基因进行了鉴定，发现大多数为转录调控因子及抗性相关蛋白等。利用qRT-PCR研究相关miRNA及靶基因在菜用大豆低温胁迫下的表达情况，获得了低温胁迫应答关键miRNA，包括miR164a、miR4411、miR169e、miR156a和miR167f等，并明确了其作用靶基因。进一步通过RLM-5’RACE验证了gma-miR156a和gma-miR164a作用的靶基因为SBP、NAC。构建了gma-miR164a前体片段过表达载体转化拟南芥，验证其在低温胁迫应答中的功能。此外，研究发现多个长链非编码RNA（lncRNA）参与菜用大豆低温胁迫响应，鉴定到2个lncRNA作为内源竞争性靶基因（eTM）与gma-miR9725和gma-miR5031结合，进而间接调控靶基因表达。