| Cordyceps militaris,as an important edible and medicinal fungus,has achieved large-scale artificial cultivation in China.Many functions are close to Ophiocordyceps sinensis.It has the functions of improving human immunity,anti-tumor and anti-oxidation.It is the best alternative to O.sinensis.However,its degradable characteristics have caused huge economic losses in industrial production.In this experiment,the inverted microscope and scanning electron microscope were used to observe the morphological characteristics of the mycelia of Cordyceps militaris degraded and normal strains,and high performance liquid chromatography(HPLC),BODIPY lipid droplet staining,and fluorescent quantitative PCR were used to determine the degradation and normality of C.militaris.The expression levels of adenosine and cordycepin content,lipid content and cell autophagy-related genes in the strain.Results: The mycelia of the degraded strain of C.militaris were branched,deformed and curved irregularly;the mycelium content of the degraded strain was 30.23% lower than that of the normal strain.There was no difference in the mycelium and fermentation broth of the degraded strains;the fluorescent brightness of the degraded strains in BODIPY staining was significantly lower than that of the normal strains;the expression levels of autophagy-related genes Atg13,Atg18,Atg22,Atg22-2 were The degraded strains showed an increased expression,and the degraded strains increased by 432.19%,170.45%,336.06%,and 61.01%,respectively,while Atg17 was down-regulated by 65.28%,compared with the normal strains.It indicates that C.militaris has undergone tremendous changes in its physiological and biochemical properties,and the relative expression levels of related genes have also changed significantly.Therefore,it is particularly necessary to use an efficient editing system to edit genes related to C.militaris degradation and explore the functions of related genes.As a new type of gene editing technology,the clustered regularly interspaced short palindromic repeat(CRISPR)/Cas9,has made prospective progress in the field of gene editing.In this study,the Cas9 gene and the gRNA gene cassettes were transformed into genome of C.militaris by employing the Agrobacterium-mediated transformation method.The ura5 gene involved in uracil synthesis was selected as the target gene to construct an efficient editing system for C.militaris.To evaluate the efficiency of gRNA promoters,U6,5S-1,5S-2 promoters were used to control gRNA transcription.It showed that all the C.militaris mutants edited by CRISPR/Cas9 could grow well in the medium containing 5-FOA,and showed uracil auxotrophy.Based on verification of the targeted ura5 gene by sequencing,it showed the U6 promoter was contributing the highest editing efficiency with 100%.A moderate editing efficiency of 70% was resulted by 5S-1 promoter.And 5S-2 promoter revealed only 50% of editing efficiency.It indicated that the U6 promoter is the most suitable promoter we tested for edited in C.militaris genome by CRISPR/Cas9. |
