Establishment Of Reverse Genetics System For Cordyceps Militaris And Mining Of Genes Related To Pigment Biosynthesis

Cordyceps militaris,as a well-known edible and medicinal fungus,contains a variety of biologically active ingredients that are beneficial to the human body and has become a research hotspot for scientists.In recent years,novel carotenoids with stronger hydrophilicity were found in C.militaris,which has broad application fields and market prospects.However,due to the low content of carotenoids in C.militaris,it is unable to meet the increasing market demand.Because the functional genes and biosynthetic pathways of C.militaris carotenoids are unknown,the promotion and application of C.militaris is severely restricted.Therefore,it is necessary to mine genes related to pigment synthesis,identifying the functions of genes,and analyzing biosynthetic pathways at the molecular level.Based on these studies,engineering strains that produce high-yield pigments can be obtained by transforming the C.militaris genome,or constructing heterologous expression engineering strains,which will help to fundamentally solve the problem of low pigment content of C.militaris.In this paper,we first mined genes related to pigment synthesis(CCM＿04119,Cm Tns gene;CCM＿05119,Cm Fhp gene;CCM＿07824,Cm Mox gene;CCM＿08296,Cm Hyp gene)by transcriptome sequencing and bioinformatics analysis.Studying the functions of the above genes requires a complete molecular genetic manipulation system.The completion of the C.militaris genome had made the advantages of the reverse genetics system more prominent.Therefore,the Cm Tns gene was used as a target gene to establish an efficient reverse genetics system(gene knockout and complementation)for C.militaris,which provides a powerful tool for studying the functions of C.militaris genes.Secondly,the functions of Cm Tns,Cm Fhp,Cm Mox,and Cm Hyp genes were studied using the reverse genetics system established in this paper.Finally,the phytoene synthase gene(Psy)was mined from the C.militaris genome,and its function was studied by gene replacement method.The main results of this paper are as follows:(1)Three mycelial samples(CM10＿WD,CM10＿WL,and CM10＿RL)of C.militaris were subjected to transcriptome sequencing and bioinformatics analysis.The results showed that there were 936 differentially expressed genes(318 up-regulated genes,618 down-regulated genes)between samples(CM10＿RL vs CM10＿WL)cultured in different media,and 1722 differentially expressed genes(866 up-regulated genes,856 down-regulated genes)between samples(CM10＿WD vs CM10＿WL)cultured under different light conditions.The alignment of C.militaris transcripts with the carotenoid biosynthetic pathway in the KEGG pathway database indicated that the CCM＿06728 and CCM＿09155 genes are involved in the biosynthesis of carotenoids.Four genes(Cm Tns,Cm Fhp,Cm Mox,and Cm Hyp)that may be involved in the synthesis of C.militaris pigment were discovered by bioinformatics analysis.(2)The conidia,blastospores,and mycelia of C.militaris were identified as mononuclear cells for the first time by fluorescence staining and microscopic observation.(3)The maximum protoplast yield of 2.25×10~7 protoplasts/g fresh weight(FW)mycelia was achieved when 4-day-old mononuclear C.militaris mycelia were incubated in an enzymolysis solution(p H 6.5)composed of 1.00%lysing enzyme,1.00%lywallzyme,and0.8 M KCl at 32°C for 3.0 h.The maximum regeneration rate(36.5%)was obtained when protoplasts prepared under the above optimal conditions were spread onto regeneration media(RM)containing 1.0 mol/L sorbitol.(4)The gene knockout system of C.militaris was established,and the split-marker approach was successfully applied to C.militaris for the first time.Glufosinate ammonium is suitable as a screening inhibitor for C.militaris,and the growth of C.militaris protoplasts was completely inhibited at a glufosinate ammonium concentration of 300μg/m L.The vector p CAMBIA0390-Bar containing the bar gene expression cassette and the knockout vector p CAMBIA0390-Bar-KOTns of the Cm Tns gene were constructed.The split-marker fragments and linear deletion cassettes of Cm Tns gene were prepared by PCR and transformed into mononuclear protoplasts of C.militaris.The Cm Tns gene of C.militaris was successfully knocked out.The frequency of targeted gene disruption by transforming the split-marker fragments was higher than that by transforming the linear deletion cassettes.The bar gene in the mutants(ΔCm Tns)can be stably inherited.(5)The gene complementation system of C.militaris was established.Benomyl is suitable as a screening inhibitor for C.militaris,and the growth of C.militaris conidia was completely inhibited at a benomyl concentration of 2μg/m L.The vector p CAMBIA0390-Ben containing the ben gene expression cassette and the vector p CAMBIA0390-Ben-Com Tns containing the Cm Tns gene expression cassette were constructed.Both the PEG-mediated transformation method and the Agrobacterium-mediated transformation method could achieve the complementation of the Cm Tns gene,but the complementary effect of the Agrobacterium-mediated transformation method was better.(6)The Cm Fhp,Cm Mox,and Cm Hyp genes were successfully knocked out by constructing the knockout vectors(p CAMBIA0390-Bar-KOFhp,p CAMBIA0390-Bar-KOMox,and p CAMBIA0390-Bar-KOHyp)and preparing the split-marker fragments.Positive target gene deletion mutants(ΔCm Fhp,ΔCm Mox,andΔCm Hyp)were obtained.(7)The complementary vectors of Cm Fhp,Cm Mox,and Cm Hyp genes(p CAMBIA0390-Ben-Com Fhp,p CAMBIA0390-Ben-Com Mox,and p CAMBIA0390-Ben-Com Hyp)were constructed.The expression cassettes of Cm Fhp,Cm Mox,and Cm Hyp genes were successfully integrated into the genomes ofΔCm Fhp,ΔCm Mox,andΔCm Hyp strains by Agrobacterium-mediated transformation,respectively,and the complementary strains Com Fhp,Com Mox,and Com Hyp were obtained.(8)The results of phenotypic analysis suggested that the deletion of Cm Tns,Cm Fhp,Cm Mox,and Cm Hyp genes all resulted in a lighter color phenotype,a lower content of carotenoids,and a lower yield of C.militaris conidia.In addition,the deletion of the Cm Tns gene resulted in a decrease in the number of fruiting bodies,a decrease in volume,an enlargement and bending at the ends of mycelia.The deletion of the Cm Fhp,Cm Mox,and Cm Hyp genes resulted in the absence of fruiting bodies in the positive mutants.(9)The phytoene synthase gene(Psy)that was mined from the C.militaris genome was cloned and used to replace the the crt B gene on plasmid p AC-BETA.The expression product of the strain Top10-p AC-BETA-Psy was detected by HPLC,and noβ-carotene was detected,indicating that the Psy gene of C.militaris could not convert GGPP into phytoene in Escherichia coli.