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Genetic variation and metabolic pathway intricacy govern the active compound content and quality of the Chinese medicinal plant Lonicera japonica thunb

Yuan Yuan1, Lipu Song2, Minhui Li14, Guiming Liu2, Yanan Chu2, Luyu Ma3, Yuanyuan Zhou2, Xiao Wang15, Wei Gao1, Shuangshuang Qin1, Jun Yu2, Xumin Wang2* and Luqi Huang1*

Author Affiliations

1 Beijing Key Laboratory of Functional Genomics for Dao-di Herbs,Institute of Chinese Materia Medica, Academy of Chinese Medical Sciences, Beijing, 100700, China

2 CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100029, China

3 Shandong Academy of Medical Sciences, Jinan, 250031, China

4 Baotou Medical College, Baotou, 014040, China

5 Shandong Analysis and Test center, Jinan, 250014, China

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BMC Genomics 2012, 13:195  doi:10.1186/1471-2164-13-195

Published: 20 May 2012

Additional files

Additional file 1:

Figure S1. Comparing volatile compound of FLJ and rFLJ using Gas chromatography-mass spectrometry. A, Bud, B, flower1, C, flower2. Black line represent FLJ and Red line represent rFLJ. FLJ, Lonicera japonica Thumb; rFLJ, Lonicera japonica Thunb. var. chinensis (Wats.). Figure S2. HPLC analysis of active compounds in FLJ and rFLJ. A, Mix of standard compounds. Chlorogenic acid (RT, 13.20; MW, 354.31); Caffeic acid (RT, 17.39; MW, 180.15); ferulic acid (RT, 24.24; MW, 194.18); Rutin (RT, 24.24; MW, 610.52); Luteoloside(RT, 25.27; MW,448.4); Hyperoside (RT, 25.35; MW, 464.37); Quercitrin (RT, 28.65; MW, 448.38); Quercetin (RT, 38.04; MW, 302.24). B, buds of rFLJ; C, buds of FLJ. FLJ, Lonicera japonica Thumb; rFLJ, Lonicera japonica Thunb. var. chinensis (Wats.). Figure S3. BlastX analysis result of contigs in FLJ and rFLJ with all non-redundant (NR) database in Genebank. Six species,Vitis vinifera, Ricinus communis, Populus trichocarpa, Glycine max, Arabidopsis lyrata, Nicotiana tabacum has highest identity with FLJ and rFLJ bud. E-value cut-off was lower than 1e-5. FLJ, Lonicera japonica Thumb; rFLJ, Lonicera japonica Thunb. var. chinensis (Wats.). Figure S4. Pathways of major chemical compounds inLonicera japonicaThumb (FLJ). All of contigs from three FLJ libraries were annotated with KEGG database. The six pathways, phenylalanine metabolism, terpenoid backbone, fatty acid biosynthesis, citric acid cycle, glycolysis and sucrose metabolism were analysis. Green rectangles repress enzymes finding in FLJ transcriptome. Figure S5. Analysis of gene differential express using MA-plot-based method. M is the Y -axis and represents the intensity ratio, and A is the X-axis and represents the average intensity for each transcript. The red points are the genes identity as differentially expressed. FLJ, Lonicera japonica Thumb; rFLJ, Lonicera japonica Thunb. var. chinensis (Wats.). Figure S6 Pathway assignment based on KEGG analysis of differential express genes between buds and other two flower developmental periods ofLonicera japonicaThumb (Group1). A, Number of contig with down-regulated and up-regulated differential express genes. B, Number of contig with only up-regulated differential express genes. Figure S7. Gene Ontology classification of differential express genes. The results are summarized in three main categories: Biological process, Cellular component and Molecular function. FLJ, Lonicera japonica Thumb; rFLJ, Lonicera japonica Thunb. var. chinensis (Wats.). A, Gene Ontology classification of differential express genes between buds and two other flower developmental period of FLJ. Bud and flower1, differential express genes between bud and flower1; Bud and flower2, differential express genes between bud and flower2; up-regulated, up-regulated express genes in both in between bud and flower1 and in between bud and flower2; down-regulated, down-regulated express genes in both in between bud and flower1 and in between bud and flower2. B, Gene Ontology classification of differential express genes between buds of FLJ and rFLJ.Group2, differential express genes between buds of FLJ and rFLJ. up-regulated, up-regulated express genes; down-regulated, down-regulated express genes. Figure S8. Gene express level in bud, flower1 and flower2 ofLonicera japonica Thumb. Square represents gene express level and nine kinds of color indicate rpkm of scaffolds. B, bud;F1, flower1; F2, flower2. rpkm of scaffolds was calculated according to Grape full-length cDNA sequence. Figure S9. Phylogenetic analysis of the predicted amino acids sequences and expression level of the Phenylalanine ammonia-lyase (PAL), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and chalcone synthase (CHS) homologues among the buds of FLJ and rFLJ and PAL,HMGR and CHS family genes in Arabidopsis and Grape. The phylogenetic tree was constructed by the neighborjoining method using ClustalW2. Identification of PAL,HMGR and CHS homologues was by searching the domain(PF00221, PF00368 and PF00195, respectively) in PFAM database. FLJ, Lonicera japonica Thumb; rFLJ, Lonicera japonica Thunb. var. chinensis (Wats).

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Additional file 2:

Table S1. The Elution Conditions of HPLC Analysis. Note: T, Retention time; A, mobile phase deionized water- formic acid (99:1, v/v); B, mobile phase methanol. Table S2. Formula of Active Compound Content. Note: Calibration plots of eight standards were constructed on the basis of peak areas (y) using seven different concentration solutions (x). All plots were linear in the examined ranges, and linear ranges had been shown as the concentration of the standard compounds (μg mL-1). The r referred to the correlation coefficient of the equation. The standard compounds Chlorogenic acid (110753), Caffeic acid(110885), ferulic acid(110773), Rutin(100080), Luteoloside(111720), Hyperoside(111521), Quercitrin(111538) and Quercetin (100081) were purchased from National institutes for food and drug control, China. Table S3. Number of Contigs in KEGG Pathways. Note: FLJ, Lonicera japonica Thumb; rFLJ, Lonicera japonica Thunb. var. chinensis (Wats.); B, bud; F1,flower1; F2, flower2. Table S4. Predicted Phosphorylated Sites in 34 Protein Sequence from Differential Express Gene. Note: Predicted Phosphorylated Sites using software online (http://kinasephos2.mbc.nctu.edu.tw/) and protein sequence was perdicted by ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html webcite). Table S5. qRT-PCR and RNA-seq Analysis of Gene Express between Buds ofLonicera japonicaThunb. var. chinensis (Wats.) andLonicera japonicaThumb. Note: RR/YR, the ratio of transcripted level in buds of Lonicera japonica Thunb. var. chinensis (Wats.) and Lonicera japonica Thumb. Table S6. Orthologs Identity of Differential Express Genes Sequence between FLJ and rFLJ. Note: FLJ, Lonicera japonica Thumb; rFLJ, Lonicera japonica Thunb. var. chinensis (Wats.) Table S7 PAL, CHS, HMGR and PK Gene Families in Arabidopsis and Grape. Table S8. Putative Enzyme Pool to Control the Active Compounds in Buds ofLonicera japonicaThumb.

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