Genomic Analysis Of Endophytic Fungi Producing Swainonine And Preliminary Study On Establishment Of CRISPR-Cas9 Gene Editing Technique

Locoweed is a general term for toxic plants of the genera Astragalus and Echinococcus in the legume family.Long-term ingestion of locoweed by grazing livestock can cause toxic diseases characterized by nervous system dysfunction.Swainonine(SW),an indoleidine alkaloid,is the major toxic component of Locosa chinensis,which is produced by endophytic fungi of Alternaria Section Undifilum spp.The number of endophytic fungi in the host plant and the ability of strain to synthesize amadozin are closely related to the virulence of Locoweed.Comparative genomics studies have shown that swnK gene is highly homologous in SW-producing fungi of different species,such as endophytic fungi of Locospina cordifolia,S.leguminicola,M.robertsii and Chaetothyriaceae spp.SWNK gene is essential for the production of S W in fungi.The swnK gene sequences of Alternaria Section Undifilum spp isolated from different hosts were more than 97%identical,but their SW synthesis ability was significantly different.Alternaria gansuense and Alternaria bornmuelleri,for example,produce only extremely trace amounts of SW.At present,the synthesis mechanism and metabolic pathway of SW in pathogenic fungi such as Rhizoctonia legume and Metarhizium scallop have been partially elucidated.The synthesis mechanism of SW in endophytic fungi of Alternaria Section Undifilum spp may be similar to those of the above two pathogenic fungi,but it is not clear yet.In this study,based on the sequences of ITS,GPD,KS and other genes,the endophytic fungi of Alternaria Section Undifilum spp isolated from different species of locoplant,which can produce a high amount of SW,and the endophytic fungi of Alternaria Section Undifilum SPP,which can produce only a very small amount of SW,were compared Genetic evolution analysis of the spp.Comparative genomics analysis was then performed on the endophytic fungus A.Oxytropis and pathogenic fungus A.gansuense,which produced only trace SW.On this basis,the CRISPR-Cas9 gene editing system for A.Oxytropis gene function study was constructed by taking swnK gene as the target gene.The results will lay the foundation for further understanding the SW synthesis mechanism and biological function of the endophytic fungi in Locoweed.Through the above research,the following results were obtained:1.The endophytic strains of the Section Undifilum spp Alternaria and pathogenic fungi Alternaria.bornmuelleri and Alternaria.gansuense isolated from Astragalus and Echinococcus cochinensis in China and the United States were analyzed by phylogenetic analysis.The endophytic fungi and plant pathogens Alternaria bornmuelleri and Alternaria gansuense isolated from Astragalus variatus,Astragalus spp.,Echinococcus glacialis and Echinococcus xanthus from different regions of China were compared with the foreign cordycepin-producing fungi based on ITS,GPD and KS The gene sequences were analyzed for sequence identity and genetic evolution.Then,the three genes were concatenated for phylogenetic analysis.The results of BLAST showed that the ITS,GPD and KS gene sequences of 13 strains of Cordyceps chinensis isolated from the Alternaria Section Undifilum spp of Cordyceps Chinensis and Cordyceps Chinensis were completely consistent with the endophytic fungal strains of Alternaria section Undifilum SPP.The sequence identity was 99.79%,99.43%and 100%,respectively,with the endophytic fungi of Alternaria Section Undifilum spp isolated from Locophala chinensis in the United States,which had a very close genetic evolution relationship.It is also highly homologous to the pathogenic fungi Alternaria a.brunmuelleri and Alternaria a.guensuense as plant pathogenic fungi.In the genetic evolution analysis of tandem genes,they had a close genetic evolution relationship.The results of phylogenetic analysis showed that although the endophytic fungi isolated from C.locosa from China were highly homologous to the endophytic fungi isolated from C.locosa from the United States and clustered into the same branch,they belonged to a different branch from Alternaria.cinerea and Alternaria.Oxytropis has the closest genetic evolutionary relationship with Alternaria.Oxytropis.Although the endophytic fungal strains of Astragalus and Echinococcus in China should be unified under the species classification of Alternaria.Oxytropis,they are significantly different from Alternaria Oxytropis isolated from the United States.The results also showed that the endophytic fungi from Astragalus and Echinococcus could not be differentiated into different species based on ITS,GPD and KS gene sequences.2.A.Oxytropis UA003 strain and A.gansuense EA strain isolated from Astragalus officinalis,which produced only trace SW,were subjected to whole-genome sequencing and genomic comparative analysis using high-throughput sequencing technology.The genome size of A.xytropis UA003 was 77.08 Mb,and the GC content was 39.81%.It contained 10,569 protein-coding genes,of which 6094(57.66%)genes were annotated by GO database and 8494 genes were annotated by KEGG database.We identified 437 carbohydrate active enzyme(CAZymes)genes,652 secreted protein synthesis genes,and 37 secondary metabolite synthesis gene clusters in eight types,including t1PKS,NRPS,terpene,T1PKS-Nprs,t3PKS and indole-NRPS.A total of 106 cytochrome genes and 661 pathogenic genes were identified.The genome size of A.gonsuense EA was 75.57Mb,and the GC content was 37.25%.It contained 7523 protein-coding genes,of which 5070 genes(67.39%)were annotated by GO database and 6883 genes were annotated by KEGG database.A total of 409 genes of carbohydrate active enzyme(CAZymes),513 genes of secreted protein synthesis,20 gene clusters of 4 types(NRPS,terpene,t1PKS and T1PKS-Nprs),96 P450 cytochrome genes and 599 pathogenic genes were identified.The results of comparative genomic analysis showed that there were 3186 unique genes in the genome of UA003 strain and 1769 unique genes in the genome of EA strain.The results of analysis of genes related to the production of swichorin showed that SDH,PIPOX and P5CR were present in the genomes of A.oxytropis and A.gasense,but they lacked the key enzymes in the L-lysine P2C degradation pathway.It is possible that the synthetic pathway of Lpipecolic acid obeys lysine→saccharopine→P6C→pipecolic acid was used to synthesize oridonin.There were secondary metabolite synthesis gene clusters with high homology to the reported SWN gene cluster in both genomes.3.In this study,using A.xytropis UA003 strain as the experimental strain and swnK gene related to SW synthesis as the targeted editing sequence,we attempted to establish an efficient CRISPR/Cas9 editing system for the endophytic fungus A.xytropis of locoweed.The swnK gene editing mutant strains were further screened.In this study,four CRISPR/Cas9 tool plasmids,PFC332-AQ,PFC332-A1,PFC332-A2 and PFC332-A3,were constructed using pFc332 and pFc334 plasmids for the four swnK editing sites screened.The PFC332-Aq,PFC332-A1 and PFC332-A3 plasmids were successfully transformed by PEG-CaC12 protoplast transformation method,and 10,2 and 6 transformed strains were obtained,respectively.However,the swnK gene of the transformed strains was not successfully edited.The obstacle of Cas9 nuclear guidance may be the main reason for the failure of gene editing.It is necessary to optimize the system to make it suitable for the study of functional genes of Alternaria oxytropis.