阳原驴MSTN基因生物信息学分析及组织表达研究

Bioinformatics Analysis and Tissue Expression of MSTN Gene in Yangyuan Donkeys

【目的】试验旨在分析阳原驴肌生长抑制素(myostatin,MSTN)基因CDS序列特征及其在不同生长发育时期和不同组织中的表达水平。【方法】采集6、12、18、24月龄阳原驴的血样及不同组织样,提取其基因组DNA及不同组织样总RNA。利用PCR技术扩增阳原驴MSTN基因CDS序列,并对其进行生物信息学分析。利用实时荧光定量PCR技术检测MSTN基因在阳原驴不同生长发育时期背最长肌、腿肌和18月龄不同组织(心脏、肝脏、脾脏、肺脏、肾脏)中的表达水平。【结果】阳原驴MSTN基因CDS序列长1128 bp,编码375个氨基酸,与马的核苷酸序列相似性最高(99.9%),编码蛋白属于不稳定的亲水性蛋白,含有转化生长因子-β(TGF-β)超家族结构域,不含跨膜区,存在1个信号肽区域,包含6个O-糖基化位点、1个N-糖基化位点和34个磷酸化位点,主要分布于细胞核中(39.1%),二级结构中无规则卷曲占比最高(50.93%)。MSTN基因在18月龄阳原驴背最长肌和腿肌中的表达水平显著高于6、12、24月龄(P<0.05);MSTN基因在18月龄阳原驴不同组织中均有表达,其中背最长肌中的表达水平显著高于其他组织(P<0.05),其次是腿肌、肺脏、脾脏、肾脏和肝脏,心脏中的表达水平最低。【结论】试验成功扩增出阳原驴MSTN基因CDS序列,MSTN基因在阳原驴不同生长发育时期背最长肌和腿肌中的表达水平均以18月龄最高,且在18月龄不同组织中以背最长肌和腿肌中表达水平较高。研究结果为进一步探讨MSTN基因在阳原驴骨骼肌生长发育中的作用机制提供参考。

【Objective】The purpose of this study was to analyze the CDS sequence of myostatin(MSTN)gene and its expression in different growth stages and different tissues of Yangyuan donkeys.【Method】Blood samples and various tissue samples were collected from Yangyuan donkeys aged 6,12,18 and 24 months old,genomic DNA and total RNA from different tissues were extracted.The CDS sequence of MSTN gene in Yangyuan donkeys was amplified by PCR and analyzed by bioinformatics.Real-time quantitative PCR was used to detect the expression of MSTN gene in longissimus dorsi muscle,leg muscle of Yangyuan donkeys at different growth and development stages,and different organizations(heart,liver,spleen,lung,kidney)of 18 months old Yangyuan donkeys.【Result】The CDS sequence of MSTN gene in Yangyuan donkeys was 1128 bp and encoded 375 amino acids,which had the highest similarity of nucleotide sequences(99.9%)with Equus caballus.The encoded protein was an unstable hydrophilic protein,containing transforming growth factorβ(TGF-β)superfamily domain,no transmembrane region,1 signal peptide region,6 O-glycosylation sites,1 N-glycosylation site and 34 phosphorylation sites,mainly distributed in nucleus(39.1%),and the proportion of random coil in the secondary structure was the highest(50.93%).The expression of MSTN gene in longissimus dorsi muscle and leg muscle of Yangyuan donkeys at 18 months old were significantly higher than that of 6,12 and 24 months old(P<0.05).MSTN gene was expressed in different tissues of 18 months old Yangyuan donkeys.The expression in longissimus dorsi muscle was significantly higher than that in other tissues(P<0.05),followed by leg muscle,lung,spleen,kidney and liver,and the expression in heart was the lowest.【Conclusion】The CDS sequence of MSTN gene in Yangyuan donkeys was successfully amplified.The expression of MSTN gene in longissimus dorsi muscle and leg muscle of Yangyuan donkeys at different growth and development stages was the highest at 18 months old,and the expression of longissimus dorsi muscle and leg muscle was higher in different tissues at 18 months old.The results provided a reference for further exploring the mechanism of MSTN gene in the growth and development of skeletal muscle in Yangyuan donkeys.