Supplemental Data for Huang et al. 2012,Molecular characterization of the pentacyclic triterpenoid biosynthetic pathway in Catharanthus roseus

Lili Huang1,2,3, Jia Li1, Hechun Ye2, Changfu Li1, Hong Wang3, Benye Liu2, Yansheng Zhang1

From 1CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, The Chinese Academy of Science, Wuhan, 430074, China;

2Key Laboratory of Plant Molecular Physiology, Institute of Botany, The Chinese Academy of Science, Beijing, 100093, China;

3Graduate University of the Chinese Academy of Science, Beijing, 100049, China

*Running Title: Catharanthus roseus triterpene synthase and C-28 oxidase

To whom correspondence should be addressed: Yansheng Zhang, CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, The Chinese Academy of Science, Wuhan, 430074, China, Tel: 86-27-87617026, E-mail: or Benye Liu, Key Laboratory of Plant Molecular Physiology, Institute of Botany, The Chinese Academy of Science, Beijing, 100093, China, Email:

Table S1. Primers used in this study. Restriction endonuclease sites are underlined, start and stop codons are indicated in bold.

Gene name designated in this study / No. / Descriptions / Primer Sequences
CrAS / 1 / 5’-end of ORF / 5'-CGCGGATCCATGTGGAAGCTAAAGATAGCAAAAGG-3'
2 / 3’-end of ORF / 5'-GCGTCGACCTACAAAGCTTTGGTTGGCCAGAC-3'
CYP71D1V1 / 3 / Tail-PCR Primer / 5'-NTCGASTWTSGWGTT-3'
4 / Tail-PCR Primer / 5'-NGTCGASWGANAWGAA-3'
5 / Tail-PCR Primer / 5'-TGAGCAAAGGTAACACCCAAAC-3'
6 / Tail-PCR Primer / 5'-ATTTACTGACCGATCTGCAACG-3'
7 / 5’-end of ORF / 5’-CCCGAATTCATGGAGTTTTCTTTCTCCTC-3'
8 / 3’-end of ORF / 5’-CCCGAGCTCCTAATCGTTAACAAGATGAG-3'
CYP716AL1 (CrAO) / 9 / 5’ Race Primer / 5'-GGCCACGCGTCGACTAGTAC(G)10-3'
10 / 5’ Race Primer / 5'-GGCCACGCGTCGACTAGTAC-3'
11 / 5’ Race Primer / 5'-CAGAATTTCCAGCCTAGCGTAT-3'
12 / 5’ Race Primer / 5'-CACACTCCAGTAAATCTTCCATCC-3'
13 / 5’-end of ORF / 5’-CGGGATCCATGGAGATCTTCTATGTCACTCT-3'
14 / 3’-end of ORF / 5'-GGGGTACCTTATGCATTAATGTGAGGATAAAGTCG-3'
CYP71D1V2 / 15 / 3’ Race Primer / 5’-CTGATCTAGAGGTACCGGATCCATA(T)17-3'
16 / 3’ Race Primer / 5’-CTGATCTAGAGGTACCGGATCCATA-3'
17 / 3’ Race Primer / 5’-ACGGGAGGTGTTTATTGATCTAAT-3'
18 / 3’ Race Primer / 5’-TCTGTTTCCGTCCTACAAGATACTTC-3'
19 / 5’-end of ORF / 5'-GGGGTACCATGGAGTTTCATGAATCTTCTCCCT-3'
20 / 3’-end of ORF / 5'-CGGCTAGCTCATGCATAGGACGTAGCGATTAAT-3'
AtLUP / 21 / 5’-end of ORF / 5'-ATGGATCCATGTGGAAGTTGAAGATAGG-3'
22 / 3’-end of ORF / 5'-GCCTCGAGTTAATTAACGATAAACACAACTT-3'
Actin / 23 / qPCR Primer / 5'-TGTGACGTGGATATCAGGAAGG-3'
24 / qPCR Primer / 5'-TCTGCCTTTGCAATCCACAT-3'
CrAS / 25 / qPCR Primer / 5'-CTACTGTCGCACAACCTAC-3'
26 / qPCR Primer / 5'-CGTCTCATAACTGGCTCA-3'
CrAO / 27 / qPCR Primer / 5'-AGGATGGAAGATTTACTGGAGTGTGA-3'
28 / qPCR Primer / 5'-TGGTACAAATGTATATGGAGCAGGTC-3'

