Muráni Eduard, Murániová Mária, Ponsuksili Siriluck, Schellander Karl, Wimmers Klaus
Research Institute for the Biology of Farm Animals (FBN), Research Unit Molecular Biology, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany.
BMC Dev Biol. 2007 Oct 1;7:109. doi: 10.1186/1471-213X-7-109.
Postnatal muscle growth is largely depending on the number and size of muscle fibers. The number of myofibers and to a large extent their metabolic and contractile properties, which also influence their size, are determined prenatally during the process of myogenesis. Hence identification of genes and their networks governing prenatal development of skeletal muscles will provide insight into the control of muscle growth and facilitate finding the source of its variation. So far most of the genes involved in myogenesis were identified by in vitro studies using gene targeting and transgenesis. Profiling of transcriptome changes during the myogenesis in vivo promises to obtain a more complete picture. In order to address this, we performed transcriptome profiling of prenatal skeletal muscle using differential display RT-PCR as on open system with the potential to detect novel transcripts. Seven key stages of myogenesis (days 14, 21, 35, 49, 63, 77 and 91 post conception) were studied in two breeds, Pietrain and Duroc, differing markedly in muscularity and muscle structure.
Eighty prominent cDNA fragments were sequenced, 43 showing stage-associated and 37 showing breed-associated differences in the expression, respectively. Out of the resulting 85 unique expressed sequence tags, EST, 52 could be assigned to known genes. The most frequent functional categories represented genes encoding myofibrillar proteins (8), genes involved in cell adhesion, cell-cell signaling and extracellular matrix synthesis/remodeling (8), genes regulating gene expression (8), and metabolism genes (8). Some of the EST that showed no identity to any known transcripts in the databases are located in introns of known genes and most likely represent novel exons (e.g. HMGA2). Expression of thirteen transcripts along with five reference genes was further analyzed by means of real-time quantitative PCR. Nine of the target transcripts showed higher than twofold differences in the expression between the two breeds (GATA3, HMGA2, NRAP, SMC6L1, SPP1, RAB6IP2, TJP1 and two EST).
The present study revealed several genes and novel transcripts not previously associated with myogenesis and expands our knowledge of genetic factors operating during myogenesis. Genes that exhibited differences between the divergent breeds represent candidate genes for muscle growth and structure.
出生后肌肉的生长很大程度上取决于肌纤维的数量和大小。肌纤维的数量以及在很大程度上它们的代谢和收缩特性（这也会影响其大小）在产前肌生成过程中就已确定。因此，鉴定控制骨骼肌产前发育的基因及其网络将有助于深入了解肌肉生长的调控机制，并有助于找到其变异的来源。到目前为止，大多数参与肌生成的基因是通过基因靶向和转基因的体外研究鉴定出来的。对体内肌生成过程中转录组变化进行分析有望获得更完整的图景。为了解决这个问题，我们使用差异显示逆转录聚合酶链反应（differential display RT-PCR）作为一种开放系统对产前骨骼肌进行转录组分析，该系统有潜力检测新的转录本。我们在皮特兰和杜洛克这两个品种中研究了肌生成的七个关键阶段（受孕后第14、21、35、49、63、77和91天），这两个品种在肌肉发达程度和肌肉结构上有显著差异。
对80个显著的cDNA片段进行了测序，其中43个显示出与阶段相关的表达差异，37个显示出与品种相关的表达差异。在得到的85个独特的表达序列标签（EST）中，52个可以被归类到已知基因。最常见的功能类别包括编码肌原纤维蛋白的基因（8个）、参与细胞黏附、细胞间信号传导和细胞外基质合成/重塑的基因（8个）、调节基因表达的基因（8个）以及代谢基因（8个）。一些在数据库中与任何已知转录本都没有同源性的EST位于已知基因的内含子中，很可能代表新的外显子（如HMGA2）。通过实时定量PCR进一步分析了13个转录本以及5个参考基因的表达。其中9个目标转录本在两个品种之间的表达差异超过两倍（GATA3、HMGA2、NRAP、SMC6L1、SPP1、RAB6IP2、TJP1和两个EST）。
本研究揭示了几个以前与肌生成无关的基因和新的转录本，扩展了我们对肌生成过程中作用的遗传因素的认识。在不同品种之间表现出差异的基因代表了肌肉生长和结构的候选基因。
