Division of Plant Biology, Bose Institute, P 1/12 CIT Scheme VII M, Kolkata, 700054, India; Ramakrishna Mission Vivekananda Centenary College, Rahara, Kolkata, 7000118, India.
Division of Plant Biology, Bose Institute, P 1/12 CIT Scheme VII M, Kolkata, 700054, India; Laboratorio de Micología y Biotecnología, Universidad Nacional Agraria La Molina, Av. La Molina s/n, Lima, 12, Peru.
Plant Sci. 2017 Apr;257:96-105. doi: 10.1016/j.plantsci.2017.01.016. Epub 2017 Jan 31.
MicroRNAs (miRNAs) are 20-24 nucleotides long non-coding RNAs known to play important regulatory roles during biotic and abiotic stresses by controlling gene expression. Blackgram (Vigna mungo), an economically important grain legume is highly susceptible to pathogenic begomovirus Mungbean Yellow Mosaic India Virus (MYMIV) and resulting in high yield loss. In this study two different leaf-small-RNA libraries were prepared from the pooled RNA at three different time points of resistant V. mungo inbred line VM84 inoculated either with viruliferous or non-viruliferous whiteflies carrying MYMIV and performed high-throughput Illumina sequencing. Sequencing followed by bioinformatics analysis of the small RNA reads indicated that the expression patterns of most of the known and novel miRNAs were altered in resistant line over mock-inoculated sample during the plant virus incompatible interaction. Highly altered miRNAs belong to the families of miR156, miR159, miR160, miR166, miR398, miR1511, miR1514, miR2118 and novel vmu-miRn7, vmu-miRn8, vmu-miRn13 and vmu-miRn14. These results were validated using qPCR, and most of the miRNAs showed similar pattern of expression like that of Illumina reads. The expression patterns of some selected known and novel miRNAs were also compared between the infected MYMIV-resistant and -susceptible genotypes and most of these were modulated after MYMIV-inoculation. Target transcripts like NB-LRR, NAC, MYB, Zinc finger, CCAAT-box transcription factor, fructose 2-6 bisphosphate, HDZIP protein that confers immune response were predicted as targets amongst identified miRNAs using psRNATarget server. Some selected target transcripts including NB-LRR, ARF, SOD, SPB, Basic blue copper protein were validated and their differential expression were demonstrated between MYMIV-resistant and -susceptible V. mungo by qPCR data analyses. In the present study we have identified miRNAs that implicate in the regulation of MYMIV-induced stress response in V. mungo; and generated genomic resources for a non-model legume with the aid of bioinformatics tools supplemented by experimental validation.
MicroRNAs (miRNAs) 是长度为 20-24 个核苷酸的非编码 RNA，已知它们通过控制基因表达在生物和非生物胁迫中发挥重要的调节作用。黑绿豆（Vigna mungo）是一种经济上重要的粮食豆类作物，对致病性的单番茄黄化曲叶病毒（MYMIV）高度敏感，导致产量损失很大。在这项研究中，从接种了携带 MYMIV 的有传染性和非传染性粉虱的抗性 V. mungo 自交系 VM84 的三个不同时间点的混合 RNA 中制备了两个不同的叶片小 RNA 文库，并进行了高通量 Illumina 测序。对小 RNA 读数进行测序和生物信息学分析表明，在植物病毒不相容互作中，抗性系相对于模拟接种样本中大多数已知和新的 miRNA 的表达模式发生了改变。高度改变的 miRNA 属于 miR156、miR159、miR160、miR166、miR398、miR1511、miR1514、miR2118 和新的 vmu-miRn7、vmu-miRn8、vmu-miRn13 和 vmu-miRn14 家族。使用 qPCR 对这些结果进行了验证，大多数 miRNA 的表达模式与 Illumina 读数相似。在感染了 MYMIV 的抗性和敏感性基因型之间，还比较了一些选定的已知和新的 miRNA 的表达模式，其中大多数在 MYMIV 接种后发生了调节。使用 psRNATarget 服务器，预测了 NB-LRR、NAC、MYB、锌指、CCAAT 盒转录因子、果糖 2-6 二磷酸、赋予免疫反应的 HDZIP 蛋白等靶转录物作为鉴定 miRNA 的靶标。通过 qPCR 数据分析，验证了一些选定的靶转录物，包括 NB-LRR、ARF、SOD、SPB、碱性蓝铜蛋白，并证明了它们在 MYMIV 抗性和敏感性 V. mungo 之间的差异表达。在本研究中，我们鉴定了 miRNA 参与了 V. mungo 中 MYMIV 诱导的应激反应的调节，并利用生物信息学工具和实验验证生成了非模式豆科植物的基因组资源。
