S1 File - Functional Analysis of the Brassica napus L. Phytoene Synthase (PSY) Gene Family

Contains the following files: S1 Figure. PSY Heterologous Complementation System. A. Two E. coli BL21-Gold strains were used, a β-carotene producer strain (DS1B) transformed with plasmid pDS1B, a pBAD33 vector carrying Erwinia uredovora carotenogenic genes crtE, crtB, crtI, crtY and CrtX and a non-producer strain (DS1B-ΔcrtB) transformed with plasmid pDS1B-ΔcrtB which has a deletion of the Eu crtB gene. B. Six pETBlue1-BnaX.PSY vectors were used, each carrying a B. napus PSY homologue, without its corresponding signal peptide, cloned into the EcoRV site. S2 Figure. Multiple nucleotide BnaX.PSY sequence alignment. S3 Figure. Homologue-specific PCR primer control reactions. A. Primer specificity was tested by PCR using plasmids containing each of the six B. napus PSY genes. Primers BnaC.PSY.a and BnaA.PSY.b could not be tested against BnaA.PSY.d and BnaC.PSY.f (clones did not include 5′UTRs). B. SSCP analysis of BnaC.PSY.a RT-PCR reactions show that only two strands exhibiting the same exact pattern as the BnaC.PSY.a plasmid control are present, confirming primer specificity. C. SSCP analysis of BnaA.PSY.b RT-PCR reactions show that only two strands exhibiting the same exact pattern as the BnaA.PSY.b plasmid control are present, confirming primer specificity. L: 100 bp ladder; BnaX.PSY.a-f: plasmid DNA controls; gDNA: B. napus genomic DNA control; WC: water control; ND. Not determined; 20 dpa: seed cDNA 20 days post anthesis; Leaf: leaf cDNA. S4 Figure. Alignment of B. napus PSY proteins with squalene synthase and carotenoid dehydrosqualene synthase templates. 2zcs: Staphylococcus aureus Dehydrosqualene synthase complexed with BPH-700; 3acx: Staphylococcus aureus Dehydrosqualene synthase complexed with BPH-673; 4e9u: Staphylococcus aureus Dehydrosqualene synthase complexed with thiocyanate inhibitor; 2zco: Staphylococcus aureus Dehydrosqualene synthase; 4hdl: PpcA F15L mutant from Geobacter sulfurreducens; 3vj8: Homo sapiens Squalene synthase. S5 Figure. Tridimensional enzyme structure prediction of B. napus PSY homoelogous pairs. Alpha-helices are shown in green and the putative active site (DXXXD) in purple with the four conserved aspartate residues shown as licorice representations [49]. S1 Table. Oligonucleotide primers used in this study. S2 Table. BnaX.PSY protein sequence identity (%). S3 Table. Ka/Ks. (DOCX)