Capturing the Alternative Cleavage and Polyadenylation Sites of 14 NAC Genes in Populus Using a Combination of 3'-RACE and High-Throughput Sequencing.
Capturing the Alternative Cleavage and Polyadenylation Sites of 14 NAC Genes in Populus Using a Combination of 3'-RACE and High-Throughput Sequencing.
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利用 3-RACE 和高通量测序相结合捕获杨属中 14 个 NAC 基因的选择性切割和多聚腺苷酸化位点
DOI：
10.3390/molecules23030608
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发表时间：
2018-03-08
期刊：
影响因子：
--
通讯作者：
Cheng Q
中科院分区：
文献类型：
--
作者：
Wang H;Wang M;Cheng Q
真核生物基因的复杂剪接位点（SSs）和聚腺苷酸化位点（PASs）的检测是阐明基因调控机制的必要条件。利用高通量测序（HTS）的转录组研究揭示了植物中普遍存在的选择性剪接（AS）和选择性聚腺苷酸化（APA）。然而，针对基因或基因家族检测的小尺度、高深度HTS却非常少且有限。我们探索了一种简便、灵活的分析ss和PASs的方法，该方法结合了3 ' -cDNA末端（3 ' -RACE）的快速扩增和HTS。采用3′-RACE-seq分析了毛杨14个NAC （NAM、ATAF1/2、CUC2）转录因子基因。基于实验可重复性、边界序列分析和反转录PCR （RT-PCR）验证，只有典型的SSs被认为是真实的。根据严格的标准，没有任何内部启动特征的候选PASs被选为真实的PASs，并被认为是富含pas的标记。通过3′-RACE-seq分析，共发现34个新的典型SSs、6个内含子/内部外显子和30个富含3′-UTR pas的标记。利用3′-RACE和实时PCR技术，我们证实了在富含pas的标记中结束的三个APA转录本在植物激素的响应中受到差异调节。我们的研究结果表明，3 ' -RACE-seq是一种强大而经济的方法来发现SSs和标记基因或基因家族受APA影响的活性区域。该方法适用于初始阶段的小规模AS和APA研究。
Detection of complex splice sites (SSs) and polyadenylation sites (PASs) of eukaryotic genes is essential for the elucidation of gene regulatory mechanisms. Transcriptome-wide studies using high-throughput sequencing (HTS) have revealed prevalent alternative splicing (AS) and alternative polyadenylation (APA) in plants. However, small-scale and high-depth HTS aimed at detecting genes or gene families are very few and limited. We explored a convenient and flexible method for profiling SSs and PASs, which combines rapid amplification of 3′-cDNA ends (3′-RACE) and HTS. Fourteen NAC (NAM, ATAF1/2, CUC2) transcription factor genes of Populus trichocarpa were analyzed by 3′-RACE-seq. Based on experimental reproducibility, boundary sequence analysis and reverse transcription PCR (RT-PCR) verification, only canonical SSs were considered to be authentic. Based on stringent criteria, candidate PASs without any internal priming features were chosen as authentic PASs and assumed to be PAS-rich markers. Thirty-four novel canonical SSs, six intronic/internal exons and thirty 3′-UTR PAS-rich markers were revealed by 3′-RACE-seq. Using 3′-RACE and real-time PCR, we confirmed that three APA transcripts ending in/around PAS-rich markers were differentially regulated in response to plant hormones. Our results indicate that 3′-RACE-seq is a robust and cost-effective method to discover SSs and label active regions subjected to APA for genes or gene families. The method is suitable for small-scale AS and APA research in the initial stage.
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