Analysis Of The Expression Pattern Of The Swainsonine Synthesis Gene Cluster And The Knockout Of SwnK Gene In Endophytic Fungus Of Locoweed

Locoweed is a general term for the toxic plants of Oxytropis and Astragalus.Its main toxic component is swainsonine,and swainsonine in locoweed is mainly produced by Alternaria Section Undifilum spp.Recent studies have shown that there is a class of structurally similar gene clusters in swainson-producing fungi,called swainsonine biosynthesis gene cluster(SWN).The SWN gene cluster of the endophytic fungus of Locoweed is mainly composed of genes such as swnA,swnR,swnN,swnH1,swnH2,and swnK.These genes may play an important role in the synthetic pathway of fungi from L-piperic acid to swainsonine,but the specific functions of endophytic fungi in Locoweed have not been verified.The swnK gene is a multifunctional enzyme-encoding gene in the SWN gene cluster.It is composed of multiple regions such as A,KS,AT,SDR,and SDRel.It may play the most important role in the synthesis of fungal swainsonine.The study of the function of swnK gene It will lay a foundation for further elucidating the mechanism of the synthesis of swainsonine by endophytic fungi of Locoweed.Swnk gene is a unique gene of swainsonine producing fungi.If a specific quantitative detection method for this gene is constructed,it will lay a foundation for further elucidating the distribution of locoweed endophytic fungi in locoweed and the relationship between swnk gene and swainsonine content in locoweed.The main findings are as follows:1.In this study,based on the KS sequence of swnk gene of locoweed endophytes,a fluorescence quantitative PCR method was constructed for quantitative analysis of swainsonine producing endophytes in locoweed plant tissues.The method was applied to detect swainsonine producing endophytic fungi in Oxytropis flavescens plant samples,and the biomass and swainsonine content of endophytic fungi in Oxytropis flavescens plant samples were preliminarily analyzed the relationship between quantity.The results show that the real-time quantitative PCR method based on the KS sequence in the swnK gene has good specificity and high sensitivity.When the initial DNA concentration of the fungus is in the range of 0.009?90 ng/p.L,the logarithm of initial DNA concentration of endophytic fungi was negatively correlated with Ct value,and the minimum detection limit of endophytic fungal DNA was 0.009 ng/?L.The results showed that there was a positive correlation between the biomass of endophytic fungi and swainsonine content.In this study,the real-time fluorescence quantitative PCR method based on KS sequence of swnK gene was more specific than the method based on ITS sequence.It can be used for quantitative analysis of swainsonine producing endophytic fungi in locoweed,which provides an important technical support for the research on the relationship between the content of swainsonine and the endophytic fungi in locoweed.2.Secondly,in this study,the endophytic fungus wild type strain UA003,Ethyl methanesulfonate(EMS)mutagenized UA003 mutant strains E02,E23 and E25 and the The low yield of swainsonine originated from Alternaria gansuensis strain EA.The expression pattern of each gene in the SWN gene cluster of them and its relationship with the yield of swainsonine were analyzed,and the mutation sites of the swnK gene of each strain and their encoded products were compared.The results show that all genes of the SWN gene cluster are expressed in the five test strains,but the expression patterns of the genes in different strains are different.compared with the wild-type strain UA003,the E02,E23 and E25 strains with significantly increased swainsonine yield after EMS mutagenesis treatment had upregulated A,KS,SDR and SDRel gene expression in the swnK gene.There is a positive correlation with the swainsonine yield of each strain.AATL,swnR,swnN,swnH1 and swnH2 were all down-regulated in E02,E23 and E25 strains,which was negatively correlated with the fungal swainsonine yield.The result of swnK gene sequence alignment shows that the swnK gene sequences of the five fungi have high homology with the swnK gene sequence of the published endophytic fungus Alternaria oxytropis(KY365741.1)(the sequence identity is 97.55%?99.97%).Compared with the wild-type strain UA003,the E02,E23 and E25 strains had nucleotide mutations in the swnK gene,but the amino acid sequence encoded by them did not change,which is a synonymous mutation.Compared with Alternaria Oxytropis(ky365741.1),EA strain had multiple nucleotide site mutations,in which there was a deletion fragment in the a region of swnK gene,and an insertion fragment in AT region.The amino acid encoded by swnk gene of EA strain was different from other strains,which belonged to frameshift mutation.3.Finally,this study used Split-Marker recombination technology to construct a swnK gene knockout cassette containing the hygromycin phosphotransferase gene(hph),and transferred it to the endophytic fungus protoplast of the Locoweed,to knock out the swnK gene of the fungus,and apply The PCR method was used to screen the transformants.In this study,Split-marker technology was used to construct the upstream segment L1H1 and downstream segment L2H2 of the gene knockout cassette after two rounds of PCR amplification.Sequence analysis results showed that L1H1 contained the upstream homology arm sequence of KS gene and the upstream of hph gene.The fragment L2H2 contains the downstream fragment of the hph gene and the downstream homology arm sequence of the KS gene,indicating that the swnK gene knockout cassette was successfully constructed.After transforming it into protoplasts and regenerating,the transformants can grow on regeneration medium containing hygromycin.Screening by PCR confirmed that the DNA template of the two transformants could simultaneously amplify the upstream and downstream homologous arm fragments of the KS gene and fragments of the hph gene,but could not amplify the KS gene,indicating that the hph gene in the two positive transformants Integrated into the fungal genome,the KS region of the swnK gene was successfully knocked out.This study will lay a foundation for further determining the function of swnK gene in swainsonine synthesis of locoweed endophytic fungus.