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 resources^1,2. Caterpillar fungus (Ophiocordyceps sinensis) is a well-known fungal TCM and is regarded as a source of innovative drugs^3,4. 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 biolo....

Protocol

NOTE: A strain of anamorph of caterpillar fungus (H. sinensis) was deposited in our laboratory. Escherichia coli DH5 were preserved by Shenzhen Hospital, Beijing University of Chinese Medicine.

1. Preparing for RNA-Seq

1. Harvesting of mycelia
   1. Prepare fermentation medium for fermentation of H. sinensis: powdered corn flour (1%), silkworm pupae (1.5%), yeast extract (0.5%), tryptone (1%), glucose (1.5%), bran (1.5%), dextrin (0.5%), KH₂PO₄ (0.02%) and MgSO₄ (0.01%).
   2. Prepare inoculation by 10% fermentation medium for scale-up culture (add 10 mL medium ....

Results

The ORF sequence of PN gene was 855 bp in length, which encoded 284 amino acids with a calculated molecular mass of 30.69 kDa and a predicted isoelectric point of 11.55, indicating that PN is an alkaline protein. Application of SignalP4.0 Server was conducted to identify signal peptide, and the results indicated that PN has no signal peptides. Moreover, the results of BLASTP search indicated that PN originated from caterpillar fungus shared the highest identity (85.06%, E value = 1e-88) with nucleoside .......

Discussion

Human health is facing a series of major medical problems such as tumor, cardiovascular, and cerebrovascular diseases^26,27. 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 ingredients^28,29. Caterpillar fungus is a fungal parasite on the larvae of Lepidoptera, and it is an invigorant .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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