【Objective】 To investigate the variation rule of carotenoids in the leaves of Anoectochilus roxburghii at different growth stages, and to analyze the molecular mechanism of carotenoid accumulation.【Method】 The carotenoids contents in the leaves of A. roxburghii at different growth stages（S3, in three months old; S6, in six months old; and S10, in ten months old）were investigated by liquid chromatograph mass spectrometer（LC-MS）and high-throughput transcriptome sequencing（RNA-seq）.【Result】 Combining the transcriptome and metabolome to analyze the leaves of A. roxburghii at different growth periods, the metabolomics results showed that there were 23 differential metabolites involved in carotenoid synthesis in the leaves, including neoxanthin, graphemexanthin, luteolin, and caroxanthin, etc., and the transcriptome results showed that there were 21 differential metabolism enzyme genes, including CYP97C1, CCD8, NCED, ZEP, and PDS, etc., which predicted that 14 transcription factors, including ERF, MYB, C2H2, WRKY and bZIP, were involved in regulating the differential carotenoid synthesis and accumulation in A. roxburghii leaves. and real-time quantitative PCR（RT-qPCR）experiments were performed to show that the expression trends of eight selected enzyme genes involved in the synthesis of carotenoids in the different phases of the process were consistent with the results of the sequencing of transcriptomics.【Conclusion】 Five transcription factors, including ERF, MYB, and C2H2, were involved in the regulation of carotenoid synthesis during different growth periods of Anoectochilus roxburghii, and were significantly associated with five key differential enzyme genes, including CYP97C1, CCD, and NCED, which together regulate six key differential metabolites, including Abscisic aldehyde, Zeaxanthin, and Lutein.
