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Dioscorea zingiberensis is a perennial herb famous for the production of diosgenin, which is a valuable initial material for the industrial synthesis of steroid drugs. Sterol C26-hydroxylases, such as Tf CYP72A616 and Pp CYP72A613, play an important role in the diosgenin biosynthesis pathway. In the present study, a novel gene, DzCYP72A12-4 , was identified as C26-hydroxylase and was found to be involved in diosgenin biosynthesis, for the first time in D. zingiberensis , using comprehensive methods. Then, the diosgenin heterogenous biosynthesis pathway starting from cholesterol was created in stable transgenic tobacco ( Nicotiana tabacum L.) harboring DzCYP90B71 (QPZ88854), DzCYP90G6 (QPZ88855) and DzCYP72A12-4 . Meanwhile, diosgenin was detected in the transgenic tobacco using an ultra-performance liquid chromatography system (Vanquish UPLC 689, Thermo Fisher Scientific, Bremen, Germany) tandem MS (Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer, Thermo Fisher Scientific, Bremen, Germany). Further RT-qPCR analysis showed that DzCYP72A12-4 was highly expressed in both rhizomes and leaves and was upregulated under 15% polyethylene glycol (PEG) treatment, indicating that DzCYP72A12-4 may be related to drought resistance. In addition, the germination rate of the diosgenin-producing tobacco seeds was higher than that of the negative controls under 15% PEG pressure. In addition, the concentration of malonaldehyde (MDA) was lower in the diosgenin-producing tobacco seedlings than those of the control, indicating higher drought adaptability. The results of this study provide valuable information for further research on diosgenin biosynthesis in D. zingiberensis and its functions related to drought adaptability.

The Functional Characterization of DzCYP72A12-4 Related to Diosgenin Biosynthesis and Drought Adaptability in Dioscorea zingiberensis