斑马鱼肌肉高表达基因近端非编码元件分析及功能检测

IDENTIFICATION OF PROXIMAL CIS-REGULATORY ELEMENT FOR MUSCLE HIGHLY EXPRESSED GENES IN ZEBRAFISH

  • 摘要: 为鉴定鱼类肌肉组织特异性顺式调控元件, 通过分析斑马鱼多个组织的转录组数据, 筛选出肌肉高表达基因及低表达基因。通过MEME对肌肉高表达基因和低表达基因非编码区序列特征进行分析, 在5个肌肉高表达基因的转录起始位点上游发现了序列保守的DNA区域, 包含6个排列顺序一致的DNA基序。将其中一段目标片段插入具有Tol2转座子元件的基础启动子驱动的eGFP编码基因的上游, 构建表达载体。注射载体至斑马鱼受精卵获得转基因胚胎, 分析胚胎中eGFP荧光的表达模式, 发现该DNA片段具有增强基因在肌肉特异性表达的功能。Tomtom预测该DNA区域可能作为Myod等多个转录因子的结合位点。研究结果有助于理解鱼类肌肉基因表达的遗传基础, 并为利用生物信息学方法预测组织特异性转录调控元件提供新思路。

     

    Abstract: The related factors to the formation and growth traits of fish muscle are important topics in aquatic biology and aquaculture research. The expression and regulation of muscle component genes are essential to its tissue function maintenance and trait control, and the identification of cis- regulatory elements in muscle tissue helps explain the genetic basis of muscle formation. Conserved DNA sequences may be found among cis-regulatory elements, whose regulating genes have similar expression patterns. To predict the regulatory elements for zebrafish muscle gene expression, we explored the conservation features for DNA sequences in proximal non-coding regions of muscle highly expressed genes. By analyzing RNA-seq data of multiple zebrafish tissues from public database, we located muscle highly expressed genes as targets and lowly expressed genes as control, respectively. The GO enrichment analysis of these highly expressed genes confirmed their functions associated with muscle development. By discriminative mode of MEME motif discovery tools and using non-coding region of lowly expressed genes as background, we found five target conserved DNA regions, including six DNA motifs of the same sequence, around 300 bp in length, close to gene start sites of five muscle highly expressed genes. Meanwhile, DNA sequences of these five target regions had high pairwise identities (78.62%—84.19%). The results of qPCR confirmed the remarkably higher expression of these five genes in muscle than other tissues. We constructed an eGFP expression reporter plasmid containing the tol2 transposon system. One of the target regions, a 334 bp fragment at upstream of zgc:9242, was cloned into the plasmid at upstream of the eGFP driven by the base promoter. After the plasmid was injected into zebrafish embryos, a greater proportion of muscle specific fluorescence was observed in embryos carrying the target DNA fragment than in the control group (odds ratio=6.487, P=0.000 at 48 hpf), indicating that the 334 bp DNA fragment may enhance muscle gene expression. Using Tomtom motif comparison tools, we also found the candidate binding sites for Myod and other transcription factors within DNA motifs. Our findings suggest that the DNA motif cluster fragments might act as transcriptional regulatory elements to specifically enhance zebrafish muscle gene expression. These results can help us better understanding the genetic basis for fish muscle gene expression and provide a new strategy for predicting tissue specific cis-regulatory elements by bioinformatics.

     

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