Transcriptome analysis of Fritillaria cirrhosa D. Don

Fritillaria cirrhosa D. Don (known as Chuan-Bei-Mu in Chinese) can synthesize isosteroidal alkaloids (ISA) with excellent medicinal value, and its bulb has become an indispensable ingredient in many patented drugs. Members of the cytochrome P450 (CYP450) gene superfamily have been shown to play essential roles in regulating steroidal alkaloids biosynthesis. However, little information is available on the P450s in F. cirrhosa. Here, we performed full-length transcriptome analysis and discovered 48 CYP450 genes belonging to 10 clans, 25 families, and 46 subfamilies. By combining phylogenetic trees, gene expression, and key F. cirrhosa ISA content analysis, we presumably identify seven FcCYP candidate genes, which may be hydroxylases active at the C-22, C-23, or C-26 positions in the late stages of ISA biosynthesis. The transcript expression levels of seven FcCYP candidate genes were positively correlated with the accumulation of three major alkaloids in bulbs of different ages. These data suggest that the candidate genes are most likely to be associated with ISA biosynthesis. Finally, the subcellular localization prediction of FcCYPs and transient expression analysis within Nicotiana benthamiana showed that the FcCYPs were mainly localized in the chloroplast. This study presents a systematic analysis of the CYP450 gene family in F. cirrhosa and provides a foundation for further functional characterization of the CYPs involved in ISA biosynthesis.川贝母（Fritillaria cirrhosa D. Don），中文习称川贝母（Chuan-Bei-Mu），可合成具有优异药用价值的异甾体生物碱（isosteroidal alkaloids，ISA），其鳞茎已成为诸多专利药品中不可或缺的组分。研究证实，细胞色素P450（cytochrome P450，CYP450）基因超家族成员在调控甾体生物碱生物合成过程中发挥关键作用，但目前关于川贝母中CYP450基因的相关报道仍较为有限。本研究通过全长转录组分析，共鉴定得到48个CYP450基因，这些基因隶属于10个族、25个家族及46个亚家族。结合系统发育树分析、基因表达谱检测及川贝母关键异甾体生物碱含量测定，我们推测鉴定出7个FcCYP候选基因，其可能参与异甾体生物碱生物合成后期阶段中C-22、C-23或C-26位的羟化反应。7个FcCYP候选基因的转录表达水平，与不同发育时期鳞茎内三种主要生物碱的积累量呈显著正相关，提示这些候选基因极有可能参与异甾体生物碱的生物合成过程。最后，通过对FcCYPs的亚细胞定位预测及在本氏烟草（Nicotiana benthamiana）中的瞬时表达分析，发现FcCYPs主要定位于叶绿体中。本研究对川贝母CYP450基因家族开展了系统性分析，为后续解析参与异甾体生物碱生物合成的CYP450基因的功能奠定了坚实基础。