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 …
Disclosures
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 |
References
- Dong, C. J. The traditional Chinese medicine fungus Cordyceps and its biotechnological production. Research Journal of Biotechnology. 8, 1-2 (2013).
- Xia, E. H., et al. The caterpillar fungus, Ophiocordyceps sinensis, genome provides insights into highland adaptation of fungal pathogenicity. Scientific Reports. 7 (1), 1806 (2017).
- Koganti, P., et al. Cordyceps sinensis increases hypoxia tolerance by inducing heme oxygenase-1 and metallothionein via Nrf2 activation in human lung epithelial cells. BioMed Research International. 2013, 569206 (2013).
- Shen, C. Y., Jiang, J. G., Li, Y., Wang, D. W., Wei, Z. Anti-ageing active ingredients from herbs and nutraceuticals used in traditional Chinese medicine: pharmacological mechanisms and implications for drug discovery. British Journal of Pharmacology. 174, (2017).
- Jiang, Y., Yao, Y. J. Names related to Cordyceps sinensis anamorph. Mycotaxon. 84, 245-254 (2002).
- Chen, Y. Q., Wang, N., Qu, L. H., Li, T. H., Zhang, W. M. Determination of the anamorph of Cordyceps sinensis inferred from the analysis of the ribosomal DNA internal transcribed spacers and 5.8S rDNA. Biochemical Systematics and Ecology. 29, 597-607 (2001).
- Liu, Z. Y., et al. Molecular evidence for the anamorph-teleomorph connection in Cordyceps sinensis. Mycological Research. 105, 827-832 (2001).
- Yu, S. J., Zhang, Y., Fan, M. Z. Analysis of volatile compounds of mycelia of Hirsutella sinensis, the anamorph of Ophiocordyceps sinensis. Applied Mechanics and Materials. 140, 253-257 (2012).
- Singh, M., et al. Cordyceps sinensis increases hypoxia tolerance by inducing heme oxygenase-1 and metallothionein via Nrf2 activation in human lung epithelial cells. BioMed Research International. 2013, 569206 (2013).
- Cha, S. H., et al. Production of mycelia and exo-biopolymer from molasses by Cordyceps sinensis 16 in submerged culture. Bioresource Technology. 98, 165-168 (2007).
- Lin, S., et al. Enhancement of cordyceps polysaccharide production via biosynthetic pathway analysis in Hirsutella sinensis. International Journal of Biological Macromolecules. 92, 872-880 (2016).
- Xia, E. H., et al. The caterpillar fungus, Ophiocordyceps sinensis, genome provides insights into highland adaptation of fungal pathogenicity. Scientific Reports. 7, 1806 (2017).
- Cha, S. H., et al. Production of mycelia and exo-biopolymer from molasses by Cordyceps sinensis 16 in submerged culture. Bioresource Technology. 98, 165-168 (2007).
- Antonioli, L., Blandizzi, C., Pacher, P., Hasko, G. Immunity, inflammation and cancer: a leading role for adenosine. Nature Reviews. Cancer. 13, 842-857 (2013).
- Lin, S., Zou, Z., Zhou, C., Zhang, H., Cai, Z. Transcriptome analysis reveals the molecular mechanisms underlying adenosine biosynthesis in anamorph Strain of caterpillar fungus. BioMed Research International. 2019, 1864168 (2019).
- Lin, S., et al. Biosynthetic pathway analysis for improving the cordycepin and cordycepic aid production in Hirsutella sinensis. Applied Biochemistry and Biotechnology. 179, 633-649 (2016).
- Allard, B., Beavis, P. A., Darcy, P. K., Stagg, J. Immunosuppressive activities of adenosine in cancer. Current Opinion in Pharmacology. 29, 7-16 (2016).
- Fredholm, B. B. Adenosine, an endogenous distress signal, modulates tissue damage and repair. Cell Death and Differentiation. 14, 1315-1323 (2007).
- Ohta, A., Sitkovsky, M. Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage. Nature. 414, 916-920 (2001).
- Allard, B., Turcotte, M., Stagg, J. CD73-generated adenosine: orchestrating the tumor-stroma interplay to promote cancer growth. Journal of Biomedicine & Biotechnology. 2012, 485156 (2012).
