Optimization Of Artificial Breeding Technology Of Ophiocordyceps Sinensis And Resequencing Of Its Infected Strains

Ophiocordyceps sinensis was a traditional Chinese medicine,which had effect on tonifying kidney and lungs,stopping bleeding and resolving phlegm,etc.Therefore,it was highly praised by Chinese consumers.For a long time,due to the crazy grab of wild Ophiocordyceps sinensis resources by people,coupled with the lack of habitat protection,resulting in a growing shortage of natural Ophiocordyceps sinensis resources,and the market demand gap is also increasing.Studies had shown that cultured Ophiocordyceps sinensiswas not only similar to wild Ophiocordyceps sinensis in morphology,but also very similar in chemical composition and pharmacological action.Therefore,it could be used as an ideal substitute for natural Ophiocordyceps sinensis and reduce the ecological pressure on wild Ophiocordyceps sinensis resources.In this study,the feeding and strain identification of the host larvae of cultured Ophiocordyceps sinensis were studied,the main results as follows:?1?The feeding conditions of the larvae were optimized,and the optimum feeding density was 50 larvae/box?36 cm×28.5 cm×12.5 cm?,the optimal substrate was 40%water content,and the optimal feeding dosage was 400-500 g.The growth process of larvae was investigated,and the growth was slower between 0-90 days,and the growth rate was the fastest between 90-150 days,and then became stable between 180-210 days,finally,the larvae begin to age between 210-240 days.?2?A strain of wild Ophiocordyceps sinensis collected from kangding was isolated.Totally,three strains,KD₂B₂01,KD₂B₂02 and KD₂B₂03,were obtained.And,the culture conditions were optimized.After infection,the results showed that KD₂B₂01 had the strongest infection ability.The morphological identification of KD₂B₂01 indicated that it was Hirsutella sinensis.Cluster analysis showed that the ITS of KD₂B₂01 was clustered with the ITS of Ophiocordyceps sinensis in the NCBI database with 100%bootstrap value support.Hence,the strain KD₂B₂01 isolated in this study was indeed Hirsutella sinensis.?3?The infection effects of spraying,feeding and injection on the host larvae of KD₂B₂01 strain were compared.The results showed that spraying method could not infect the larvae of xiaojin Hepialus larvae.However,the yield of sclerotic larva and Ophiocordyceps sinensis of injection method?29.2%and 14.6%,respectively?were higher than that of feeding method?24.2%and 13.8%,respectively?.?4?The strain KD₂B₂01 was resequenced,and obtained Raw data 11.886 Gb,and then filtered to 11.862 Gb Clean data,accounting for 99.80%of the total,so the sequencing quality was very high.The sequencing data were mapped to the reference genome,and the comparison rate was 98.67%,the average depth was 92.72X.The polymorphism of genome sequence was analyzed,and identified 607339 SNPs,43322InDels,7326 SVs and4084 CNVs.The G protein-coding genes were identified,and 109 genes were obtained,and no CNV variation was found in the genes encoding G??four?and G??one?.The remaining 104 were G?WD-40 repeat domains encoding genes,two of them had no CNV variation,and three of them had CNV variation in the intergenic region,and the other 99showed CNV variation in exon regions to different degrees,and 10 of them had CNV deletion variation.