Research On The Diversity Of Cicada Fungi And The Biosynthetic Pathway Of Cordycepi

Cordyceps cicadae Shing(Chanhua)is one of a parasitic fungus that grows on cicada flammata larvae,this fungus-host insect complex is a rare traditional Chinese medicinal material that has been used to relieve exhaustion and treat numerous diseases such as epilepsy,heart palpitations,children’s night crow.C.cicadae possesses a variety of medicinal bioactive components,such as adenosine,ergosterol,cordycepin,cordycepic acids,polysaccharides,macrolides,and other metabolites.Cordycepin(3’-deoxyadenosine,an adenosine analog)is a major bioactive nucleoside antibiotic compound that found in Cordyceps species,possesses a wide range of pharmacological activities,such as antitumor,pro-immunity,antibacterial effects.However,most studies related to Chanhua have focused on detecting active components,and fungal diversity associated with in different regions remains unclear.Less research about biosynthetic pathway of cordycepin has been conducted on Chanhua(C.cicadae)compared to C.militaris and C.sinensis,further studies need to be done for revealing the molecular mechanisms of cordycepin biosynthesis.In this study we conducted comprehensive researches on C.cicadae.The PCR-DGGE and Illumina MiSeq-based methods were used to investigate the C.cicadae fungal community structures.We artificially cultured C.cicadae successfully on the results of fungal community structures,and we determined the cordycepin contents from the mycelium,fruiting bodies,and sclerotium using calibration curves of standard reference compounds.And then we investigated the possible key genes involved in the cordycepin biosynthesis pathway in the mycelium,fruiting body,and sclerotium of artificially cultured C.cicadae by using the Illumina HiSeq4000 platform.The Agrobacterium tumefaciens-mediated transformation(ATMT)method was applied to confirmed the gene functions of selected key enzyme genes,and the prokaryotic and eukaryotic expressions were conducted to verifing the catalytic activity of the key enzymes.The specific contents and results are as follows:(1)The PCR-DGGE method was used to investigate the Chanhua fungal diversity.Samples were collected from 10 different geographical regions including Yibing in Sichuan Province,Jiangsu Province,Jiangxi Province,Anhui Province,Guangdong Province,and Guangxi Autonomous Region.Fungal DNA was isolated from fungal samples and PCR-DGGE(Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis)technology was adopted to analysis the fungal community structure.DGGE profiles shown that 16 specific bands were separated,these bands were cloned and sequenced.The result shows that C.cicadae from different zones displayed significant difference in Shannon-Wiener index respectively.Sequencing analysis of representative of cloned DGGE bands show that Cordyceps militaris/Isaria tenuipes was the dominant strains,and other Uncultured Wallemia,Uncultured eukaryote,Eurotium athecium,Magicicada cassini,Myrothecium roridum,Aspergillus penicillioides strains were also existed.The fungal diversity indices of Guangxiyyulin was 16 bands and Shannon-Wiener index 2.679.Community structure similarity was higher in Guangdong,Sichuan beichuan,Sichuan mianyang.(2)The Illumina MiSeq-based method was used to investigate the Chanhua fungal community structures.Based on 18S rDNA of 10 different geographical regions which was amplified by nested PCR.The bio-informatic analysis results showed high fungal diversity,with 22 families found in the different samples,Eurotiomycetes was the dominant fungal family associated with Chanhua(C.cicadae)(composing 36.69%of the sample from Sichuan Mianyang),whereas Cordycipitaceae only composed 0.51%(Sichuan Yibing)to 0.33%(Guangdong)of these samples.Surprisingly,the proportion of Capnodiales was 5.27%in Sichuan Mianyang,whereas the percentage in other regions ranged from 4.35%(Sichuan Yibing)to 2.55%(Zhejiang),and the remaining proportion(more than 80%)was made up of unidentified fungus.(3)The process of artificially cultured C.cicadae was successfully established.We isolated dominant fungi from wild C.cicadae,and inject conidia of C.cicadae into living Chinese Tussah silkmoth pupae and the strain C.cicadae produced fruiting bodies in Chinese Tussah silkmoth pupae as wild C.cicadae.We determined the cordycepin and adenosine contents from the fruiting bodies and sclerotium(normal cultured group,buried in wet soil group),using calibration curves of standard reference compounds.The contents of cordycepin in the sclerotium were higher than in the fruiting bodies,and the contents of cordycepin the sclerotium is about 2 times of fruiting body.Meanwhile the contents of cordycepin in the groups of buried in wet soil were higher then in normal cultured groups,and the contents of adenosine have obvious difference in four groups.(4)The decoction of artificially cultured and wild C.cicadae were extracted by water and tested its effects on the proliferation of in high glucose-cultured HMCs.The decoction components were soaked in water of 70℃ for 5 hours and then decocted to an extract solution(0～1000 μg/mL)with RPMI 1640 medium,and four groups were fruiting body(normal cultured),sclerotium(normal cultured),sclerotium(buried in wet soil),wild type(Anhui).HMC cells treated with high glucose RPMI 1640 medium were divided into six groups(HG,HG+1000,HG+750,HG+500 and HG+250,HG+125 μg/mL)and treated as 5%CO2 at 37℃for 48h.Cell proliferation was evaluated by CCK-8 assay.The proliferation of HMCs under HG conditions(HG group)was significantly greater than that under normal glucose condition(NC group),all groups can inhibit the proliferation of HMCs treated with high glucose RPMI 1640 medium.The sclerotium group significantly suppressed HMCs proliferation.