Target genes for differentially expressed miRNAs involved in terpenoids biosynthesis.

<p>Target genes for differentially expressed miRNAs involved in terpenoids biosynthesis.</p

Additional file 1: Table S1. of Improvement of betulinic acid biosynthesis in yeast employing multiple strategies

Primers used in this study. Figure S1. The mass fragmented patterns of the products lupeol (LUP), betulin (BN) and betulinic acid (BA) as well as their corresponding chemical standards. Figure S2. The mass spectrums of the unknown peaks 1–3 shown in the Fig. 1 of the main text. Figure S3. GC-MS analysis of the products extracted from the lupeol C28-oxidase (LO) alone-expressed yeast cultures fed with lupeol. Figure S4. The production of betulinic acid (BA) was mostly found in the yeast culture mediums of both CEN.PK-ATR1-LB and WAT11-406-LB strains with relatively much less BA being detected inside the cells. Figure S5. Confirmation of the Gal80p gene disruption by diagnostic PCRs. Figure S6. Comparison of the BPLO transcripts between the wild type strain WAT11-LB and the mutant WAT11-LB-△Gal80 under 2 % galactose as the carbon source. (DOCX 831 kb

RT-qPCR data for the transcript abundance of some miRNAs in the leaves and glandular trichomes.

<p>The miRNA levels were normalized to an internal control (actin) and expressed relative to the values of leaves (control), which were given an arbitrary value of 1. Error bars indicate the standard deviation of three biological replicates.</p

Distribution of small RNAs among different categories in leaves and glandular trichomes of <i>X</i>. <i>strumarium</i>.

<p>^aThe reads that were matched to the <i>X</i>. <i>strumarium</i> transcriptome.</p><p>Distribution of small RNAs among different categories in leaves and glandular trichomes of <i>X</i>. <i>strumarium</i>.</p

GO functional classification for the predicted targets by the differentially expressed miRNAs.

<p>X-axis, the three main GO categories and 47 GO terms assigned for the differentially expressed miRNA targets; Y-axis, the gene numbers corresponding to the GO terms.</p

Statistics of small RNA sequencing.

<p>^aThe percentage of the tissue_specific unique reads for the respective tissue source.</p><p>Statistics of small RNA sequencing.</p

Nucleotide preference at each position of novel miRNAs.

<p>(A) miRNA nucleotide bias of novel miRNAs in leaves; (B) miRNA nucleotide bias of novel miRNAs in glandular trichomes.</p

Phylogenetic tree of terpene synthases.

