SMRT and Illumina RNA sequencing reveal the complexity of terpenoid biosynthesis in Zanthoxylum armatum

Sichuan pepper (Zanthoxylum armatum DC) is a popular spice and is often prescribed in traditional Chinese medicine to treat vomiting, diarrhea, ascariasis and eczema, among other conditions. Volatile oils from Z. armatum leaves contain active ingredients, with terpenoids being one of the main components. In the present study, the combination of sequencing data of Z. armatum from PacBio single molecule real time (SMRT) and Illumina RNA sequencing (RNA-Seq) platforms facilitated an understanding of the gene regulatory network of terpenoid biosynthesis in pepper leaves. The leaves of three developmental stages from two Z. armatum cultivars, 'Rongchangwuci' (WC) and 'Zhuye' (ZY), were selected as test materials to construct sequencing libraries. A total of 143,122 predictions of unique coding sequences, 105,465 simple sequence repeats, 20,145 transcription factors and 4719 long non-coding RNAs (lncRNAs) were identified, and 142,829 transcripts were successfully annotated. The occurrence of alternative splicing events was verified by reverse transcription PCR, and quantitative real-time PCR was used to confirm the expression pattern of six randomly selected lncRNAs. A total of 96,931 differentially expressed genes were filtered from different samples. According to functional annotation, a total of 560 candidate genes were involved in terpenoid synthesis, of which 526 were differentially expressed genes (DEGs). To identify the key genes involved in terpenoid biosynthesis, the module genes in different samples, including structural and transcription factors genes, were analyzed using the weighted gene co-expression network method, and the co-expression network of genes was constructed. Thirty-one terpenoids were identified by gas chromatography-mass spectrometry. The correlation between 18 compounds with significantly different contents and genes with high connectivity in the module was jointly analyzed in both cultivars, yielding 12 candidate DEGs presumably involved in the regulation of terpenoid biosynthesis. Our findings showed that full-length transcriptome SMRT and Illumina RNA-Seq can play an important role in studying organisms without reference genomes and elucidating the gene regulation of a biosynthetic pathway.