Table_11_Transcriptome analysis to identify candidate genes related to mammary gland development of Bactrian camel (Camelus bactrianus).XLSX

IntroductionThe demand for camel milk, which has unique therapeutic properties, is increasing. The mammary gland is the organ in mammals responsible for the production and quality of milk. However, few studies have investigated the genes or pathways related to mammary gland growth and development in Bactrian camels. This study aimed to compare the morphological changes in mammary gland tissue and transcriptome expression profiles between young and adult female Bactrian camels and to explore the potential candidate genes and signaling pathways related to mammary gland development.
MethodsThree 2  years-old female camels and three 5  years-old adult female camels were maintained in the same environment. The parenchyma of the mammary gland tissue was sampled from the camels using percutaneous needle biopsy. Morphological changes were observed using hematoxylin-eosin staining. High-throughput RNA sequencing was performed using the Illumina HiSeq platform to analyze changes in the transcriptome between young and adult camels. Functional enrichment, pathway enrichment, and protein–protein interaction networks were also analyzed. Gene expression was verified using quantitative real-time polymerase chain reaction (qRT-PCR).
ResultsHistomorphological analysis showed that the mammary ducts and mammary epithelial cells in adult female camels were greatly developed and differentiated from those in young camels. Transcriptome analysis showed that 2,851 differentially expressed genes were obtained in the adult camel group compared to the young camel group, of which 1,420 were upregulated, 1,431 were downregulated, and 2,419 encoded proteins. Functional enrichment analysis revealed that the upregulated genes were significantly enriched for 24 pathways, including the Hedgehog signaling pathway which is closely related to mammary gland development. The downregulated genes were significantly enriched for seven pathways, among these the Wnt signaling pathway was significantly related to mammary gland development. The protein–protein interaction network sorted the nodes according to the degree of gene interaction and identified nine candidate genes: PRKAB2, PRKAG3, PLCB4, BTRC, GLI1, WIF1, DKK2, FZD3, and WNT4. The expression of fifteen genes randomly detected by qRT-PCR showed results consistent with those of the transcriptome analysis.
DiscussionPreliminary findings indicate that the Hedgehog, Wnt, oxytocin, insulin, and steroid biosynthesis signaling pathways have important effects on mammary gland development in dairy camels. Given the importance of these pathways and the interconnections of the involved genes, the genes in these pathways should be considered potential candidate genes. This study provides a theoretical basis for elucidating the molecular mechanisms associated with mammary gland development and milk production in Bactrian camels.

引言 骆驼乳具有独特的治疗特性，市场需求日益增长。乳腺是哺乳动物中负责乳汁合成与品质调控的器官。然而，目前针对双峰驼（Bactrian camels）乳腺生长发育相关基因及信号通路的研究仍较为匮乏。本研究旨在比较青年与成年雌性双峰驼的乳腺组织形态学变化及转录组表达谱，以期挖掘与乳腺发育相关的潜在候选基因及信号通路。
方法 本研究选取3只2岁龄雌性双峰驼与3只5岁龄成年雌性双峰驼，在相同饲养环境下进行管理。采用经皮穿刺针活检法采集乳腺实质组织样本。通过苏木精-伊红（hematoxylin-eosin）染色观察组织形态学变化。利用Illumina HiSeq平台开展高通量RNA测序，分析青年与成年双峰驼间的转录组差异。此外，还进行了功能富集分析、通路富集分析及蛋白质-蛋白质相互作用（protein–protein interaction）网络分析。采用实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）验证基因表达水平。
结果 组织形态学分析显示，成年雌性双峰驼的乳腺导管与乳腺上皮细胞较青年个体出现显著发育与分化。转录组分析结果表明，与青年骆驼组相比，成年骆驼组共筛选得到2851个差异表达基因，其中1420个基因上调表达、1431个基因下调表达，2419个基因编码蛋白质。功能富集分析显示，上调表达基因显著富集于24条信号通路，其中包括与乳腺发育密切相关的刺猬信号通路（Hedgehog signaling pathway）。下调表达基因显著富集于7条信号通路，其中Wnt信号通路（Wnt signaling pathway）与乳腺发育显著相关。通过蛋白质-蛋白质相互作用网络按基因交互度对节点进行排序，最终筛选得到9个候选基因：PRKAB2、PRKAG3、PLCB4、BTRC、GLI1、WIF1、DKK2、FZD3及WNT4。随机选取15个基因进行qRT-PCR验证，结果与转录组测序分析结果一致。
讨论 本研究初步表明，刺猬信号通路、Wnt信号通路、催产素信号通路、胰岛素信号通路及类固醇生物合成信号通路对泌乳双峰驼的乳腺发育具有重要调控作用。鉴于上述通路的重要性及其所涉及基因间的相互关联，这些通路中的基因可被视为潜在候选基因。本研究为阐明双峰驼乳腺发育与泌乳相关的分子机制提供了理论依据。