Liu Xiaomeng, Tang Ning, Xu Feng, Chen Zexiong, Zhang Xian, Ye Jiabao, Liao Yongling, Zhang Weiwei, Kim Soo-Un, Wu Peiyin, Cao Zhengyan
College of Horticulture and Gardening, Yangtze University, Jingzhou 434025, Hubei, China.
College of Landscape Architecture and Life Science, Chongqing University of Arts and Sciences, Chongqing 402160, China.
Tree Physiol. 2022 Mar 9;42(3):664-683. doi: 10.1093/treephys/tpab114.
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.
花椒（竹叶花椒）是一种广受欢迎的香料，在传统中药中常用于治疗呕吐、腹泻、蛔虫病和湿疹等病症。竹叶花椒叶片中的挥发油含有活性成分，萜类化合物是主要成分之一。在本研究中，结合来自PacBio单分子实时（SMRT）测序和Illumina RNA测序（RNA-Seq）平台的竹叶花椒测序数据，有助于了解花椒叶片中萜类生物合成的基因调控网络。选取两个竹叶花椒品种‘荣昌无刺’（WC）和‘竹叶’（ZY）三个发育阶段的叶片作为试验材料构建测序文库。共鉴定出143,122个独特编码序列预测、105,465个简单序列重复、20,145个转录因子和4719个长链非编码RNA（lncRNA），并成功注释了142,829个转录本。通过逆转录PCR验证了可变剪接事件的发生，并使用定量实时PCR确认了6个随机选择的lncRNA的表达模式。从不同样本中筛选出96,931个差异表达基因。根据功能注释，共有560个候选基因参与萜类合成，其中526个为差异表达基因（DEG）。为了鉴定参与萜类生物合成的关键基因，采用加权基因共表达网络方法分析不同样本中的模块基因，包括结构基因和转录因子基因，并构建基因共表达网络。通过气相色谱-质谱法鉴定出31种萜类化合物。联合分析两个品种中18种含量差异显著的化合物与模块中高连通性基因之间的相关性，得到12个可能参与萜类生物合成调控的候选DEG。我们的研究结果表明，全长转录组SMRT和Illumina RNA-Seq在研究无参考基因组的生物以及阐明生物合成途径的基因调控方面可发挥重要作用。
