Molecular Biology Reports

Molecular Biology Reports

Characterization and functional analysis of the genes encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase and 1-deoxy-D-xylulose-5-phosphate synthase, the two enzymes in the MEP pathway, from Amomum villosum Lour.

Jinfen Yang, Megha Nath Adhikari, Hui Liu, Hui Xu, Guozhen He, Ruoting Zhan, Jieshu Wei,Weiwen Chen

The affiliations and addresses of the authors:

J. Yang, H. Liu, H. Xu, R. Zhan, J. Wei, W. Chen

Research Center of Chinese Herbal Resource Science and Engineering, Guangzhou University of Chinese Medicine, 232 Waihuandong Road, Guangzhou Higher Education Mega Center, Guangzhou 510006, People’s Republic of China

M. N. Adhikari, G. He

College of Chinese Medicine, Guangzhou University of Chinese Medicine, 232 Waihuandong Road, Guangzhou Higher Education Mega Center, Guangzhou 510006, People’s Republic of China

corresponding author:

Prof. Dr. Weiwen Chen

E-mail: or

Phone: 8620-39358331, Fax: 8620-39358066

Online Supplementary Table 1Primers used in this study

Primers / Nucleotide sequences
Primers for the isolation of AvDXR
AD1
AD2 / 5’-ATTCCTGGGGAGCAAGGTGT(C/T)ATAG-3’
5’-GCAATTGAAGCTGG(G/A/T)AAAGACATAG-3’
AD3 / 5’-CCAGGTG ACCTC(G/A/T)GAGCAATAGA-3’
AD4 / 5’-CTGAGCCA(A/G)(G/A/C)ACAGATGAATCCTG-3’
AD51 / 5’-GCTTCAGAGCATCAGCAACCTTCACATC-3’
AD52 / 5’- TCACATTGTGTTTGTGCGCAAGTGGTAGCAC-3’
AD31 / 5’-CACTGTGGATTCTGCTACCCTGTTC-3’
AD32 / 5’- CGATAATCCATTCCATGGTCGAGACT-3’
ADO1 / 5’-GTGCGGGTTGCAAAGATGACACTC-3’
ADO3 / 5’-GCATAAAGAAAAAATGTCTCAAGC-3’
Primers for the isolation of AvDXS
AS1 / 5’-CT(G/T/C)CACTA(C/T)GTCTTCAATGC(G/A/T)CC-3’
AS2 / 5’-ACAAGATGCCGAC(G/A/T)ATGCG(G/A/T)CAG-3’
AS3 / 5’-GAT(G/T)GCGCAGAA(G/A/C)GGCTT(A/G)AGGCC-3’
AS4 / 5’-AAGCGGCGrAGGAAGTAGTT(G/A/C)AG-3’
AS51 / 5’-CGGGACCTGTCGTCTTTGTGCTCTTG-3’
AS52 / 5’-GTGCTCTTGACCTCTTTCAGGATGGTG-3’
AS31 / 5’-CACCATCCTGAAAGAGGTCAAGAGCAC-3’
AS32 / 5’-GGTCAAGAGCACAAAGACGACAGGTCC-3’
ASO1 / 5’-GTGATCTTCCTCATGTCTCTTTC-3’
ASO2 / 5’-TGTGCTTTCCTACGACATCATTTA-3’
Primers for theconstruction of pTrc-AvDXR and pTrc-AvDXS
ADP1 / 5’-GTGAGATCTaGCATGCbATGACACTCAG-3’
ADP2 / 5’-CGCGAATTCcCTCAAGCAGCAACAG-3’
ASP1 / 5’- CTTCCGCATGCbATGACTGAAC-3’
ASP2 / 5’- CGATTTGTGAATTCcCTACGACA-3’
a: BglⅡrestriction site, b: SphⅠrestriction site, c: EcoRⅠrestriction site.
Primers for semi-quantitative RT-PCR
ADR1 / 5’-GCTATTGAGGCTGGGAAAGACATA-3’
ADR2 / 5’-TTGAGCCAATATAGATGAGTCCTG-3’
ASR1 / 5’-GGAGGTGGCCAAGGGAGTGACAA-3’
ASR2 / 5’-GATGGCGCAGAAAGGCTTGAGGC-3’
18SF / 5’-CGCTCTGGATACATTAGCATGG-3’
18SR / 5’-GACAAATCGCTCCACCAACTAAG-3’
Primers for theconstruction of pBI-AvDXR
ADP3 / 5’-GTGCGGTCTAGAdAAGATGACACTC-3’
ADP4 / 5’-GCATCCCGGGeAAAATGTCTCAAGC-3’
d: XbaⅠrestriction site, e: SmaⅠrestriction site.
Primers for thescreening of AvDXR transgenic tobbaco
35S2 / 5’-GTGATATCTCCACTGACGTAAGGG-3’
AD53 / 5’-TGGCAGAATGTTCTGAATCAGCAGG-3’
ADR3 / 5’-AGTCCAGCCGAGGTACCTTTACC-3’
ADR4 / 5’-CCTGATGCAGTCAAAATTATGCGAC-3’

