Cloning And Functional Verification Of RNR Gene Related To Cordycepin Biosynthesis

Cordyceps is a complex of cordyceps fungus,which is parasitic on the sclerotia of insects,spiders and Elaphomyces Nees,and produces fruiting bodies under certain conditions.Among them,Cordyceps sinensis and Cordyceps militaris are the most widely studied.Cordyceps sinensis is a valuable and traditional Chinese medicine in China.Cordyceps militaris has similar medicinal value to Cordyceps sinensis and can be used as a substitute for Cordyceps sinensis.Cordycepin is one of the common active components of Cordyceps sinensis and Cordyceps militaris.Because of its good antitumor effect,Cordycepin has always been the focus of research on the active components of Cordyceps sinensis,but the mechanism of Cordycepin synthesis in Cordyceps sinensis is not clear.It affects its large-scale popularization and application.In this study,the gene of the key enzyme of cordycepin synthesis,ribonucleotide reductase(RNR),which was deduced from the transcriptome analysis of the fruiting body of Cordyceps sinensis,was used as the target gene,and its gene function was investigated by genetic transformation.Due to the long growth cycle and harsh growth conditions of Cordyceps sinensis,there are few reports on its genetic transformation,while the methods of Agrobacterium tumefaciens mediated genetic transformation of Cordyceps militaris spores have been widely reported,and the RNR subunit sequences of Cordyceps sinensis and Cordyceps militaris were highly conserved.Therefore,Cordyceps militaris was selected as the research object.Functional analysis of the key gene RNR for Cordycepin Synthesis Verification and verification.The whole length of RNR gene of Cordyceps sinensis fruiting body was obtained by cDNA cloning technique.Three recombinant plasmids were successfully constructed by designing primers with FLAG expression tags and corresponding restriction sites,and by means of genetic engineering,through enzyme digestion,ligation,transformation and sequencing.Three recombinant plasmids,pCAMBIA1300-35S-RNRMN,pCAMBIA1300-35S-RNRL,pCAMBIA1300-35S-RNRM-35S-RNRL.The T-DNA fragment of the three recombinant plasmids were inserted into the chromosomal genome of Cordyceps militaris,and three recombinant strains were successfully obtained by screening and PCR identification.The recombinant strains were identified by fluorescence quantitative PCR and Western Blot.The results showed that the recombinant strains were expressed at gene and protein levels.HPLC was used to detect the difference of Cordycepin expression between the three recombinant bacteria and the untreated original strain(negative control).The results showed that the content of Cordycepin in CM-M was significantly higher than that of the control group,and there was no significant change in CM-L compared with the control group,and the content of Cordycepin in CM-ML was lower than that in the control group.It is speculated that RNRM gene plays an important role in the synthesis of Cordyceps sinensis,and RNRM binds with RNRL to form ribonucleotide reductase RNR.The RNR enzyme is the key enzyme in the metabolic catabolism of adenosine to form 2'-deoxyadenosine,and the content of adenosine is more decomposed to form 2'-deoxyadenosine.Because adenosine is also a precursor of Cordyceps.The content of Cordycepin was reduced.