豹纹鳃棘鲈生长差异分化相关lncRNA与mRNA加权基因共表达网络分析

Weighted gene co-expression network of growth differentiation-related lncRNAs and mRNAs in Plectropomus leopardus

关键词:  豹纹鳃棘鲈  生长差异分化  WGCNA  共表达网络

Key words:  Plectropomus leopardus  Growth differentiation  WGCNA  Co-expression network

摘要:

为揭示豹纹鳃棘鲈(Plectropomus leopardus)生长差异分化的分子调控作用机制，该研究利用豹纹鳃棘鲈中间培育过程中不同生长阶段(42、70和91日龄)的18个幼鱼肌肉组织样本进行全转录组测序，获得lncRNA和mRNA基因表达谱数据，采用加权基因共表达网络分析(WGCNA)方法构建生长差异分化相关lncRNA和mRNA的共表达网络。通过特异性基因模块中差异表达基因的GO和KEGG富集对模块生物学功能进行分析，运用Cytoscape软件构建基因互作网络，挖掘豹纹鳃棘鲈生长差异分化相关的关键候选基因。结果显示，基因差异表达分析共筛选到6 252个差异表达mRNA和367个差异表达lncRNA；共表达网络分析获得MEmagenta、MEgreenyellow和MEblack共3个生长差异分化特异性基因模块；GO和KEGG富集分析显示，特异性模块中差异表达基因显著富集于肌肉蛋白形成、横纹肌组织发育调控、主轴组织形成等生物学过程，参与了蛋白酶体、肌动蛋白细胞骨架调控及肌醇磷酸代谢等与生长差异分化相关的代谢通路；筛选3个特异性模块中权重和连通度高的lncRNA和mRNA构建基因互作网络，得到包括psmd6、nmt1、als2、brcc3、kank1、ada12、at131和hdac3等在内的生长差异分化关键基因和MSTRG.15660、MSTRG.8694、MSTRG.1896等20个lncRNA，为进一步解析豹纹鳃棘鲈生长差异分化的分子机制提供了新思路。

Abstract:

The leopard coral grouper (Plectropomus leopardus) exhibits captivating chromatic patterns and boasts substantial nutritional content. This species garners significant consumer preference owing to its aesthetic appeal and nutritional richness, thereby conferring considerable economic and aquacultural significance. The seedling and intermediate breeding of the P. leopardus, much like many other groupers, undergoes a process involving the screening of fries and acclimatization to artificial feed. During this process, repeated occurrences of growth differentiation among the same batch of fries, juveniles, and fingerlings are observed, which constitutes one of the principal factors contributing to prolonged cultivation cycles and impacting the economic efficiency of aquaculture. To address this issue, a majority of grouper species undergo a “size grading” process during seedling cultivation, wherein fries are segregated according to different size categories post-screening, thereby enhancing fry survival rates and shortening the overall cultivation cycle. However, “size grading” effectiveness is not only significantly influenced by human factors but also by inherent variations among fry themselves. Despite the considerable influence of environmental factors on this phenomenon of growth differentiation, undefined potential genetic factors remain. Yet, there is a noticeable absence of comprehensive research addressing this pressing issue. However, the quality of growth traits serves as a critical indicator for evaluating the economic value of fish aquaculture. Influenced by both genetic predispositions and environmental factors, these traits warrant investigation into the genetic mechanisms and regulatory strategies governing growth-related characteristics in farmed fish. Such research offers insights into variety improvement and breeding practices within the field. Here, during the artificial breeding and intermediate rearing process of P. leopardus, early juveniles exhibited relatively minor susceptibility to non-genetic effects such as water temperature and quality changes, along with significantly differentiated growth and shorter intervals of divergence. Consequently, we selected muscle tissues of individuals exhibiting differential divergence within three consecutive time points of size screening for early juveniles (42, 70 and 91 days post-hatching) for whole transcriptome sequencing, aiming to elucidate genetic mechanisms. The expression profiles of lncRNAs and mRNAs were acquired, and a Weighted Gene Co-expression Network Analysis (WGCNA) method was used to construct a co-expression network of growth differentiation related lncRNAs and mRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of differentially expressed genes within specific gene modules were conducted to analyze the biological functions of the modules. Additionally, Cytoscape software was used to construct a gene interaction networks to identify key candidate genes associated with growth differential differentiation in P. leopardus. The results showed that a total of 6,252 differentially expressed mRNAs and 367 differentially expressed lncRNAs were identified through gene differential expression analysis. Co-expression network analysis revealed three growth differentiation specific gene modules (MEmagenta, MEgreenyellow, and MEblack). GO and KEGG analyses indicated that differentially expressed genes in these specific modules were significantly enriched in biological processes related to muscle protein formation, regulation of skeletal muscle tissue development, and axial tissue formation, participating in metabolic pathways associated with growth differential differentiation such as proteasome, cytoskeletal regulation by actin proteins, and inositol phosphate metabolism. High-weight and high-connectivity lncRNAs and mRNAs were screened from the three specific modules to construct a gene interaction network, revealing key genes associated with growth differentiation including psmd6, nmt1, als2, brcc3, kank1, ada12, at131, hdac3, as well as 20 lncRNA transcription factors such as MSTRG.15660, MSTRG.8694, and MSTRG.1896. Wherein, Psmd6 in zebrafish is encoded by the Volvox mutant, which regulates cellular function by degrading polyubiquitinated proteins, impacting the proliferation of zebrafish lens epithelial cells and the differentiation of lens fiber cells. Mmt1 is an essential gene for mammalian development, serving as a major N-myristoyltransferase during early embryogenesis. Als2 plays a significant role in normal muscle development, and mutations in this gene can lead to autosomal recessive amyotrophic lateral sclerosis and related disorders. The Brcc3, as a cell cycle regulatory gene, participates in the generation of phosphorylated cyclin-dependent kinase activator and biological processes such as cell proliferation. Kank1 regulates actin polymerization, actin stress fiber formation, and cell migration through RhoA signaling. Ada12 (Adam12) plays a crucial role in murine myogenesis and adipogenesis. Adam12 synthesis sequence and amino acid sequence in zebrafish are consistent with mammals and is strongly expressed in the cardiovascular system, erythroid progenitor cells, brain, and jaw cartilage. Zebrafish with knocked out Ada12 genes exhibited reduced body size in infancy without significant morphological defects. At131 (Atp13a1) plays a crucial role in stabilizing the MAVS virus protein in mice. At131 knockout in mice resulted in issues such as growth retardation and embryonic lethality. Hdac3 is essential for liver formation in zebrafish, primarily by inhibiting the growth differentiation factor 11 (Gdf11), which is a negative regulator of cell proliferation and a specific transcription target of Hdac3. Ada12 and At131 knockout leads to defects in growth in zebrafish and mice, directly affecting body size and growth rate. Therefore, Ada12 and At131 are considered as important candidate genes for further research on the molecular regulatory mechanisms of growth differentiation in P. leopardus. This study provides insights into the molecular mechanisms underlying growth differentiation in P. leopardus.