Online Supplementary Fig. 1 Phylogenetic analysis of AvDXR with DXRs from 11 other plant species

The species source of sequences used for generating the phylogenetic tree and their GenBank accession numbers are indicated in the right. The bar represents the branch length equivalent to 0.05 amino acid changes per residue.

Online Supplementary Fig.2Multiple alignment of AvDXR with DXRs from other plants

AvDXR, ZmDXR, OsDXR, HvDXR, HbDXR and CsDXR indicate DXR from Amomum villosum, Zea mays (ACG33012), Oryza sativa (BAB78606), Hordeum vulgare (CAE47438), Hevea brasiliensis (ABD92702) and Croton stellatopilosus (ABO38177), respectively.Black regions show the homologous regions. The cleavage site of chloroplast transit peptide is indicated with an arrowhead. The Pro-rich region of PPPAWPG(R/T)A is marked with the double horizontal line and two putative NADPH-binding domains (GSTGSIGT and LAAGSNV) are marked with the horizontal line on the top. Two substrate-binding domains (LPADSEHSAI and NKGLEVIEAHY) are indicated with asterisks.

Online Supplementary Fig.3Conserved domains and protein classification of AvDXR

The deduced amino acid sequence of AvDXR(GenBank protein accession no. ACS26204)was used to predict the conserved domain and it is classified as DXR (PRK05447) based on the conserved domain database on GenBank.

Online SupplementaryFig. 4 Phylogenetic analysis of AvDXS with DXSs from 18 other plant species

The species source of sequences used for generating the phylogenetic tree and their GenBank accession numbers are indicated in the right.The bar represents the branch length equivalent to 0.05 amino acid changes per residue.

Online SupplementaryFig.5 Multiple alignment of AvDXS with DXSs from other plants.

AvDXS, EgDXS, MtDXS, PmDXSand ZmDXS indicate DXS from Amomum villosum,Elaeiguineensis (AAS99588), Medicago truncatula (CAE47438), Pueraria montana var. lobata (AAQ84169) and Zea mays (ABP88134),respectively.Black regions show the homologous regions. The cleavage site of chloroplast transit peptide is indicated with an arrowhead. The histidine residue for DXS catalysis is marked by a star and the invariant glutamic acid residue is indicated with a triangle. The TPP-binding domain is marked with the horizontal line and the pyridine binding DRAG domain is indicated with the double horizontal line on the top.

Online SupplementaryFig.6Conserved domains and protein classification of AvDXS

The deduced amino acid sequence of AvDXS(GenBank protein accession no. ACR02668)was used to predict the conserved domain and it is classified as DXS (PRK05444) based on the conserved domain database on GenBank.

Online Supplementary Fig.7 EI-MSchromatograms of caryophyllene, santalol and m-Mentha-4,8-diene