Abstract
BackgroundBletilla striata has been widely used in the pharmacology industry. To effectively produce the secondary metabolites through suspension cultured cells of B. striata, it is important to exploring the full-length transcriptome data and the genes related to cell growth and chemical producing of all culture stages. We applied a combination of Real-Time Sequencing of Single Molecule (SMRT) and second-generation sequencing (SGS) to generate the complete and full-length transcriptome of B. striata suspension cultured cells.MethodsThe B. striata transcriptome was formed in de novo way by using PacBio isoform sequencing (Iso-Seq) on a pooled RNA sample derived from 23 samples of 10 culture stages, to explore the potential for capturing full-length transcript isoforms. All unigenes were obtained after splicing, assembling, and clustering, and corrected by the SGS results. The obtained unigenes were compared with the databases, and the functions were annotated and classified.Results and conclusionsA total of 100,276 high-quality full-length transcripts were obtained, with an average length of 2530 bp and an N50 of 3302 bp. About 52% of total sequences were annotated against the Gene Ontology, 53,316 unigenes were hit by KOG annotations and divided into 26 functional categories, 80,020 unigenes were mapped by KEGG annotations and clustered into 363 pathways. Furthermore, 15,133 long-chain non-coding RNAs (lncRNAs) were detected. And 68,996 coding sequences were identified based on SSR analysis, among which 31 pairs of primers selected at random were amplified and obtained stable bands. In conclusion, our results provide new full-length transcriptome data and genetic resources for identifying growth and metabolism-related genes, which provide a solid foundation for further research on its growth regulation mechanisms and genetic engineering breeding mechanisms of B. striata.
Highlights
Bletilla striata is a traditional Chinese medicinal (TCM) herb, spelled in Chinese as ”Baiji,”which is widely used in the treatment of pulmonary and gastric diseases such as pulmonary and gastric bleeding, silicosis, tuberculosis, gastric ulcers, etc. (Xu et al, 2019)
The full-length transcripts expressed in suspension cultured callus were sequenced by Illumina and PacBio Sequel platform separately, which were combined to establish a database profiling throughout the whole growing period of the callus suspension culturing system of B. striata
0–9th days is the lag period, during which the dry weight of suspension cultured cells changes slowly, which is a process in which the cells gradually adapt to the environment; 9–18th days is the exponential phase, during which the cell growth rate gradually accelerates to the maximum; 18–24th days is the deceleration period, during which the cell growth rate gradually slows down; on the 24th day, the dry weight reached the maximum, gradually decreases as the days increase
Summary
Bletilla striata is a traditional Chinese medicinal (TCM) herb, spelled in Chinese as ”Baiji,”which is widely used in the treatment of pulmonary and gastric diseases such as pulmonary and gastric bleeding, silicosis, tuberculosis, gastric ulcers, etc. (Xu et al, 2019). Bletilla striata is a traditional Chinese medicinal (TCM) herb, spelled in Chinese as ”Baiji,”which is widely used in the treatment of pulmonary and gastric diseases such as pulmonary and gastric bleeding, silicosis, tuberculosis, gastric ulcers, etc. (Liao et al, 2018; Jiang S. et al, 2019) It is a traditional and precious TCM in China, which was first recorded in Shennong’s Materia Medica (Shen Nong Ben Cao Jing) and has been used for more than two thousand years (Xu et al, 2019). To effectively produce the secondary metabolites through suspension cultured cells of B. striata, it is important to exploring the full-length transcriptome data and the genes related to cell growth and chemical producing of all culture stages. We applied a combination of Real-Time Sequencing of Single Molecule (SMRT) and secondgeneration sequencing (SGS) to generate the complete and full-length transcriptome of B. striata suspension cultured cells
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