The Breeding Of Hirsutella Sinensis With High Yield Of Nucleosides And The Expression Of Key Enzyme Genes

Cordyceps sinensis is a complex stroma of Ophiocordyceps fungus and the parasitic larvae of Hepialidae larva.Hirsutella sinensis is the only asexual form of C.sinensis.Its function and composition are highly similar to those of C.sinensis,and it has become a substitute of wild C.sinensis.Nucleosides,as an important indicator of H.sinensis products,are essential for improving product quality and enhancing market competitiveness.The preparation and regeneration of H.sinensis protoplasts was first studied in this article.A compound protoplast mutagenesis technique of atmospheric and room temperature plasma?ARTP?and ethyl methanesulfonate?EMS?was established and a high-yield nucleoside-producing strain was screened.The gene expression differences of key enzymes of nucleoside metabolism pathway in the mutated strain were developed.First,the preparation and regeneration of H.sinensis ZJB18002 protoplasts were conducted.The effects of osmotic pressure stabilizers,lytic enzymes,digesting temperature,digestion time and other factors on protoplast preparation were investigated.The results showed that the optimal protoplast preparation conditions for H.sinensis were as follows:suspending the mycelium cultured for 7 days in 5 m L reaction solution,containing 0.6 mol/L KCl and 3 mg/m L Lysing Enzyme,4 mg/m L Yatalase and 3 mg/m L Driselast in 0.1 mol/L potassium phosphate buffer?p H=5.5?,and the solution was reacted for 0.5 h at 18?,120 rpm.Then,the components of regeneration medium,digestion conditions and spreading concentration during the protoplast regeneration process were further optimized.The results showed that12.89%of protoplast regeneration rate was obtained when the initial protoplast concentration of 5×10⁴/m L was spreaded on the regeneration medium and cultured in the dark at 16?for 21 d.Second,the mutagenesis was carried out by the combination of ARTP and EMS.The optimal mutagenesis conditions for ARTP and EMS in H.sinensis were obtained.The optimal ARTP treatment time was 80 s and the EMS mutagenic dose was 40?L/m L.The ARTP-mutated positive mutant strains were further mutated by EMS,a high-yield strain was screened and named ZJB19050.The adenosine content was 3.36mg/g,guanosine content was 2.7 mg/g and uridine content was 6.46 mg/g,which were 160.2%,67.4%,and 113.5%higher than that of the wild strain,respectively.Finally,the quantitative detection of the key enzyme expression of involved in the nucleoside metabolism pathway was carried out using real-time quantitative PCR technology.The biological metabolic pathways of adenosine,guanosine and uridine in H.sinensis were predicted and constructed,and the sequences of eight key enzymes and their corresponding functional genes involved in the metabolic pathway were verified and successfully cloned and expressed.According to the analysis of the relative quantitative results of 2^-??Ct,it was found that there were four key enzyme genes with up-regulation and three down-regulation genes in the nucleoside metabolism pathway.Among them,the activities of 5'-nucleotidase and transformylase were increased by 658.8%and 222.5%,respectively,while the expression of adenosine dehydrogenase was only 31.2%of the original strain;hypoxanthine nucleotide dehydrogenase and GMP synthetase in the guanosine metabolism pathway are up-regulated genes,which were 195.6%and 179.6%higher than the original strain,respectively;the expression levels of uridine kinase and orotate nucleotide decarboxylase in the uridine metabolic pathway were 36.1%and23.5%of the original strain.