Figure 1.
The putative biosynthetic pathway of salidroside and Rhodiola crenulata.
The arrows indicated the cloned genes and the dashed arrows showed the unidentified genes; the background photo was flowering Rhodiola crenulata. TYDC: tyrosine decarboxylase; UDPGT: UDP-glucosyltransferase.
Figure 2.
The full-length cDNA of RcTYDC and its deduced amino acids.
The coding sequence of RcTYDC was shown with capital letters in bold fonts; the untranslated regions were in small letters; and the stop codon was marked with an asterisk.
Figure 3.
Sequence comparison of plant TYDCs and TDCs.
RvTDC: Rauvolfia verticillata tryptophan decarboxylase; CrTDC: Catharanthus roseus tryptophan decarboxylase; PcTYDC: Petroselinum crispum tyrosine decarboxylase; RcTYDC: Rhodiola crenulata tyrosine decarboxylase.
Figure 4.
The phylogentic analylsis of plant and cyanobacterium TYDCs.
AcTYDC: Anabaena cylindrical TYDC (AFZ60767); AtTYDC: Arabidopsis thaliana TYDC (CAB56038); CrTYDC: Citrus reticulata TYDC (ACX29991); MtTYDC: Medicago truncatula TYDC (XP_003625397); PcTYDC: Petroselinum crispum TYDC (Q06086); PsTYDC: Papaver somniferum TYDC (AAC61843); RcTYDC: Rhodiola crenulata TYDC (AFN89854.1); TfTYDC: Thalictrum flavum TYDC (AAG60665); VvTYDC: Vitis vinifera TYDC (CAN61896).
Figure 5.
A: recombinant protein of RcTYDC; B: enzymatic assay of recombinant RcTYDC.
The blue line was authentic tyramine HPLC trace, the red line was tyramine HPLC trace with feeding tyrosine to recombinant RcTYDC; the grey line was HPLC trace without feeding tyrosine to recombinant RcTYDC.
Figure 6.
Establishment of transgenic hairy root cultures of R. crenulata (genomic PCR detection included).
A: bacteria-free seedlings; B: induction of hairy roots; C: subcultured hairy root; D: hairy roots cultured in liquid medium; E: genomic PCR analysis of genes of interest.
Figure 7.
Gene expression levels and salidroside accumulations in different tissues/under different treatments.
A: tissue profile of RcTYDC; B: tissue profile of RcUDPGT; C: the content of salidroside in different tissues of R. crenulata; D: elicitor-induced profile of RcTYDC; E: elicitor-induced profile of RcUDPGT; C: the salidroside content of hairy root cultures of R. crenulata in different treatments. MeJA: methyl jasmonate; ABA: abscisic acid; SA: salicylic acid; G: glucose. Different letters indicated significant differences (P<0.05) given by Duncan’s multiple range test. ** represented significant difference at P<0.01 given by one sample t test. Results represented means±standard deviation (n = 3).
Figure 8.
Relative gene expression level of RcTYDC in hairy root cultures of R. crenulata.
C: nontransgenic hairy root lines; T: transgenic hairy root lines with overexpressing RcTYDC. Different letters indicated significant differences (P<0.05) given by Duncan’s multiple range test. Results represented means±standard deviation (n = 3).
Figure 9.
Analysis of metabolites including tyramine, tyrosol and salidroside in hairy root cultures of R. crenulata.
C: nontransgenic hairy root lines; T: transgenic hairy root lines with overexpressing RcTYDC. Different letters in tyramine, tyrosol and salidroside contents indicated significant differences (P<0.05) given by Duncan’s multiple range test. Results represented means±standard deviation (n = 3).