(5)Identification of cordycepin biosynthesis-related genes through comparative de novo transcriptome assembly and analysis of Cordyceps cicadae Shing.We conducted the comparative de novo transcriptomic data of artificially cultured C.cicadae and obtained 54G raw data in the mycelium,fruiting body,and sclerotium groups by using the Illumina HiSeq4000 platform and we obtained high quality of sequencing data.A total of 70,097 unigenes were generated and within the average length was 717 bp.And annotated C.cicadae genes had the greatest number of matches with genes of Beauveria bassiana and C.militaris.The GO analysis results revealed that the unigenes were categorized into 52 functional subgroups belonging to three main GO groups,i.e.,cellular components,molecular functions,and biological processes,We found that the most frequent GO terms in the groups were metabolic process,cellular process and catalytic activity.The annotated sequences were searched against the KEGG database were assigned to five main categories.The largest group was genes responsible for metabolism,genetic information processing,organismal systems,cellular processes,and environmental information processing.The number of unigenes involved carbon metabolic pathways other secondary metabolites was 2,376 and 305 respectively.We examined the number of DEGs for each of these three comparisons,i.e.mycelium(control)versus fruiting body,mycelium(control)versus sclerotium,fruiting body(control)versus sclerotium,and the greatest number of upregulated genes was found in a comparison between the mycelium and sclerotium libraries,and a total of 3,876 DEGs were detected,with 1,576 upregulated and 2,300 downregulated genes,the mycelium versus fruiting body and fruiting body and sclerotium groups,with 1,604 and 1,474 upregulated genes and 1,365 and 1,320 downregulated genes,respectively.The Venn diagram indicated that 372 genes were significantly differentially expressed between the three comparisons.In the mycelium versus fruiting body group,19 participating genes were identified,and 28 and 16 participating genes were identified in the mycelium versus sclerotium and fruiting body versus sclerotium groups,respectively.To validate the transcriptome analysis data and provide a better understanding of the molecular basis of the metabolic pathways involved in purine biosynthesis,we selected six gene encoding key enzymes,including surE(c19447g1,Down),RRM1(c19459g1,down),CRCE1(c34980g1,Down),ADA2(c35629g1,Up),E3.1.3.5(c62060g1,Up),and NDK(c76121g1,Down),and we examine differences in gene expression levels by using qRT-PCR,the results showed that the six genes expression levels were consistent with those of the transcriptome analysis.These results revealed that adenosine deaminase and 5’-nucleotidase were important enzymes involved in cordycepin biosynthesis in C.cicadae.(6)A method of Agrobacterium tumefaciens-mediated transformation in C.cicadae was established.In order to verified the functions of 5’-nucleotidase and adenosine deaminase which involved the biosynthesis pathway of cordycepin,the ORF genes of c62060g1 and c35629g1 were cloned from cDNA,then pCambial-1303 gpdA expression vectors were reconstructed by homelogens DNA technology.And we transformed the recombinant plasmids into C.cicadae by ATMT.We obtained two transformants c35629-C.ciacadae and c62060C.cicadae within the condition of hygromycinB(500 μg/mL)on PDA medium.We examined the expression of c62060 and c35629 genes at the mRNA level by using Real-time PCR analysis.And the results shown the expression levels of the two genes in transformants C.cicadae were over 40 times than that in wild-type strain.Finally,to confirm that the genes play a significant reaction in cordycepin metabolism,using HPLC technique to detect cordycepin content of the genetically modified strains.Results shown that the cordycepin content of transgenic c3 5629 group and transgenic c62060 group increased compared with negative control group about 6.68 and 15 times respectively.(7)The prokaryotic and eukaryotic expressions were conducted for candidate key enzymes.The c62060,c35629 and c19447 genes were successfully amplified by PCR from cDNA of C.cicadae.Sequence analysis revealed that the nucleotide sequence of these genes contains 1866,1899 and 957 bp,and the c62060 ORF gene encoding 621 amino acid residues,with of 64.78 kDa and PI of 4.92,c35629 ORF gene encoding 632 amino acid residues,with of 71.48 kDa and PI of 4.83,c19447 ORF gene encoding 319 amino acid residues,with of 33.78 kDa and PI of 5.00.The genes of c62060,c35629 and c19447 were into two conventional vectors,pET28b and pPIC9α,and expressed in E.coli BL21 and Saccharomyces cerevisiae BY4742.Then isopropyl-d-thiogalactopyranoside(IPTG)with a fnal concentration of 0.2 mM was added to the LB medium for further heterologous expression by E.coli BL21.The positive Saccharomyces cerevisiae transformant with was cultivated for fermentation in a 100-mL Erlenmayer flask with a fnal concentration of 1%methyl alcohol added to the YPD medium.Unfortunately,SDS-PAGE analysis indicated that the target proteins was not expressed.Consequently,our research accelerates substantial understanding of Chanhua(C.cicadae Shing)fungal communities and will provide a foundation for further research on fungal community structure.We conducted first large-scale transcriptome RNA-seq analysis of the mycelium,fruiting bodies,and sclerotium of artificially cultured C.cicadae.Our data provided comprehensive coverage of the C.cicadae transcriptome and revealed specific genes involved in the development of C.cicadae.We also initially established the Agrobacterium tumefaciens-mediated transformation(ATMT)method and the overexpressing the 5’nucleotidase and adenosine deaminase genes in C.cicadae.The cordycepin content of transgenic C.cicadae increased significantly.Our results are expected to facilitate further pharmaceutical and industrial applications of C.cicadae and could provide insights into the study of the molecular mechanisms of cordycepin biosynthesis in C.cicadae.