Table S2. The analysis of Catharanthus roseusleaf epidermome ESTs related to cytochrome P450

monoxygenase in GenBank database

Contig name designated in this study / EST accession number in GenBank / Subfamily / Putative function description
CrContig1 / FD660792.1,FD660876.1, FD660944.1, FD661056.1 / CYP71 / Geraniol-10-hydroxylasein indole alkaloids biosynthesis
CrContig2 / FD660784.1, FD661158.1 / CYP98 / p-coumaroyl-3-hydroxylase in Lignin biosynthesis
CrContig3 / FD660751, FD660752, FD660762, FD661072, FD660823, FD660827, FD660859, FD660886, FD660892, FD660923, FD660942, FD660968, FD660975, FD661038, FD661097, FD661214, FD661129, FD661288 / CYP71 / unknown
CrContig4 / FD660831, FD661068, FD661229 / CYP716 / unknown
CrContig5 / FD660807, FD661212 / CYP716 / unknown
CrContig6 / FD660792, FD660876, FD660916, FD661056 / CYP71 / Geraniol-10-hydroxylase in indole alkaloids biosynthesis
CrContig7 / FD660753, FD661166, FD661225, FD661266 / CYP71 / Tabersonine-16-hydroxylase in indole alkaloids biosynthesis
CrContig8 / FD660872, FD660896, FD660936, FD660972, FD661030, FD661188, FD661254 / CYP71 / unknown
CrContig9 / FD660812, FD660891, FD661110, FD661131, FD661125 / CYP77 / unknown
CrContig10 / FD660733, FD660964, FD661019, FD661224, FD661291 / CYP96 / unknown
CrContig11 / FD660746, FD660767, FD660790, FD660813, FD660825, FD660836, FD660841, FD660845, FD660847, FD660867, FD660873, FD660893, FD660899, FD660924, FD660926, FD660948, FD660958, FD660960, FD660980, FD661016, FD661028, FD661037, FD661040, FD661084, FD661111, FD661113, FD661142, FD661156, FD661189, FD661211, FD661231, FD661237, FD661258, FD661269, FD661281, FD661290 / CYP72 / Secologanin synthase in indole alkaloids biosynthesis
CrContig12 / FD660813, FD660825, FD660836, FD660845, FD660847, FD660899, FD660926, FD660924, FD660948, FD661113, FD660958, FD660980, FD661084, FD661111, FD661156, FD661189, FD661211, FD661269, FD661281 / CYP72 / Secologanin synthase in indole alkaloids biosynthesis
CrContig13 / FD660813, FD660825, FD660836, FD660845, FD660847, FD660899, FD660924, FD660926, FD660958, FD660980, FD661084, FD661111, FD661113, FD661156, FD661189, FD661211, FD661269, FD661281 / CYP71 / Geraniol-10-hydroxylase in indole alkaloids biosynthesis
CrContig14 / FD661231, FD660960, FD661237, FD660867, FD660841, FD661040, FD660767, FD661142, FD661016, FD660790, FD661258, FD661037, FD660873, FD660948, FD661290, FD660893, FD660746, FD661028 / CYP72 / Secologanin synthase in indole alkaloids biosynthesis
CrContig15 / FD660813, FD660825, FD660836, FD660845, FD660847, FD660899, FD660924, FD660958, FD660980, FD660926, FD661084, FD661111, FD661113, FD661156, FD661189, FD661211, FD661269, FD661281 / CYP72 / Secologanin synthase in indole alkaloids biosynthesis

Fig. S1EI-mass spectra of the products detected in the in vitro enzyme assaysof CrAO with α-amyrin (a), β-amyrin (b) and lupeol (c). Peaks 3-8 was corresponding to the authentic uvaol, ursolic acid, erythrodiol, oleanilic acid, betulin and betulinic acid, which wasshown in the right column, respectively