Running Head: Mevalonate Pathway Genes from Catharanthus Roseus
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Peroxisomal localisation of the final steps of the mevalonic acid pathway in planta
Andrew J. Simkin a,*, Grégory Guirimand a, Nicolas Papon a, Vincent Courdavault a, Insaf Thabet a,b, Sadok Bouzid b, Nathalie Giglioli-Guivarc’h a, Marc Clastre a
(a) Université François-Rabelais, EA 2106, Biomolécules et Biotechnologies végétales, 31 avenue Monge, 37200 Tours, France.
(b) Laboratoire de Biotechnologie et Physiologie Végétale, Faculté des Sciences de Tunis, Département des Sciences Biologiques, 2092 El-Manar II, Tunis, Tunisie.
* Corresponding author:
Supplementary Methods
Cloning of C. roseus cDNAs encoding enzymes of the MVA pathway
The sequences of the primers used for the isolation of the periwinkle cDNAs MVK, PMK and MVD are given in supplementary Table S1.
1. Mevalonate kinase
A partial length cDNA of 962 bp was recovered from seedling leaves by RT-PCR using degenerate primers CrMVKdegF2 and CrMVKdegR2 corresponding to the GEHAVVHG and SKLTGAGG conserved protein sequences of plant MVKs (Genbank accession numbers: AF429384, AC150244 and AF141853). PCR reactions were carried out using the GoTaq polymerase (Promega, France) and PCR conditions are described in (Simkin et al. 2006). Briefly, the PCR program was as follows: 9 amplification cycles of 95°C/30 sec, 1 min at 60-50°C (decreasing by 1°C per cycle) and 1 min at 72°C followed by 25 amplification cycles of 95°C/30 sec, 1 min at 50°C and 1 min at 72°C. The PCR product was cloned into pGEM-T Easy vector (Promega, France) to generate the plasmid pGEM-MVK962. A partial-coding sequence of 918 bp “minus degenerate primer regions” was obtained. An additional 48 bp of the 5’ sequence was recovered by Nested PhageWalker using a C. roseus cDNA library (Simkin AJ, unpublished data) constructed with the ZAP Express System (Stratagene, France). The first PCR reaction (50 µl) contained 200 nM of reverse primer GWMVK1 and forward primer corresponding to the phage plasmid T3 primer. PCR reaction conditions are as described for GenomeWalker (Simkin et al. 2008). Nested PCR was carried out using 1µl of a 1/50 dilution of the first PCR with primers GWMVK2 and T3. A band of approximately 550 bp was recovered, cloned into pGEM-T Easy vector to generate pGEM-PWMVK1 and sequenced. The remaining 21 bp, including the start ATG codon, were recovered by using the GenomeWalker kit (Clontech, France). PCR amplification of genomic DNA extracted from periwinkle leaf was done with primers GWMVK4 and AP1 (from the GenomeWalker kit) followed by nested PCR using primers GWMVK5 and AP2 (from the GenomeWalker kit). This generated a fragment of 523 bp, which was cloned into pGEM-T Easy vector to generate pGEM-GWMVK1. The resulting insert was sequenced giving the remaining 21 bp of the coding sequence and 476 bp upstream of the start ATG codon containing a part of the promoter region. The 3’ coding sequence of MVK was recovered by 3’RACE-PCR according to the manufacturer’s instructions (Invitrogen, France). Reverse transcription was made with primer AP followed by PCR amplification with the primer MVK_F and the reverse primer AUAP (from the 3’RACE-PCR kit) generating a fragment of 688 bp including the polyA tail.
2. Phosphomevalonate kinase
A partial length cDNA of 1336 bp was recovered from seedlings by RT-PCR using degenerate primers CrPMKdegF1 and CrPMKdegR1 corresponding to the MAVVASAP and GVPGAGGF conserved protein sequences of plant PMKs (Genbank accession numbers: AB294694 and AK221797). The PCR product was cloned into pGEM-T Easy vector to generate pGEM-PMK1336. A partial coding sequence of 1291 bp “minus degenerate primer regions” was obtained.
Using the Genomewalker kit, the 5’ coding sequence of the periwinkle PMK was obtained using PCR reaction conditions described above and primers GWPMK1 and AP1 followed by nested PCR using primers GWPMK2 and AP2. This generated a fragment of 582 bp, which was cloned into pGEM-T Easy vector to generate pGEM-GWPMK1. The resulting insert was sequenced giving the remaining 104 bp of the coding sequence, including an intron of 132 bp between bases 9 and 10 of the coding sequence. A region of 346 bp upstream of the start ATG codon was also recovered. The 3’ coding sequence of PMK was recovered by 3’RACE-PCR using primer PMK_F and reverse primer AUAP. This generated a fragment of 531 bp including the polyA tail.
3. Mevalonate 5-diphosphate decarboxylase
A partial cDNA clone of 760 bp was recovered from C. roseus seedlings using degenerate primers CrMVDdegF1 and CrMVDdegR1 corresponding to the DRMWLNGKE and DAGPNAV conserved protein sequences of plant MVDs (Genbank accession numbers: AB294695, AM433143 and Y17593). The PCR product of 760 bp was cloned in pGEM-T Easy vector to generate pGEM-MVD760. A partial coding sequence of 716 bp “minus degenerate primer regions” was obtained.
Using the Genewalker kit, the 5’ coding sequence of the periwinkle MVD was obtained using primers GWMVD1 and AP1 followed by nested PCR using primer GWMVD2 and AP2. This generated a fragment of 706 bp, which was cloned into pGEM-T Easy vector to generate pGEM-GWMVD1. The resulting insert was sequenced giving the remaining 296 bp of the coding sequence, including an intron of 144 bp between bases 217 and 218 of the coding sequence. A region of 266 bp upstream of the start ATG codon was also recovered. The 3’ coding sequence of periwinkle MVD was recovered by 3’RACE-PCR using primer MVD_F and reverse primer AUAP. This generated a fragment of 827 bp including the polyA tail.
References
Simkin, A.J., Qian, T., Caillet, V., Michoux, F., Ben Amor, M., Lin, C., Tanksley, S. and McCarthy, J. (2006) Oleosin gene family of Coffea canephora: quantitative expression analysis of five oleosin genes in developing and germinating coffee grain. J. Plant Physiol. 163, 691-708.
Simkin, A.J., Moreau, H., Kuntz, M., Pagny, G., Lin, C., Tanksley, S. and McCarthy, J.. (2008) An investigation of carotenoid and apocarotenoids biosynthesis in Coffea canephora and C. arabica. J. Plant Physiol. 165, 1087-1106.