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Here, we cloned a phytoene desaturase (PDS) gene from Rehmannia glutinosa, and realized RgPDS1 knock out in R. glutinosa resulted in the generation of albino plants. Rehmannia glutinosa is a highly important traditional Chinese medicine (TCM) with specific pharmacology and economic value. R. glutinosa is a tetraploid plant, to date, no report has been published on gene editing of R. glutinosa. In this study, we combined the transcriptome database of R. glutinosa and the reported phytoene desaturase (PDS) gene sequences to obtain the PDS gene of R. glutinosa. Then, the PDS gene was used as a marker gene to verify the applicability and gene editing efficiency of the CRISPR/Cas9 system in R. glutinosa. The constructed CRISPR/Cas9 system was mediated by Agrobacterium to genetically transform into R. glutinosa, and successfully regenerated fully albino and chimeric albino plants. The next-generation sequencing (NGS) confirmed that the albino phenotype was indeed caused by RgPDS gene target site editing, and it was found that base deletion was more common than insertion or replacement. Our results revealed that zCas9 has a high editing efficiency on the R. glutinosa genome. This research lays a foundation for further use of gene editing technology to study the molecular functions of genes, create excellent germplasm, accelerate domestication, and improve the yield and quality of R. glutinosa.

Rehmannia glutinosa L., a perennial plant of Scrophulariaceae, is one of the most commonly used herbs in traditional Chinese medicine (TCM) that have been widely cultivated in China. However, to date, the biosynthetic pathway of its two quality-control components, catalpol and acteoside, are only partially elucidated and the mechanism for their tissue-specific accumulation remains unknown. To facilitate the basic understanding of the key genes and transcriptional regulators involved in the biosynthesis of catalpol and acteoside, transcriptome sequencing of radial striation (RS) and non-radial striation (nRS) from four R. glutinosa cultivars was performed. A total of 715,158,202 (~107.27 Gb) high quality reads obtained using paired-end Illumina sequencing were de novo assembled into 150,405 transcripts. Functional annotation with multiple public databases identified 155 and 223 unigenes involved in catalpol and acteoside biosynthesis, together with 325 UGTs, and important transcription factor (TF) families. Comparative analysis of the transcriptomes identified 362 unigenes, found to be differentially expressed in all RS vs. nRS comparisons, with 143 upregulated unigenes, including those encoding enzymes of the catalpol and acteoside biosynthetic pathway, such as geranyl diphosphate synthase (RgGPPS), geraniol 8-hydroxylase (RgG10H), and phenylalanine ammonia-lyase (RgPAL). Other differentially expressed unigenes predicted to be related to catalpol and acteoside biosynthesis fall into UDP-dependent glycosyltransferases (UGTs), as well as transcription factors. In addition, 16 differentially expressed genes were selectively confirmed by real-time PCR. In conclusion, a large unigene dataset of R. glutinosa generated in the current study will serve as a resource for the identification of potential candidate genes for investigation of the tuberous root development and biosynthesis of active components.

Tyrosine decarboxylase (TyrDC) is an important enzyme in the secondary metabolism of several plant species, and was hypothesized to play a key role in the biosynthesis of phenylethanoid glycosides. Based on the transcriptome data, we cloned the full-length cDNA (GenBank accession NO. KU640395) of RgTyDC gene from Rehmannia glutinosa, and then performed bioinformatic analysis of the sequence. Further, we detected the expression pattern in different organs and hair roots treated with four elicitors by qRT-PCR. The results showed that the full length of RgTyDC cDNA was 1 530 bp encoding 509 amino acids. The molecular weight of the putative RgTyDC protein was about 56.6 kDa and the theoretical isoelectric point was 6.25. The RgTyDC indicated the highest homology with Sesamum indicum SiTyDC and Erythranthe guttata EgTyDC, both of them were reached 88%. RgTyDC highly expressed in R. glutinosa leaf, especially in senescing leaf, and rarely expressed in tuberous root. After the treatment of SA and MeJA, the relative expression level of RgTyDC mRNA was substantially increased. The results provide a foundation for exploring the molecular function of RgTyDC involved in phenylethanoid glycosides biosynthesis.