RESUMEN
Objective To clone the full-length of cDNA protein marker cyanase (IP4) of Ophiocordyceps sinensis and predict the antigenic sites. Methods The information of protein marker of O. sinensis was obtained by proteomics technology. The transcriptome database of O. sinensis and mycelium constructed in our lab were used to analyze IP4 unigenes and appropriate primers designed to amplify IP4, which was then cloned and sequenced. The conserved sequence of IP4, homology comparison, and the antigenic site were analyzed by DNAMAN6.0 and DNAStar software. Results Two alternative splice variants of IP4 were discovered from the transcriptome data and belonged to the invariant alternative splicing. The protein marker of O. sinensis was identified as cyanate hydratase, 465 bp, encoding 154 aa. The analysis of the conserved and functional regions showed that catalytic site and binding site mainly lied in C-terminal of IP4. Analysis of DNAMAN 6.0 showed that species 1-39 aa and 55-81 aa were of higher species specificity, and DNAStar results showed that the epitope region of IP4 protein is distributed from 25 to 90 aa. Conclusion The optimal antigen region of IP4 lied in the region 25-39 and 55-81 aa, which lays a foundation for the subsequent large-scale preparation of IP4 protein and eutilizing ELISA to identify authenticity of O. sinensis.
RESUMEN
Objective Recent years, some studies have been studied on the biosynthesis of cordycepin, but it is not clear. To sequence the transcriptomes of the Ophiocordyceps sinensis which could provide the basis for revealing the bio-synthesis mechanism of cordycepin. Methods In this study, by Illumina/Solexa HiSeq 2500 technology, the transcriptomes of the O. sinensis fungus (anamorph) and the fruiting body (teleomorph) was sequenced, assembled and analyzed. By RT-PCR, the full lengths of RNRL (RNR large subunit) and RNRM (RNR small subunit) cDNA were cloned from the fresh O. sinensis fruit body. Results The pathway and the genes involved in cordycepin biosynthesis were predicted. Among of them, RNR was the critical enzyme in the metabolism of adenosine, also predicted to play an important role in the biosynthesis of cordycepin. From the transcriptome data, one large, one small subunits, and four similar sequences of RNR were found. RNRL mRNA was 2 733 bp, encoding 910 aa and RNRM mRNA 1 257 bp, encoding 418 aa. The analysis of the conserved and functional regions showed that catalytic site and binding site mainly lied in RNRL, RNRM contained a ferritin-like conserved sequence. Conclusion This study would be established for revealing the bio-synthesis mechanism of cordycepin.