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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 others. 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 and Illumina 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 4,719 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 DEGs. To identify the key genes involved in terpenoid biosynthesis, the module genes in different samples, including structural and TFs 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 sequencing can play an important role in studying organisms without reference genomes and elucidating the gene regulation of a biosynthetic pathway.

The WD40 transcription factor family is a superfamily found in eukaryotes and implicated in regulating growth and development. In this study, 167 WD40 family genes are identified in the Ginkgo biloba genome. They are divided into 5 clusters and 16 subfamilies based on the difference analysis of a phylogenetic tree and domain structures. The distribution of WD40 genes in chromosomes, gene structures, and motifs is analyzed. Promoter analysis shows that five GbWD40 gene promoters contain the MYB binding site participating in the regulation of flavonoid metabolism, suggesting that these five genes may participate in the regulation of flavonoid synthesis in G. biloba. The correlation analysis is carried out based on FPKM value of WD40 gene and flavonoid content in 8 tissues of G. biloba. Six GbWD40 genes that may participate in flavonoid metabolism are screened. The biological functions of the WD40 family genes in G. biloba are systematically analyzed, providing a foundation for further elucidating their regulatory mechanisms. A number of WD40 candidate genes involved in the biosynthesis and metabolism of G. biloba also predicted. This study presents an important basis and direction for conducting further research on the regulatory network of flavonoid synthesis and metabolism.

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