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. 2023 Aug 10;10(9):uhad162. doi: 10.1093/hr/uhad162

Figure 4.

Figure 4

Subcellular localization of CsS40 gene and the effect of overexpression of CsS40 on the accumulation of caffeine precursors xanthine and hypoxanthine in tobacco. (a) Schematic of the vectors used to assess CsS40 subcellular localization. (b) Detection of GFP fluorescence intensity. 35S: GFP, empty vector: GFP expression driven by the 35S promoter; 35S: CsS40-GFP, CsS40-GFP fusion protein driven by the 35S promoter. (c) RT-qPCR was used to evaluate the expression levels of CsS40 in tobacco. CK, transgenic tobacco overexpressing pSH737-35S-GUS as a control; NtpSH, transgenic tobacco overexpressing pSH737-35S-CsS40-GUS. (d) HPLC was used to evaluate the xanthine content of NtpSH tobacco. (e) HPLC was used to evaluate the hypoxanthine content of NtpSH tobacco (*P < 0.05; **P < 0.01). (f) CsS40 was silenced using VIGS; RT-qPCR was performed to evaluate the relative expression of CsS40 in transformed seedlings. (g) CsS40 was silenced using VIGS; HPLC was performed to evaluate the accumulation of caffeine in transformed seedlings (*P < 0.05; **P < 0.01).