关键词: 美洲大蠊, 肢体再生, SP1, 转录组分析, GSEA

Abstract: Animal limb regeneration refers to the ability of certain animals to regrow a complete limb with morphology and function equivalent to those of the original limb following amputation. However, studies of animal limb regeneration have long relied on a limited number of vertebrate models, primarily salamanders (e.g., Ambystoma mexicanum) and zebrafish (Danio rerio), each of which has inherent limitations. Genetic manipulation systems in salamanders remain underdeveloped, and their reproductive cycles are relatively long. Although genetic manipulation is comparatively convenient in D. rerio, their regenerative capacity is largely restricted to the caudal fin and therefore cannot fully represent the mechanisms underlying regeneration of typical tetrapod limbs. Moreover, both models are associated with complex genomes, high experimental costs, and substantial ethical considerations. Consequently, existing model systems are insufficient to meet the demands of in-depth mechanistic studies. The development of new model organisms that combine robust limb-regenerative capacity with genetic tractability has therefore become an important research priority. Periplaneta americana exhibits stable and robust limb-regenerative capacity, a high-quality genome, and high sensitivity to RNA interference (RNAi), facilitating gene-function screening. In addition, it is readily maintained under laboratory conditions, and its experimental materials are inexpensive and readily available. These characteristics make P. americana an excellent model for studying limb regeneration. In this study, we used P. americana to investigate the mechanistic role of the transcription factor SP1 (specificity protein 1) in limb regeneration; to our knowledge, the involvement of this gene in insect limb regeneration has not previously been reported. RT-qPCR analysis revealed that SP1 expression was significantly upregulated during limb regeneration. RNAi-mediated knockdown of SP1 resulted in pronounced regenerative defects, preventing the formation of normal leg structures. Functional assays demonstrated that SP1 is required for insect limb regeneration. Transcriptomic analysis indicated that SP1 may drive the regenerative process by regulating gene sets associated with anteroposterior axis specification and germ-layer segmentation. Loss of SP1 function suppressed both gene sets, thereby leading to regenerative failure. Collectively, our findings validate the value of P. americana as an invertebrate model for studying limb regeneration and reveal that SP1, an evolutionarily conserved transcription factor, regulates the regenerative process through the control of gene sets associated with anteroposterior axis specification and germ-layer segmentation. This study expands the phylogenetic diversity of limb-regeneration models and provides new theoretical support for understanding the evolutionarily conserved mechanisms of regeneration-related genes across species.

Key words: Periplaneta americana, limb regeneration, SP1, transcriptomic analysis, GSEA