| Cordyceps sinensis(renamed as Ophiocordyceps sinensis(Berk.)Sacc),one of the well-known traditional Chinese medicines(TCM)in China for thousand years,is a composite consisting of the stroma of Ophiocordyceps fungus and the parasitic larvae of Hepialidae larva,which has a variety of pharmacological effects including the effectiveness of moistening lung,anti-arrhythmias,regulating and enhancing immunity,and antitumor.In recent years,the increased domestic and international market requirement of wild O.sinensis caused prices to soar,excessive excavation and severe damage of wild resources,in this case,mycelia fermentation of Cordyceps fungal species became a feasible and urgent mean for producing the medicinal fungus and easing the market demand.This paper focused on the annotation information of functional genes by genome and transcriptome sequencing of the isolated Hisrtella sinensis strain,and investigated in terms of the chitinases involved in infection process,the biosynthetic pathways and fermentation regulation of cordycepic acid,cordycepin and cordyceps polysaccharide in details.The process of isolating H.sinensis from wild O.sinensis was studied firstly.A strain of L0106,which had high similarity of colonial morphology to H.sinensis,was isolated from wild O.sinensis sclerotia.The strain was identified by electron microscopy,morphology,molecular biology,Biolog system and phylogenesis analysis,the results indicated that 18 S rDNA sequence of the strain had the highest homology of 99% with that of O.sinensis.Phylogenetic tree analysis found that it had a close genetic relationship with O.sinensis and H.liboensis in the evolutionary relationships,they formed a relatively independent branch.Biolog identification indicated that its carbon source fingerprint was highly similar to that of O.sinensis.Therefore,it was certain that the strain was identified as H.sinensis.The submerged fermentation of H.sinensis was conducted,and the main active ingredients in the obtained mycelia were detected,the results indicated that the average contents of cordycepic acid,cordycepin,crude protein,total polysaccharides and crude fat were 10.90%,0.308 mg/g,383.4 mg/g,3.85% and 5.66%,respectively,each index was in or higher than the corresponding index range of wild O.sinensis,indicating the strain H.sinensis by submerged fermentation had a high similarity and alternative with wild O.sinensis in terms of active ingredients and efficacy.In order to better investigate the functional genes in molecular levels and save the biological information of this precious TCM,genome and transcriptome sequencing were carried out.23198 Contigs and 655 Scaffolds were obatianed after genome sequencing and reads assembly.10200 protein coding genes were obtained after gene prediction and glean software merging,the GC content was 60.19% and the average lenght was 1506 bp.The functional annotation(KEGG pathway classification,COG function classification histogram and GO classification histogram)was accomplished by analysis of protein sequences and alignment of genes to databases,BLAST was applied to accomplish all functional annotations by combination with different databases.Three transcriptomes of H.sinensis at different cultivation time(growth period 3D,pre-stable period 6D and stable period 9D)were sequenced by RNA_seq,and 25511 Unigenes(3D),25214 Unigenes(6D)and 16245 Unigenes(9D)were assembled and obtained,respectively,the Unigenes of the three samples were further assembled into 20822 Unigenes(All).The annotation results of the Uningenes(All)were obtained through alignment with the selected databases(KEGG,COG and GO),and the functions of the corresponding functional genes were cleared.Furthermore,differential expression analysis of H.sinensis transcriptome were investigated and the differentially expressed genes(DEGs)were detected,764 DEGs between 3D and 6D,1869 DEGs between 3D and 9D,and 770 DEGs between 6D and 9D were found,respectively.Moreover,this study mined and verified chitinase genes(chiA and chiH)from the transcriptome,chiA shared the highest identity(52%)with a known chitinase from A.fumigatus Af293,and chiH shared the highest identity(83%)with a known chitinase from O.unilateralis.Subsequently,chi A and chiH were cloned and expressed in E.coli BL21,the recombinant chitinases were further purified by nickel-affinity chromatography,and the purified chitinase chiA and chi H were obtained for enzymatic properties,the results indicated that chi A activity reached the peak at 55 °C while chiH at 50 °C,the activities both reached their peak at pH 5.0,the activity of chiA was stimulated by Mn2+,K+ and Fe3+,while chiH was stimulated by Ba2+.Subsequently,substrate specificity and catalytic properties were investigated,it showed that the highest activities of chiA and chi H were both determined towards colloidal chitin,the analyses of Vmax and kcat values showed that the chiA(Vmax=1.94 ?mol/?g/h,kcat=1.443 S-1)showed higher catalytic efficiency towards colloidal chitin compared with chiH(Vmax=1.63 ?mol/?g/h,kcat=1.175 S-1).To improve cordycepin and cordycepic acid production in H.sinensis,the biosynthetic pathways of cordycepin and cordycepic acid were predicted based on the annotation results of KEGG pathway,and verified by cloning and expressing the genes involved in the pathways,respectively.3 enzymes(9 functional genes)involved in condycepic acid biosynthesis and 12 enzymes(16 functional genes)involved in condycepin biosynthesis were cloned and expressed.Furthermore,differential expression analysis were conducted by real-time PCR,the results indicated that there were 15 up-regulated and 4 down-regulated genes in cordycepin biosynthesis,and 5'-nucleotidase gene was accompanying with significantly up-regulated 15.03-fold,in addition,there were 6 up-regulated and 1 down-regulated genes in cordycepic acid biosynthesis,hexokinase and glucose phosphate isomerase genes were accompanying with significantly up-regulated 5.27-fold and 3.94-fold,respectively.Based on the above information,the effects of adding different concentrations of substrates of the key enzymes involved in the pathways were investigated,the results indicated that cordycepin production reached 1.09 mg/g when additional 4 mg/mL AMP was added resulting in an increase of 201.1% compared with the control,while the cordycepin production only reached 0.71 mg/g when 5 mg/mL adenosine was added resulting in an increase of 96%,indicating cordycepin prefers to be generated from ADP instead of adenosine.Moreover,cordycepic acid production reached 26.6% or 23.4% by adding D-glucose or D-glucose-6-phosphate,leading to a rise of 77.3% or 55.1%,respectively.The addition of various sulfates for enhanced cordyceps polysaccharide(CP)production in submerged cultivation of H.sinensis was investigated.The results indicated that 2 mmol/L of manganese sulfate on 0D was investigated as the optimal adding condition,and the CP production reached optimum of 5.33%,increasing by 93.3% compared with the control.Dynamic profiles of medium pH,residual sugar,mycelia biomass and CP production under the optimal culture conditions of fermenting H.sinensis were investigated,the results indicated that the CP production reached a peak of 5.04% on 8D with adding manganese sulfate,increasing by 76% compared with the control,suggesting that manganese ion could more effectively promote CP biosynthesis.The content change of precursors of CP over entire cultivation under two conditions was investigated,the results indicated that mannose content decreased from 65.32 mg/g to 37.17 mg/g decreasing by 43.1%,indicating that mannose was considered as the most prefered precursor of CP.Subsequently,mannose biosynthetic pathway which constituted the important part of CP biosynthesis was constructed,and the functional genes were verified by cloning and expression.Furthermore,the transcriptional levels of the biosynthetic genes were studied,cpsA gene was significantly up-regulated with 5.35-fold on 6D,consistent with the peak time of CP production,indicating it played an important role in mannose or CP biosynthesis. |
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