地中海富盐菌中非编码RNA的鉴定与分析

Identification of non-coding RNA in Haloferax mediterranei

摘要:

[目的]通过转录组高通量测序技术（即RNA-seq），结合生物信息学分析和分子生物学方法，在组学水平鉴定极端嗜盐菌中可能的非编码RNA（ncRNA）。[方法]将培养至对数中期的地中海富盐菌在不同盐浓度下处理30分钟，提取RNA，进行链特异的转录组测序和5'端区分的转录组测序，通过生物信息学分析在全基因组范围内鉴定ncRNA，预测其转录边界；然后通过Northern blot和环化RNA反转录聚合酶链式反应（CR-RT-PCR）对部分预测的ncRNA进行实验验证。[结果]比较两种RNA-seq技术在不同培养条件下的RNA测序结果和对转录单元的精细分析，共鉴定到105个高可信度的ncRNA，并发现4个在不同盐度下表达差异较大的ncRNA，通过Northern blot和CR-RT-PCR验证了incRNA1436和incRNA1903的表达情况、转录本、转录起始位点及终止位点等。[结论]首次在组学水平鉴定了地中海富盐菌中的ncRNA，不同盐浓度刺激下部分ncRNA的转录差异暗示其有可能参与地中海富盐菌对盐胁迫的适应，高可信度ncRNA的组学发现为今后全面开展嗜盐古菌ncRNA的功能机制研究提供了基础数据及重要的切入点。

关键词:地中海富盐菌;非编码RNA;盐度刺激;转录组测序

Abstract:

[Objective] To identify non-coding RNAs in Haloferax mediterranei through high-throughput RNA sequencing, bioinformatics analysis and molecular techniques.[Methods] After H. mediterranei cells under log phase of growth were treated with different salt concentrations for 30 minutes, total RNA was extracted for the following strand-specific RNA sequencing and differential RNA sequencing. These RNA-seq data were used to identify the genome-wide ncRNAs and to predict the 5' and 3'-ends of the transcripts by bioinformatics analysis. A few selected ncRNAs were further confirmed by Northern blotting and Circularized RNA reverse transcription-PCR analysis.[Results] We identified 105 highly credible ncRNAs. Expression of four ncRNAs showed difference in different salt concentrations. We confirmed the expression, length of transcripts, transcription start and termination sites of incRNA1436 and incRNA1903 by Northern blotting and CR-RT-PCR.[Conclusion] We identified the ncRNAs of H. mediterranei in a genome-wide scale, including identification of a few ncRNAs involved in the responses of H. mediterranei to different salt concentrations. Our results have provided fundamental data and novel insights for future study of the function of ncRNA in haloarchaea.

Key words:Haloferax mediterranei;non-coding RNA;salinity stimulation;RNA-sequencing