全州禾花鲤鳞片下层组织的结构及全长转录组分析

Tissue structure and full-length transcriptome analysis of the scaly sublayer of Cyprinus carpio var. Quanzhounensis

中文关键词: 全州禾花鲤  皮肤  性状  全长转录组

英文关键词: Cyprinus carpio var. Quanzhouensis  Skin  Character  Full-length transcriptom

中文摘要:

      为研究全州禾花鲤(Cyprinus carpio var. Quanzhounensis)皮肤组织结构和转录组差异、丰富鲤鱼皮肤转录组信息，选用建鲤(Cyprinus carpio var. Jian)和全州禾花鲤鳞片下层组织，利用透射电镜观察结构差异，发现禾花鲤与建鲤相比组织缺乏层叠排列的鸟嘌呤结晶且黑色素颗粒增多；利用牛津纳米孔(ONT)测序技术分析了转录组特征，获得高质量数据18.49 Gb，识别为全长序列17 182 183条，检测出可变剪接(AS)事件3 075个、可变多聚腺苷酸化(APA) 57 624个，预测新基因编码区序列15 615个、长链非编码RNA (lncRNA) 771个。其中，可变剪接事件中外显子跳跃、内含子保留数量在种间差异极显著(P<0.01)，具有5个多聚腺苷酸化位点的转录本数量在种间差异极显著(P<0.01)，表明可变剪接和多聚腺苷酸化参与性状形成相关的调控过程；共筛选出差异表达转录本841个，KEGG通路分析表明其在细胞外基质–受体相互作用、粘着斑等通路富集，GO分析发现其在细胞外隙、转录因子复合体、整合素复合物等词条显著富集，且最显著富集的KEGG通路和GO词条均与细胞外基质紧密关联，推测这些转录调控的变化可能导致皮肤细胞外基质的组成、密度和构象变化。后续研究应更多关注鸟嘌呤结晶缺失造成的皮肤性状变化，有助于全州禾花鲤种质资源开发与利用。

英文摘要:

      Cyprinus carpio var. Quanzhounensis, native to Quanzhou County, Guilin City, Guangxi, has a dark brown body color, translucent gill cover, and abdominal skin, and is an important farmed species in the local integrated rice-fishery industry. A comparative study on the skin of Cyprinus carpio var. Quanzhounensis revealed a lack of reflective guanine crystals on the body surface and a significantly higher melanin content than that of C. carpio var. Jian, which was tentatively considered the direct cause of variations in body color of this group. Iridocytes are a pigment cell species that contain regularly arranged guanine crystals, which are the key material basis for the metallic luster of the fish body surface. In species such as medaka (Oryzias latipes) and zebrafish (Danio rerio), the absence of guanine crystals is considered a manifestation of the absence of iridocyte differentiation and therefore is ideal for studying the mechanism of pigment cell differentiation. The pnp4a, Gbx2, sox10, tfec genes and other iridocyte-related genes have been mined using mutant materials, and we have uncovered the differentiation mechanism that regulates the formation of iridescent cells. C. carpio var. color has abundant genetic variation in body color and is a good material for studying the mechanism of body color determination in fish. The roles of ASIP and MC1R in the aggregation and distribution of melanin and formation of black spots in C. carpio var. color were verified. The guanine crystalline deletion trait of C. carpio var. Quanzhounensis may be loaded with regulatory mechanism diversity and mutation loci related to guanine crystal formation or iridescent cell differentiation. In addition, as a rice-fish culture species, the living environment of C. carpio var. Quanzhounensis harvestmen differ significantly from pond and net-pen culture species, requiring high resistance to disease, adversity, and transport. It is unclear whether the absence of guanine crystals in their skin leads to changes in their basal physiological state, and in-depth studies are beneficial for accurate assessment of culture performance. To reveal the structural basis and transcriptomic characteristics of guanine crystalline deficiency traits in the skin of C. carpio var. Quanzhounensis in this study, we selected the subscale tissues with the most significant differences in guanine crystalline distribution as the control material, and transmission electron microscopy was used to observe the tissue structure and full-length transcriptome sequencing to understand the structural and transcriptomic characteristics of guanine crystalline deficiency in the skin of C. carpio var. Quanzhounensis. The results of this study provide information for the analysis of body color traits, identification of economic traits, and utilization of germplasm resources. Transmission electron microscopy of the subscale tissue sections revealed two significant differences in the histological structure of C. carpio var. Quanzhounensis, and C. carpio var. Jian. First, guanine crystals were absent in C. carpio var. Quanzhounensis, whereas guanine crystals were widely present in the tissues of C. carpio var. Jian and cascading cavities were observed in the sections after guanine crystals were dislodged. Second, the number and density of melanin particles in the tissues of C. carpio var. Quanzhounensis harvestmen were significantly higher than those of C. carpio var. Jian, showing smaller, darker, and more numerous particles, which is consistent with the darker color and lack of silvery reflective material on the body surface of C. carpio var. Quanzhounensis harvestmen. The transcriptome characteristics were analyzed using Oxford Nanopore (ONT) sequencing technology, and 2.88~3.26 Gb of high-quality data were obtained for each sample. The number of full-length sequences after filtering ribosomal RNA for all sample data was 2 203 826~2 412 500, and the proportion of full-length sequences for each sample was 87.06%~88.57%. The comparison rate was 90.35%~92.46%. Variable splicing events in the transcripts were counted; 3 075 variable splicing events and 57 624 variable polyadenylation events were detected; and 15 615 new coding region sequences and 771 long-stranded non-coding RNAs were predicted. The number of exon jumps and intron retention in variable splicing events differed significantly (P<0.01) between species, and the number of transcripts with five polyadenylation sites differed significantly (P<0.01) between species, indicating that variable splicing and polyadenylation are involved in regulatory processes related to trait formation. A total of 15 615 open reading frames (ORFs), including 9 890 complete ORFs, were predicted in this study. A total of 841 differentially expressed transcripts were screened in this study; 183 transcripts were upregulated and 658 transcripts were downregulated in C. carpio var. Quanzhounensis compared to C. carpio var. Jian (JH), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed enrichment in extracellular matrix-receptor interactions and adherent spots. KEGG pathway analysis showed that it was enriched for extracellular gaps, transcription factor complexes, integrin complexes, and other terms. The most significantly enriched KEGG pathway and GO terms were closely associated with the extracellular matrix, and it is speculated that these transcriptional changes may lead to changes in the composition, density, and conformation of the skin extracellular matrix.