利用Solexa测序技术鉴定甜橙miRNAs

Novel and conserved MiRNAs identification from Citrus sinensis by solexa sequencing

【目的】预测鉴定甜橙miRNA及其作用机制和功能,为深入研究挖掘甜橙miRNA及其抗病相关miRNA的作用等提供翔实的基因信息。【方法】利用高通量测序技术、相关的生物信息学软件和数据库,构建甜橙小RNA文库,然后以甜橙基因组为参考基因组,进行甜橙miRNA鉴定和靶基因预测;利用GO和KEGG数据库对靶基因进行功能分析。【结果】成功构建了甜橙小RNA文库:获得12 617 944条小RNA序列分属于3 065 625种小RNA序列,获得序列中24 nt长的序列最多,占到序列的49.3%。鉴定出2 199 561条miRNA,其中已知miRNA638 722条,分属52种miRNA家族,候选miRNA1 560 839条,分属204种miRNA。已知的52种保守miRNA基因预测出927个靶基因位点,候选miRNA中有154种预测出1 777个靶基因位点。GO数据库对靶基因分类显示,甜橙miRNA调控的靶基因主要参与多细胞互作、转录本调控和细胞组成等生物学途经和分子功能方面。KEGG数据库中病原物与寄主互作亚库分析结果显示,靶基因主要与抗性蛋白、转录因子和抗病酶等关联。【结论】预测鉴定出一批甜橙miRNA及其调控的靶基因,为进一步研究提供了参考信息。

[Objective]Finding out the Citrus sinensis miRNAs and predicting their mechanism of action and function will provide a lot of information for developing new varieties of C. sinensis with disease resis- tance and studying further plant-pathogen interaction. The interaction mechanism between C. sinensis and pathogens will lay a solid foundation for the further study of the miRNA. [Method]Small RNA sequences library was obtained by high-throughout sequencing technology. With respect to C. sinensis reference genome, miRNA identification and prediction of miRNAs target genes were carried out by bioinformatics software and databases. The target genes of miRNA were classified by search against the GO and the KEGG databases. [Results]12617944 small RNA sequences were acquired accounting for 99.32% of the total sequences and 3065625 were unique sequences. The 24 nt length small RNA is the major RNAs in the total sequences, accounting for 49.03% of the total sequences. A total of 2199561 miRNA sequences were obtained including 638722 conserved and 21030839 novel miRNA sequences. 1777 target genes of unique novel miRNAs and 927 target genes of 52 unique conserved miRNAs were found. Based on Gene Ontology database, the target unigenes may be involved in the multicellular organismal process and the nucleic acid binding transcription factor activity molecular function. The target genes of miRNA are asso-ciated with transcription factors, disease resistance proteins and receptor kinase on the mapping of plant- pathogen intraction map in KEGG database, etc. [Conclusion]A number of C. sinensis miRNAs and their target genes were found in this study. The results will facilitate the further study.