蒙古冰草干旱胁迫相关microRNA及其靶基因鉴定与调控机制研究

Agropyron mongolicum is diploid perennial grasses of Gramineae Triticeae. Due to stronger drought resistance, it is a dominant species of desertification grassland, and has important value for feed, ecological and breeding utilization, as well as have valuable genetic resources for the genetic improvement of wheat crops. microRNA (miRNA) are one class of noncoding RNAs and ubiquitous in plants. As one of the key regulators for mRNA expression, its have been involved in response to a wide variety of stresses including drought, salinity, chilling temperature et al. However, the genetic regulatory networks and signaling pathways of Agropyron mongolicum involved in drought remain to be elucidated. Based on the previous study, miRNA and their targets of A. Mongolicum in response to drought stress are experimentally identified via degradome and small RNA sequencing, and the expression patterns between miRNA and its target genes were analyzed. Based on the profiles of differentially expressed mRNA, miRNA and targets, an co-expression regulatory network of miRNA-mRNA are constructed using A. mongolicum roots, stems and leaves subjected to different drought stress treatment. Some characteristics of the miRNA-mRNA mediated regulatory network are analyzed, and the regulatory relationship between the drought-responsive hub genes and their miRNAs are analyzed. The expression vectors of one or two hub genes are constructed and transformed into Arabidopsis thaliana, and selected the transgenic plants to clarify the function and regulatory mechanism of drought-responsive microRNA and hub genes in A. mongolicum. These results would be provide basis for the development and utilization of drought resistant genes from A. Mongolicum.

蒙古冰草（Agropyron mongolicum）属禾本科小麦族二倍体多年生牧草，是荒漠草原上抗旱性强的优势草种，蕴含麦类作物遗传改良的宝贵基因资源，具有重要的饲用、生态和育种利用价值。microRNA（miRNA）是植物体内普遍存在的非编码RNA，参与调控干旱、盐害、寒冷等多种逆境胁迫响应，是mRNA表达的关键调控因子之一，但蒙古冰草抗旱相关miRNA调控机制尚待研究。本项目在前期研究基础上，基于小RNA和降解组测序技术，深度挖掘鉴定蒙古冰草抗旱相关miRNA及其靶基因，并分析miRNA及其靶基因表达模式；同时利用转录组-小RNA-降解组联合分析，构建miRNA-mRNA共表达调控网络，分析枢纽基因及其miRNA间调控关系，并对1～2个抗旱相关枢纽基因进行功能验证，以阐释蒙古冰草miRNA及其重要靶基因干旱应答中的调控机制，为蒙古冰草抗逆性研究及优异基因的开发利用提供理论基础。

蒙古冰草（Agropyron mongolicum）是生活在干旱半干旱沙漠地区的一种极耐旱优良禾本科牧草，但其抗旱调控机制尚不清晰。本研究利用转录组-小RNA-降解组联合分析和WGCNA技术，获得干旱胁迫下蒙古冰草差异表达基因及miRNA-抗旱枢纽基因调控网络。通过高通量测序共鉴定出41792个unigenes及1104个miRNAs；通过miRNA、转录组和降解组联合分析，发现干旱胁迫下差异表达负调控的miRNA有99个，共调控1476个靶基因，其中有8个差异表达负调控的miRNA是蒙古冰草特有的，对应调控的靶基因有36个。从全部unigene构建的39个模块中鉴定出4个抗旱相关模块并找到了5个miRNA—抗旱枢纽靶基因对，抗旱枢纽靶基因通过参与油菜素类固醇介导的信号通路(osa-miR444a-3p.2—MADS47）、钠、钾离子运输交换(bdi-miR408-5p_1ss19TA—CCX1、氧化还原过程(ata-miR169a-3p—PAO2）、水解酶活性调控(tae-miR9774_L-2R-1_1ss11GT—carC）、对缺水、对脱落酸的反应及脱落酸激活的信号通路(bdi-MIR528-p3_2ss15TG20CA—NST1-like）等功能调控干旱胁迫。深入研究抗旱枢纽基因的调控网络，发现与其互作的基因，如BPM2（TRINITY_DN19691_c0_g7）、Os06g0107700（TRINITY_DN22022_c0_g3）、CLPC1（TRINITY_DN21467_c0_g3）和XB3（TRINITY_DN22959_c0_g1）等，主要对缺水、氧化还原、植物激素、蛋白泛素化等功能和反应起调控，通过枢纽靶基因的核心调控及互作基因的响应，形成蒙古冰草体内应对干旱胁迫的复杂调控网络。随后，对其中的抗旱hub基因MADS转录因子基因进行了基因克隆、亚细胞定位、过表达载体构建和遗传转化水稻，获得了过表达转基因水稻T1代株系，并验证蒙古冰草MADS转录因子基因参与干旱应答中的功能。这些结果为进一步研究蒙古冰草抗旱功能提供候选基因，同时推动了禾本科作物抗旱基因资源的开发利用。