Research Of Transcriptome And Biosynthetic Pathway Of TIAs In Catharanthus Roseus

Catharanthus roseus is not only the sole source of anticancer drugs-vinblastine and vincristine, but also the model plant for the study of terpenoid indole alkaloids (TIAs) biosynthesis and their regulations. In this study, we sequenced and analyzed the transcriptome of three C. roseus tissues (flower, leaf and root) using next-generation sequencing technology (Illumina) and next-next-generation sequencing technology (PacBio). About 140 million short reads were generated using Illumina platform and assembled into a total of 191,167 unigenes with an average length of 674 bp. Comprehensive functional annotation revealed the representation of many genes involved in different biological processes and molecular functions. In total,56.12% of C. roseus transcripts showed homology with sequences available in 7 public databases. Gene ontology (GO) analysis discovered 261 genes possibly involved in secondary metabolic process, while euKaryotic Ortholog Groups (KOG) analysis discovered 1809 genes involved in secondary metabolites biosynthesis, transport and catabolism. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis discovered 604 genes involved in metabolism of terpenoids and polyketides and 12 genes involved in indole alkaloid biosynthesis. Differential expression analysis showed that most of characterized genes involved in TIAs biosynthesis are expressed higher in roots and leaves than flowers. Interestingly, genes in the branch pathway of vindoline biosynthesis are expressed higher in aerial tissues. In general, the genes in the same biosynthetic pathway are co-expressed. Based on the co-expression analysis, eight cytochrome P450s (CYP450s) and five functional genes were selected as the candidates most likely involved in TIAs biosynthesis. In addition, a total of 786,008 long reads were generated from the three tissues using PacBio platform, including 264,553 from the flowers,262,140 from the roots and 259,315 from the leaves. Pre-processing resulted in 378,581 full-length reads (123,665 in flower,120,510 in root and 134,406 in leaf) and 311,081 non-full-length reads (105,613 in flower,106,459 in root and 99,009 in leaf). Some possible gene isoforms of enzymes and transcription factors involved in TIAs pathway were found using the full-length reads, indicating alternative splicing might take part in the regulation of TIAs biosynthesis. Such large-scale transcriptome study by different platforms provides a rich source for understanding C. roseus-specialized secondary metabolism, and the long reads generated by PacBio technology will improve our understanding on the roles of alternative splicing in the regulation of TIAs biosynthesis.We also explored the feasibility of production of TIAs in heterologous hosts such as yeast. Firstly, codon usage bias of C. roseus was analyzed based on transcriptome data. Rare codons of yeast are seldom used in C. roseus, indicating that yeast may be a suitable host for heterologous expression of genes from C. roseus. All complete CDS of the ten genes involved in iridoid pathway were cloned and no differences were found compared with the corresponding sequences deposited in GenBank, except CPR, DLGT, IRS and LAMT. Expression cassettes of GES, G10H and CPR were built using SOE-PCR strategy. A vector harboring the three cassettes will be constructed by yeast in vivo recombination. We try to set up a procedure from part selection, device construction to multi-cassette construction by in vivo recombination, thus laying the foundation for the reconstruction of a complete TIA biosynthetic pathway in yeast.