School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, 141004, India.
Department cum National Centre for Human Genome Studies and Research, Punjab University, Chandigarh, India.
Interdiscip Sci. 2018 Dec;10(4):762-770. doi: 10.1007/s12539-017-0235-6. Epub 2017 May 22.
MicroRNAs (miRNAs) are a large family of 19-25 nucleotides, regulatory, non-coding RNA molecules that control gene expression by cleaving or inhibiting the translation of target gene transcripts in animals and plants. Despite the important functions of miRNAs related to regulation of plant growth and development processes, metabolism, and abiotic and biotic stresses, little is known about the disease-related miRNA. Here, we present a new pipeline for miRNA analysis using expressed sequence tags (ESTs)-based bioinformatics approach in Kinnow mandarin, a commercially important citrus fruit crop. For this, 56,041 raw EST sequences of Citrus reticulata Blanco were retrieved from EST database in NCBI through step-by-step filtering and processing methods and 130 miRNAs were predicted. Upon blast with Citrus sinensis transcriptome data, these produced potential targets related to disease resistance proteins, pectin lyase-like superfamily proteins, lateral organ boundaries (LOB) domain-containing proteins 11, and protein phosphatase 2C family proteins, protein kinases, dehydrogenases, and methyltransferases. Majority of the predicted miRNAs were of 22, 23, and 24 nucleotides in length. To validate these computationally predicted miRNA, poly(A)-tailed Reverse Transcription-PCR was applied to detect the expression of seven miRNA which showed disease-related potential targets, in citrus greening diseased leaf tissues in comparison to the healthy tissues of Kinnow mandarin. Our study provides information on regulatory roles of these potential miRNAs for the citrus greening disease development, miRNA targets, and would be helpful for future research of miRNA function in citrus.
微小 RNA（miRNAs）是一类由 19-25 个核苷酸组成的、调控性的非编码 RNA 分子，在动植物中通过切割或抑制靶基因转录本的翻译来控制基因表达。尽管 miRNAs 在调节植物生长发育过程、代谢以及非生物和生物胁迫方面具有重要功能，但与疾病相关的 miRNA 知之甚少。在这里，我们提出了一种使用基于表达序列标签（EST）的生物信息学方法分析柑橘 miRNA 的新途径。为此，我们从 NCBI 的 EST 数据库中检索了 56041 条柑橘属 Blanco 的原始 EST 序列，并通过逐步过滤和处理方法，预测了 130 个 miRNA。将这些 miRNA 与柑橘属转录组数据进行比对，产生了与抗病蛋白、果胶裂解酶样超家族蛋白、侧生器官边界（LOB）结构域包含蛋白 11 和蛋白磷酸酶 2C 家族蛋白、蛋白激酶、脱氢酶和甲基转移酶相关的潜在靶标。预测的 miRNA 大多为 22、23 和 24 个核苷酸长。为了验证这些计算预测的 miRNA，我们应用 Poly(A)-尾反转录 PCR 检测了七个 miRNA 在柑橘黄龙病叶片组织中的表达情况，这些 miRNA 与健康的 Kinnow 橘叶片组织相比，具有潜在的疾病相关靶标。我们的研究提供了这些潜在 miRNA 在柑橘黄龙病发展过程中调控作用的信息，miRNA 靶标，并将有助于柑橘 miRNA 功能的未来研究。
