De Novo Transcriptome and Expression Profile Analysis to Reveal Genes and Pathways Potentially Involved in Cantharidin Biosynthesis in the Blister Beetle Mylabris cichorii.

The dried body of Mylabris cichorii is well-known Chinese traditional medicine. The sesquiterpenoid cantharidin, which is secreted mostly by adult male beetles, has recently been used as an anti-cancer drug. However, little is known about the mechanisms of cantharidin biosynthesis. Furthermore, there is currently no genomic or transcriptomic information for M. cichorii. In this study, we performed de novo assembly transcriptome of M. cichorii using the Illumina Hiseq2000. A single run produced 9.19 Gb of clean nucleotides comprising 29,247 sequences, including 23,739 annotated sequences (about 81%). We also constructed two expression profile libraries (20-25 day-old adult males and 20-25 day-old adult females) and discovered 2,465 significantly differentially-expressed genes. Putative genes and pathways involved in the biosynthesis of cantharidin were then characterized. We also found that cantharidin biosynthesis in M. cichorii might only occur via the mevalonate (MVA) pathway, not via the methylerythritol 4-phosphate/deoxyxylulose 5-phosphate (MEP/DOXP) pathway or a mixture of these. Besides, we considered that cantharidin biosynthesis might be related to the juvenile hormone (JH) biosynthesis or degradation. The results of transcriptome and expression profiling analysis provide a comprehensive sequence resource for M. cichorii that could facilitate the in-depth study of candidate genes and pathways involved in cantharidin biosynthesis, and may thus help to improve our understanding of the mechanisms of cantharidin biosynthesis in blister beetles

Length distribution of assembled unigenes.

<p>The number of y-axis has been transfer into logarithmic scale.</p

Gene Ontology analysis of genes in the Illumina <i>de novo</i> transcriptome of <i>M</i>. <i>cichorii</i>.

<p>The results are summarized in three main categories: biological process, cellular component, and molecular function. The righty-axis indicates the number of genes in a category. The left y-axis indicates the percentage of a specific category of genes in that main category.</p

Homology search of Illumina sequences against the Nr database.

<p><b>(A)</b> E-value distribution of BLAST hits for each unique sequence with a cut-off E-value of 1.0E^-5. <b>(B)</b> Species distribution of the top BLAST hits for each sequence. <b>(C)</b> Species distribution as a percentage of the total homologous sequences with an E-value ≥1.0E^-5. The first hit of each sequence was used for analysis.</p

“Terpenoid backbone biosynthesis” pathway of <i>M</i>. <i>cichorii de novo</i> transcriptome.

<p>The red box indicates the homologous enzyme of the unigenes have been found in the transcriptome databases of <i>M</i>. <i>cichorii</i>, while the white box represents there is no unigene has been found.</p

RT-qPCR analysis of 13 unigenes differentially-expressed between 20–25 day-old female and male groups.

<p>Line diagrams represent the expression patterns of selected unigenes from the <i>M</i>. <i>cichorii</i> transcriptome. Expression was compared between female and male groups. Error bars represent standard error. *Significant difference between gene expression (based on 20–25 day-old females, using Student’s <i>t</i>-test with p < 0.05).</p

COG functional annotation of putative proteins.

<p>Of 23,739 Nr hits, 8,580 sequences had COG classifications among the 25 categories. The y-axis indicates the number of sequences in a category.</p