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)本数据集包含以下文件： 图版S1：PSY异源互补系统（PSY Heterologous Complementation System） A. 本研究使用两株大肠杆菌BL21-Gold（E. coli BL21-Gold）菌株：其一为β-胡萝卜素（β-carotene）生产菌株DS1B，该菌株转化有携带欧文氏菌（Erwinia uredovora）类胡萝卜素合成基因crtE、crtB、crtI、crtY及CrtX的质粒pDS1B；另一株为非生产菌株DS1B-ΔcrtB，其转化有缺失欧文氏菌crtB基因的质粒pDS1B-ΔcrtB。 B. 本研究使用六株pETBlue1-BnaX.PSY载体，每株载体均带有1株甘蓝型油菜（B. napus）PSY同源基因，且去除了其对应信号肽，克隆至EcoRV酶切位点。 图版S2：BnaX.PSY多核苷酸序列比对（Multiple nucleotide BnaX.PSY sequence alignment） 图版S3：同源特异性PCR引物对照反应（Homologue-specific PCR primer control reactions） A. 通过PCR验证引物特异性，所用模板为分别携带六株甘蓝型油菜PSY基因的质粒。引物BnaC.PSY.a与BnaA.PSY.b无法针对BnaA.PSY.d及BnaC.PSY.f进行验证（对应克隆未包含5′非翻译区（5′UTR））。 B. 针对BnaC.PSY.a的逆转录聚合酶链式反应（Reverse Transcription Polymerase Chain Reaction, RT-PCR）产物的单链构象多态性（Single-Strand Conformation Polymorphism, SSCP）分析显示，仅存在两条与BnaC.PSY.a质粒对照完全一致的条带，证实了引物特异性。 C. 针对BnaA.PSY.b的RT-PCR产物的SSCP分析显示，仅存在两条与BnaA.PSY.b质粒对照完全一致的条带，证实了引物特异性。 其中，L：100 bp DNA分子量标准；BnaX.PSY.a-f：质粒DNA对照；gDNA：甘蓝型油菜基因组DNA（genomic DNA, gDNA）对照；WC：无菌水对照；ND：未测定；20 dpa：开花后20天的种子互补DNA（cDNA）；Leaf：叶片cDNA。 图版S4：甘蓝型油菜PSY蛋白质与鲨烯合酶、类胡萝卜素脱氢鲨烯合酶模板的序列比对（Alignment of B. napus PSY proteins with squalene synthase and carotenoid dehydrosqualene synthase templates） 各模板信息如下：2zcs：结合BPH-700的金黄色葡萄球菌（Staphylococcus aureus）脱氢鲨烯合酶复合物；3acx：结合BPH-673的金黄色葡萄球菌脱氢鲨烯合酶复合物；4e9u：结合硫氰酸盐抑制剂的金黄色葡萄球菌脱氢鲨烯合酶；2zco：金黄色葡萄球菌脱氢鲨烯合酶；4hdl：硫还原地杆菌（Geobacter sulfurreducens）来源的PpcA F15L突变体；3vj8：人（Homo sapiens）鲨烯合酶。 图版S5：甘蓝型油菜PSY同源对的三维酶结构预测（Tridimensional enzyme structure prediction of B. napus PSY homologous pairs） 其中α螺旋（Alpha-helices）以绿色显示，推定活性位点（DXXXD基序）以紫色显示，四个保守天冬氨酸残基以棍状模型展示[49]。 表S1：本研究使用的寡核苷酸引物（Oligonucleotide primers used in this study） 表S2：BnaX.PSY蛋白质序列同源性（%）（BnaX.PSY protein sequence identity (%)） 表S3：Ka/Ks比值（Ka/Ks） 文件格式：DOCX