Gene Mining and Sequence Analysis of Purine Nucleosidase Based on RNA-Seq
Summary
In this protocol, a method for gene mining and sequence analysis of purine nucleosidase (PN, EC:3.2.2.1) based on RNA-Seq was described. ProtProm analysis was applied to show the unique secondary and tertiary structures of PN. Furthermore, the PN gene was cloned from transcriptome to verify the reliability of RNA-Seq results.
Abstract
Caterpillar fungus (Ophiocordyceps sinensis) is one of the most valued fungal Traditional Chinese medicine (TCM), and it contains plenty of active ingredients such as adenosine. Adenosine is considered as a biologically effective ingredient that has a variety of anti-tumor and immunomodulatory activities. In order to further elucidate the mechanism of purine nucleosidase (PN) in adenosine biosynthesis, a gene encoding PN was successfully mined and further analyzed based on the RNA-Seq database of caterpillar fungus. The full-length cDNA of PN was 855 bp, which encoded 284 amino acids. BLAST analysis showed the highest homology of 85.06% with nucleoside hydrolase in NCBI. ProtProm analysis showed that the relative molecular weight was 30.69 kDa and the isoelectric point was 11.55. The secondary structure of PN was predicted by Predict Protein; the results showed that alpha helix structure accounted for 28.17%, strand structure accounted for 11.97%, and loop structure accounted for 59.86%. Moreover, PN gene was further cloned from transcriptome and detected by agarose gel electrophoresis for verification. This study provides more sufficient scientific basis and new ideas for the genetic regulation of adenosine biosynthesis in fungal TCM.
Introduction
Fungal Traditional Chinese medicine (TCM) has abundant species resources1,2. Caterpillar fungus (Ophiocordyceps sinensis) is a well-known fungal TCM and is regarded as a source of innovative drugs3,4. Caterpillar fungus is a worm and fungus combined mixture that is found on the Tibetan plateau in southwestern China, where Hirsutella sinensis is parasitic on the caterpillar body5. Currently, H. sinensis is reported as the only anamorph of caterpillar fungus according to molecular and morphological biology evidence6,7, and it has less associated toxicity and similar clinical efficacy compared to wild caterpillar fungus8. It was revealed that H. sinensis possesses a variety of biologically effective ingredients, such as nucleosides, polysaccharides, and ergosterols, with extensive pharmacological effects such as repairing a liver injury9,10,11. Adenosine is a typical active ingredient isolated from caterpillar fungus, and it is a kind of purine alkaloid12. Adenosine has a variety of biological activities: anti-tumor, antibacterial, and immunomodulatory activities13,14. Unfortunately, the biosynthetic mechanism of adenosine as well as the key genes involved is still unclear15,16.
Adenosine mainly shows its anti-tumor effect through immunosuppressive actions in the tumor microenvironment17. It was reported that adenosine showed immunosuppressive functions, which was critical to initiate tissue repair after injury and to protect tissues against excessive inflammation18,19. Moreover, it was demonstrated that adenosine-mediated repression of immunity could severely impair cancer immunosurveillance as well as promote tumor growth20. Thus, it is urgent to study the mechanism of adenosine biosynthesis for its wide application in anti-tumor.
It was reported that a complete view of expressed genes and their expression levels could be systematically conducted by next-generation sequencing of transcriptome21. Furthermore, transcriptome sequencing and analysis was applied to predict the genes involved in the biosynthetic pathway of the active ingredients, and further investigate the interaction of different biosynthetic pathways22. Purine nucleosidase (PN, EC 3.2.2.1) is a class of nucleosidase with substrate specificity for purine nucleosides, which can hydrolyze the glycoside bonds of purine nucleosides into sugars and bases23. It typically plays important roles in adenosine biosynthesis. It was reported that the biosynthetic pathway of adenosine in fungal TCM was predicted; qPCR and gene expression showed that the increased adenosine accumulation is a result of down-regulation of PN gene, indicating that the PN gene may play an important role in adenosine biosynthesis15. Therefore, the mechanism of PN in adenosine biosynthesis must be urgently clarified. However, the sequence information and protein structure of PN as well as other key genes involved in adenosine biosynthesis of fungal TCM have not been further studied.
In this study, a novel sequence of PN gene was mined from RNA-Seq data of caterpillar fungus and verified by gene cloning. Furthermore, the molecular characteristics and protein structure of PN were comprehensively analyzed, which could provide new directions and ideas for the gene regulation of adenosine biosynthesis.
Protocol
Representative Results
Discussion
Human health is facing a series of major medical problems such as tumor, cardiovascular, and cerebrovascular diseases26,27. TCM has been regarded as the source of research and development of innovative medicine, because of its rich species resources and diverse structure and functions of active ingredients28,29. Caterpillar fungus is a fungal parasite on the larvae of Lepidoptera, and it is an invigorant …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This study was supported by National Natural Science Foundation of China (31871244, 81973733, 81803652), Natural Science Foundation of Guangdong Province (2019A1515011555, 2018A0303100007), Shenzhen Foundation of Health and Family Planning Commission (SZBC2018016), Special Fund for Economic and Technological Development of Longgang District of Shenzhen City (LGKCYLWS2020064, LGKCYLWS2019000361).
Materials
| RNase-free DNase I | TaKaRa | 2270B | |
| PolyATtract mRNA Isolation Systems | Promega | III | |
| Random hexamer-primers | Thermo Scientific | SO142 | |
| NEBNext1 Ultra RNA Library Prep Kit | NEB | E7530S | |
| PCR extraction kit | QiaQuick | ||
| Agarose | TransGen Biotech | GS201-01 | |
| High-throughput sequencer | Illumina | HiSeq™ 4,000 | |
| LTF Viewer | LTF | V5.2 | |
| ORF program | NCBI | ||
| ProtParam tool | SIB Swiss Institute of Bioinformatics | ||
| SignalP Server | DTU Health Tech | 5.0 | |
| BLAST | NCBI | ||
| Clustal X program | UCD Dublin | ||
| MEGA | Center for Evolutionary Medicine and Informatics | 4.0 | |
| InterProScan | European Molecular Biology Laboratory | ||
| Predict Protein | Technical University of Munich | ||
| WISS-MODEL | Swiss Institute of Bioinformatics | ||
| Primer Express | Applied Biosystems | 3.0 | |
| EcoRI | NEB | R0101V | |
| NotI | NEB | ER0591 | |
| pMD18-T Vector | TaKaRa | 6011 | |
| agarose | Sigma-Aldrich | GS201-01 | |
| Trans2K® Plus II DNA Marker | Sigma-Aldrich | BM121-01 | |
| 6×DNA Loading Buffer | Sigma-Aldrich | GH101-01 | |
| GelStain | Sigma-Aldrich | GS101-02 | |
| 50 x TAE | Sigma-Aldrich | T1060 | |
| Gel imaginganalysis system | Syngene | G:BOX F3 | |
| E. coli JM109 | Promega | ||
| T4 DNA ligase | EarthOx | BE004A-02 | |
| pPIC9K | Genloci | GP0983 |
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