目的 挖掘蟾蜍二烯酸内酯（bufadienolides，BDs）生物合成途径的关键基因。方法 使用激光捕获显微切割技术捕获中华大蟾蜍分泌腺[耳后浆液腺（CE）、皮肤浆液腺（CK）和皮肤粘液腺（CN）]为实验材料，在DNBseq测序平台进行转录组测序，并进行生信分析及对转录组结果进行验证。结果 共得到63.01 Gb原始数据和4 550个差异基因，其中CE和CK共有上调差异基因880个，其GO和KEGG富集显示主要在甾类激素生物合成、胆汁酸合成和C₂₁类固醇生物合成等功能；CN上调差异基因主要富集在蛋白质糖基化、细胞黏结、体液免疫应答等功能。在CE、CK组上调的差异基因富集结果中发现有28种与BDs合成相关的物质的代谢、合成及运输等功能，且参与调控的基因有94个。对上述基因进行蛋白互作网络分析（protein-protein interaction network，PPI）分析，结果显示，94个基因中有34个存在互作关系，其中基因AMACR、HSD17B4、SCP2及ACOT8参与调控胆汁酸合成，基因LOC122926609参与调控石胆酸结合，结合文献分析，作者认为初级胆汁酸合成途径是BDs合成的下游途径，石胆酸可能是BDs类成分的重要前体。进一步将存在互作关系的基因关联到表达量矩阵中，结果显示大部分基因在样品CE和CK组中的表达量均大于CN组，且上述5个基因的表达趋势一致（CE、CK＞CN），认为这几个基因是调控BDs的下游合成途径的关键基因。在上述基础上选取9个与BDs合成相关的基因进行qRT-PCR分析，结果与转录组测序基因表达趋势一致，认为该结果具有准确性。结论 完成了中华大蟾蜍分泌腺的转录组研究，推测出BDs下游合成途径的关键基因，为蟾毒内酯类化合物的生物合成途径研究提供了丰富的参考数据，也为深入研究该类化合物生物合成的分子机制奠定了基础。

Objective To explore the key genes of bufadienolides (BDs) biosynthesis pathway. Methods The secretory glands of Bufo bufo gargarizans [retroauricular serous glands (CE), dermal serous glands (CK) and dermal mucous glands (CN)] were captured by laser capture microdissection technology as experimental materials. Transcriptome sequencing and biogenic analysis were performed on DNBseq sequencing platform, and the transcriptome results were analyzed and verified. Result A total of 63.01Gb of original data and 4 550 differential genes were obtained. There were 880 up-regulated differential genes of CE and CK, and their GO and KEGG enrichment showed that they were mainly involved in steroid hormone biosynthesis, bile acid synthesis and C21 steroid biosynthesis. CN up-regulated differential genes were mainly concentrated in protein glycosylation, cell bonding, humoral immune response and other functions. In the up-regulated differential gene enrichment results of CE and CK groups, 28 kinds of substances related to BDs synthesis were found to have functions such as metabolism, synthesis and transportation, and 94 genes were involved in regulation. PPI analysis of the above genes showed that 34 of the 94 genes had interaction, among which AMACR, HSD17B4, SCP2 and ACOT8 were involved in the regulation of bile acid synthesis, and LOC122926609 was involved in the regulation of lithic acid binding. Combined with literature analysis, the authors suggest that the primary bile acid synthesis pathway is the downstream pathway of BDs synthesis and lithic acid may be an important precursor of BDs components. The genes with interaction relationship were further associated into the expression matrix, the results showed that the expression levels of most of the genes in the CE and CK groups were higher than those in the CN group, and the expression trend of the above five genes was consistent (CE, CK > CN). It was considered that these genes were the key genes regulating the downstream synthesis pathway of BDs. Any 9 genes related to BDs synthesis were selected for qRT-PCR analysis, and the results were consistent with the gene expression trend of transcriptome sequencing, which suggested that the transcriptome results were accurate. Conclusion The transcriptome of the secretory gland of Bufo bufo gargarizans was studied, and the key genes of the downstream synthesis pathway of BDs were speculated, which provided rich reference data for the biosynthesis pathway of bufalin compounds, and laid a foundation for further study of the molecular mechanism of the biosynthesis of bufalin compounds.

国家自然科学基金资助项目（81903757）；四川省科技厅科技计划（22CXTD0009，2020ZYD058）；四川省药用动物规范化生产现状及可持续利用研究（2023JDR0053）