- Zhang, Y., Wang, X., Nan, P., Li, J., Jin, L. De novo transcriptome sequencing of genome analysis provides insights into Solidago canadensis invasive capability via photosynthesis. Journal of Plant Interactions. 14, 572-579 (2019).
- Liu, Z. Q., et al. Transcriptome sequencing and analysis of the entomopathogenic fungus Hirsutella sinensis isolated from Ophiocordyceps sinensis. BMC Genomics. 16, 106 (2015).
- Ogawa, J., et al. Purification, characterization, and gene cloning of purine nucleosidase from Ochrobactrum anthropi. Applied and Environmental Microbiology. 67, 1783-1787 (2001).
- Konstantin, A., et al. The swiss-model workspace: a web-based environment for protein structure homology modelling. Bioinformatics. 16, 195-201 (2006).
- Chung, C., Niemela, S. L., Miller, R. H. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proceedings of the National Academy of Sciences. 86, 2172-2175 (1989).
- Lin, S., et al. Association between aldose reductase gene C(-106)T polymorphism and diabetic retinopathy: A systematic review and Meta-Analysis. Ophthalmic Research. 63, 1-10 (2020).
- Peng, Y., et al. Inguinal subcutaneous white adipose tissue (ISWAT) transplantation model of murine islets. Journal of Visualized Experiments: JoVE. (156), (2020).
- Zhang, T. T., Jiang, J. G. Active ingredients of traditional Chinese medicine in the treatment of diabetes and diabetic complications. Expert Opinion on Investigational Drugs. 21, 1625-1642 (2012).
- Lin, S., Zhou, C., Zhang, H., Cai, Z. Expression, purification and characterization of 5′-nucleotidase from caterpillar fungus by efficient genome-mining. Protein Expression and Purification. 168, 105566 (2020).
- Kinjo, N., Mu, Z. Morphological and phylogenetic studies on Cordyceps sinensis distributed in southwestern China. Mycoence. 42, 567-574 (2001).
- Xiao, J. H., Ying, Q., Xiong, Q. Nucleosides, a valuable chemical marker for quality control in traditional Chinese medicine Cordyceps. Recent Patents on Biotechnology. 7, 2 (2013).
- Tu, P., Yong, J., Guo, X. Discovery, research and development for innovative drug of traditional Chinese medicine under new situations. Zhongguo Zhong Yao Za Zhi. 40, 3423-3428 (2015).
- Zheng, P., et al. Genome sequence of the insect pathogenic fungus Cordyceps militaris, a valued traditional Chinese medicine. Genome Biology. 12, 116 (2011).
- Covarrubias, R., et al. Role of the CD39/CD73 purinergic pathway in modulating arterial thrombosis in mice. Arteriosclerosis, Thrombosis, and Vascular Biology. 36, 1809-1820 (2016).
- Ogawa, Y., Murayama, N., Yanoshita, R. Molecular cloning and characterization of ecto-5′-nucleotidase from the venoms of Gloydius blomhoffi. Toxicon. 54, 408-412 (2009).
- Lin, S., et al. Mining and characterization of two novel chitinases from Hirsutella sinensis using an efficient transcriptome-mining approach. Protein Expresion and Purification. 133, 81-89 (2017).
- Ueda, M., Hirano, Y., Fukuhara, H. Gene cloning, expression, and X-ray crystallographic analysis of a β-mannanase from Eisenia fetida. Enzyme and Microbial Technology. 117, 15-22 (2018).
- Rebets, Y., Kormanec, J., Luzhetskyy, A., Bernaerts, K., Anné, J. Cloning and expression of metagenomic DNA in Streptomyces lividans and subsequent fermentation for optimized production. Methods in Molecular Biology. 1539, 99 (2017).
- Wang, S. S., Ning, Y. J., Wang, S. N., Zhang, J., Chen, Q. J. Purification, characterization, and cloning of an extracellular laccase with potent dye decolorizing ability from white rot fungus Cerrena unicolor GSM-01. International Journal of Biological Macromolecules. 95, 920-927 (2016).
.