Gene Mining and Sequence Analysis of Purine Nucleosidase Based on RNA-Seq.

Fenfang Wu, Shiping Hu, Yun Ran, Xiaoni Chen, Shan Lin

Journal of visualized experiments : JoVE 2020 Oct 20

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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.

Citation

Fenfang Wu, Shiping Hu, Yun Ran, Xiaoni Chen, Shan Lin. Gene Mining and Sequence Analysis of Purine Nucleosidase Based on RNA-Seq. Journal of visualized experiments : JoVE. 2020 Oct 20(164)