Based On RNR-seq Of Ophiocordyceps Sinensis, Studying On The Putative Genes Involved In Cordycepin Biosynthesis, Cloning And Expression Of RNR Gene

Ophiocordyceps sinensis?O.sinensis?is one of the most valuable Chinese traditional medicines,which has been used for centuries.Recent years,some studies have been studied on the biosynthesis of Cordycepin,but it is not clear.In this study,by Illumina/Solexa HiSeq 2500 technology,the transcriptomes of the O.sinensisfungus?anamorph?andthe fruitingbody?teleomorph?was sequenced,assembled and analyzed.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.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.RNRL mRNA was 2733 bp,encoding 910 aa and RNRM m RNA 1257 bp,encoding418 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.By RT-PCR,the RNRL and RNRM were cloned with flag primers.The gene RNRL was inserted to the plasmid YEp351 and RNRM was inserted to the plasmid YEp352.Sequencs showed that it were recombinant plasmid YEp351-RNRL and YEp352-RNRM.The recombinant plasmid YEp351-RNRL and YEp352-RNRM were transformed into Sacchaminyces cerevisiae BY4741 by LiAc conversion method,positive colonies which could grow on the medium lack of uracil and leucine were selected.Real-time PCR and Western Blot were used to identify the expressed products.The results showed that RNRL and RNRM has were expressed.HPLC was used to detect the cordycepin,and UPLC-MS/MS was used to detect the putative product 3'-dADP.The experimental results show that the peak mass spectrum of the sample is[M+H]+?m/z 412.04202?,and the molecular formula is C₁₀H₁₅N₅O₉P₂,which is consistent with that of 3'-dADP.Because the chemical properties of 3'-dADP have not been reported,3'-dADP and 2'-dADP are isomers.Subsequent studies will be further confirmed 3'-dADP and confirmed the predicted metabolic pathways.