Screening and validation of the key genes for muscle growth and development in Guanling cattle based on transcriptome sequencing
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摘要: 【目的】基于转录组测序挖掘出贵州关岭牛肌肉生长发育关键基因，为后续培育贵州优质肉牛品种提供理论依据。【方法】选取3头健康且体重相近的24月龄成年关岭牛，屠宰后采集其心脏、肝脏、脾脏、肺脏、肾脏、背最长肌、大腿肌及肩峰等组织样品，采用TRIzol法提取各组织总RNA，构建cDNA文库后进行转录组测序，通过生物信息学手段筛选出不同样品间的差异表达基因（DEGs），结合GO功能注释分析和KEGG信号通路富集分析挖掘出关岭牛肌肉生长发育相关候选基因，再经实时荧光定量PCR对候选基因进行验证。【结果】测序样品有效序列（Clean reads）比对至参考基因组序列的平均比对率为90.76%，检测到26367个新转录本（有编码潜能的转录本19659个，非编码转录本6708个）；在各组织中检测到共表达基因53912个，其中已知基因51688个、预测新基因2224个；GO功能注释分析发现这些共表达基因主要发挥生物调节、代谢过程、生物膜组成、催化活性和转录调节活性等功能。通过DEGseq检测，最终筛选获得16个与肌肉生长发育相关的DEGs（QKI、CHKB、MYBPC1、MBNL1、MYH11、MYLK2、TPM3、NEXN、EZH2、TNNT3、PPARGC1A、SGCA、DMPK、FOXP1、FLNB和TNS2），主要参与内吞作用、心肌收缩、癌症、代谢及MAPK信号通路。实时荧光定量PCR验证结果显示，筛选出的16个DEGs在关岭牛背最长肌或肩峰中高表达，与转录组测序结果一致。【结论】基于转录组测序从关岭牛各组织中筛选出16个与肌肉生长发育相关的DEGs，主要在生物调节、代谢过程、细胞成分、催化和转录调节等方面发挥功能作用，可作为开展关岭牛肌肉生长发育遗传机制研究的候选基因。
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关键词:
- 关岭牛 /
- 肌肉生长发育 /
- 肌肉性状 /
- 差异表达基因(DEGs) /
- 转录组测序
Abstract: 【Objective】The purpose of the study was to excavate the key genes related to muscle growth and development of Guizhou Guanling cattle based on transcriptome sequencing， so as to provide a theoretical basis for the subsequent breeding of high-quality Guizhou beef cattle breeds. 【Method】Three healthy 24-month-old adult Guanling cattle with similar body weight were selected， and their heart， liver， spleen， lung， kidney， longissimus dorsi muscle， thigh muscle and acromion tissues were collected after slaughter. TRIzol method was used to extract total RNA from each tissue， and transcriptome sequencing was performed after constructing cDNA library. The differentially expressed genes （DEGs） among different samples were screened by bioinformatics， and candidate genes related to muscle growth and development of Guanling cattle were extracted by combining GO functional annotation analysis and KEGG signaling pathway enrichment analysis， and then the candidate genes were verified by real-time fluorescence quantitative PCR. 【Result】 The average mapped rate of clean reads to the reference genome sequence of the sequenced samples was 90.76%， and 26367 new transcripts （19659 with coding potential and 6708 with non-coding transcripts） were detected. A total of 53912 co-expressed genes were detected in each tissue， including 51688 known genes and 2224 predicted new genes. GO functional annotation analysis showed that these co-expressed genes mainly played the functions of biological regulation， metabolic process， membrane part， catalytic activity and transcriptional regulation activity. Through the DEGseq test， 16 DEGs related to muscle development were finally screened and obtained （QKI， CHKB， MYBPC1， MBNL1， MYH11， MYLK2， TPM3， NEXN， EZH2， TNNT3， PPARGC1A， SGCA， DMPK， FOXP1， FLNB and TNS2）. They were mainly involved in endocytosis， myocardial contraction， cancer， metabolism and MAPK signaling pathway. The results of realtime fluorescence quantitative PCR showed that all the 16 selected DEGs were highly expressed in longissimus dorsi muscle and acromion of Guanling cattle， which was consistent with transcriptome sequencing results. 【Conclusion】Sixteen DEGs related to muscle development are selected from various tissues of Guanling cattle based on transcriptome sequencing， which mainly play functions in biological regulation， metabolic process， cell components， catalysis and transcriptional regulation， and can be used as candidate genes for conducting the study of genetic mechanism of muscle growth and development of Guanling cattle. -
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