大麦分蘖相关miRNA的鉴定与筛选.pdf

摘 要 大麦（Hordeum vulgare L ）是在全世界范围内广泛种植的重要粮食作物。分蘖是影 响大麦产量的重要要素之一，而miRNA 在分蘖发生发展过程中具有重要调控作用。本 课题以大麦分蘖突变体 GSHO1990 和野生型 Bowman 为实验材料，通过高通量测序和 生物信息学鉴定筛选了与大麦分蘖相关的 miRNA ，并开展了初步的功能验证工作。主 要的结果如下： 1. 在两个材料从二叶期到五叶一心期中一共鉴定到 1608 个miRNA 。去除数据冗余 后比对 miRBase 数据库中已知 miRNA 的信息鉴定得到在大麦分蘖节中有 505 个已知 miRNA ，再利用miRDeep 和psRobot 软件鉴定到207 个新的候选miRNA ； 2 ．以Log |FC| 1 和FDR 0.01 为筛选条件，在两个材料中筛选出15 个miRNA 在不同发育时期均显著差异表达；根据miRNA 作用的下游靶基因功能筛选到37 个影响 分蘖的候选miRNA ，其中chr4H:400959067..400959145:+ （miR400H ）预测的下游靶基 因和分蘖相关，表达趋势一致且是大麦的新miRNA ，确定为进一步功能验证的目标； 3. 以Log|FC|0.5 为条件筛选了13 个基因作为大麦分蘖miRNA 表达检测的候选内 参基因，经过软件geNorm 、Normfinder 和 Bestkeeper 综合分析并验证，确定miR5051 和miR5048a 是研究大麦分蘖相关miRNA 的最佳内参基因； 4. 通过构建miR400H 的过表达载体，进行水稻遗传转化并获得了23 株T0 代水稻 转基因苗；同时进行大麦遗传转化，一共侵染了100 个大麦幼胚，已经有愈伤组织分化 出绿芽。 关键词：大麦；分蘖；miRNA ；转基因；内参基因 I Abstract Barley (Hordeum vulgare L) is one of the most important cereal crops in the world. Tiller is one component of yield for barley and controlled by miRNA. In order to identify tiller-related miRNAs in barley, low-tillering mutant GSHO1990 and wild type Bowman in this study were used as experimental materials based on high-throughput sequencing and bioinformatics prediction. The main results were as follows: 1. From first leaf unfolded stage to five leaf unfolded stage, in total 1608 miRNAs were identified in two barley materials. And 505 known miRNAs were identified after removing redundancy and comparing to miRBase database. 207 novel miRNAs were identified under miRDeep and psRobot software. 2. To set Log|FC|1 and FDR0.01 as conditions, a total of 15 miRNAs significantly expressed at different developmental stages and the expression trend was unanimous. And acc