<p>Phylogenetic analysis of 14 putative <i>W</i>. <i>ugandensis</i> WuTPS protein sequences with their homologs from other plants indicates that they are clustered into three main clades including: monoterpenoid synthase (WuMts), sesquiterpenoid synthase (WuSps), and diterpenoid synthase (WuDts). WarbTPS-c (ACJ46047.1, putative sesquiterpene synthase, <i>W</i>. <i>ugandensis</i>); WarbTPS-g (ACJ46048.1, putative sesquiterpene synthase, <i>W</i>. <i>ugandensis</i>); WuSps1, CL29873Contig1; WuSps2, CL29511Contig1; WuSps3, CL3178Contig1; WuSps4, CL4160Contig1; WuSps5, comp68897_c0_seq1; WuSps6, CL24969Contig1; WuSps7, CL30258Contig1; WuMts1, CL27268Contig1; WuMts2, CL27339Contig1; WuMts3, CL30385Contig1; WuMts4, CL276Contig2; WuMts5, CL1Contig9269; WuMts6, CL29966Contig1; WuMts7, CL14869Contig1; WuDts1, CL9128Contig1; WuDts2, CL28648Contig1; Citsi_Germacrene_D (XP_006494713.1, (-)-germacrene D synthase-like isoform X2, <i>Citrus sinensis</i>); Popeu_Valencene (XP_011015484.1, valencene synthase-like, <i>Populus euphratica</i>); Nelnu_Germacrene_D (XP_010258444.1, (-)-germacrene D synthase-like, <i>Nelumbo nucifera</i>); Eletr_Copaene (ADK94034.1, alpha-copaene synthase, <i>Eleutherococcus trifoliatus</i>); Gosar_Germacrene_D (KHG04103.1, (-)-germacrene D synthase, <i>Gossypium arboretum</i>); Vitvi_Germacrene_D (XP_010644711.1, (-)-germacrene D synthase, <i>Vitis vinifera</i>); Vitvi_Germacrene_A (ADR66821.1, Germacrene A synthase, <i>Vitis vinifera</i>); Citja_Elemene (BAP74389.1, delta-elemene synthase, <i>Citrus jambhiri</i>); Theca_Cadinene (EOY12648.1, Delta-cadinene synthase isozyme A, <i>Theobroma cacao</i>); Ricco_Cadinene (EEF38721.1, (+)-delta-cadinene synthase isozyme A, <i>Ricinus communis</i>); Aqusi_Guaiene (AIT75875.1, putative delta-guaiene synthase, <i>Aquilaria sinensis</i>); Vitvi_Caryophyllene (AEP17005.1, (E)-beta-caryophyllene synthase, <i>Vitis vinifera</i>); Maggr_Cubebene (ACC66281.1, beta-cubebene synthase, <i>Magnolia grandiflora</i>); Cinos_Linalool (AFK09265.1, S-(+)-linalool synthase, <i>Cinnamomum osmophloeum</i>); Nelnu_Nerolidol (XP_010248179.1, (3S,6E)-nerolidol synthase 1-like, <i>Nelumbo nucifera</i>); Vitvi_Nerolidol (XP_010646919.1, (3S,6E)-nerolidol synthase 1, chloroplastic-like isoform X1, <i>Vitis vinifera</i>); Vitvi_Linalool (ADR74212.1, (3S)-linalool/(E)-nerolidol synthase, <i>Vitis vinifera</i>); Actpo_Linalool (ADD81295.1, linalool synthase, <i>Actinidia polygama</i>); Nelnu_Ent-copalyl (XP_010277558.1, ent-copalyl diphosphate synthase, chloroplastic-like, <i>Nelumbo nucifera</i>); Theca_Ent-copalyl (XP_007050589.1, Copalyl diphosphate synthase, <i>Theobroma cacao</i>); Morno_Ent-copalyl (XP_010090409.1, Ent-copalyl diphosphate synthase, <i>Morus notabilis</i>); Gosar_Ent-copalyl (KHG01750.1, Ent-copalyl diphosphate synthase, chloroplastic, <i>Gossypium arboreum</i>); Nelnu_Ent-kaurene (XP_010260722.1, ent-kaur-16-ene synthase, chloroplastic isoform X1, <i>Nelumbo nucifera</i>); Phoda_Ent-kaurene (XP_008809130.1, ent-kaur-16-ene synthase, chloroplastic, <i>Phoenix dactylifera</i>); Ricico_Ent-kaurene (XP_002533694.1, Ent-kaurene synthase B, chloroplast precursor, <i>Ricinus communis</i>); Popeu_Ent-kaurene (XP_011014299.1, ent-kaur-16-ene synthase, chloroplastic, <i>Populus euphratica</i>); Nicta_Epi-Aristolochene (3M02.A, 5-Epi-Aristolochene Synthase, <i>Nicotiana tabacum</i>); Soltu_Vetispiradiene (Q9XJ32.1, vetispiradiene synthase 1, <i>Solanum tuberosum</i>); Litcu_Ocimene (AEJ91554.1, trans-ocimene synthase, <i>Litsea cubeba</i>); Litcu_Thujene (AEJ91555.1, alpha-thujene synthase, <i>Litsea cubeba</i>); Citli_Limonene (AAM53946.1, (+)-limonene synthase 2, <i>Citrus limon</i>); Vitvi_Ocimene/Myrcene (ADR74206.1, (E)-beta-ocimene/myrcene synthase, <i>Vitis vinifera</i>); Queil_Pinene (CAK55186.1, pinene synthase, <i>Quercus ilex</i>).</p

Summary of unigenes related to lipid and terpenoid metabolism.

<p>Summary of unigenes related to lipid and terpenoid metabolism.</p

Statistic of sequencing and <i>de novo</i> assembling of transcriptome in <i>W</i>. <i>ugandensis</i>.

<p>Statistic of sequencing and <i>de novo</i> assembling of transcriptome in <i>W</i>. <i>ugandensis</i>.</p