Mutagenesis Screening Of-Undifilum Oxytropis Endophytic Fungi And Synthesis Of Swainsonine

U.oxytropis is an endophytic fungus isolated from locoweed in northwestern China.U.oxytropis metabolite Swainsonine(SW)has good resistance on the one hand.Tumor activity,on the other hand,can lead to locoweed poisoning,causing serious damage to the grassland animal husbandry.Therefore,the elucidation of SW biosynthetic mechanism in U.oxytropis can lay a foundation for the subsequent biological fermentation to obtain a large number of SW for anti-tumor mechanism research and clinical application and detoxification of Locoweed.Studies have shown that U.oxytropis is closely related to the SW content in locoweeds,but the mechanism of SW biosynthesis of U.oxytropis is not yet clear.This study is intended to use U.oxytropis mutagenesis to screen high-throughput sequencing methods.Comparing and analyzing the genetic differences between mutagenized strains and U.oxytropis,the preliminary research on SW biosynthesis mechanism was conducted at the molecular level.There are mainly the following aspects:1.Select the same number of U.oxytropis strains for fermentation culture,take a group every 4 days,record 8 groups,measure the growth of the strain for 32 days,record the change of U.oxytropis mycelium content in different growth cycle,draw The growth curve of U.oxytropis,through the extraction of SW in the mycelium and fermentation broth,preliminary preliminary thin layer tests,qualitative tests by gas phase detection,determination of SW content by enzyme linked reaction,determination of the growth cycle of U.oxytropis,The results showed that the mycelial yield and SW content of the strain were the highest at 24 days of growth.2.Mutagenesis screening of U.oxytropis by means of physical chemical mutagenesis.U.oxytropis was used as the starting strain,U.oxytropis was induced by UV,nitrosoguanidine,UV+nitrosoguanidine,and the lethal rate was determined for the induced strains.The best induction conditions were selected and the SW was detected by fermentation.For the content,the target strain with a large change in SW content was selected.The results showed that the SW production of mutant strains after mutagenesis was changed,and the growth morphology and the original strain also had slight changes,but some strains were unstable after passage and were induced by The results showed that the yields of the two strains SW changed greatly.The yield of the mutant strain UD1 SW increased by 21.87% and the yield of the mutant strain D4 SW decreased by 23.35%.3.Through the high-throughput sequencing platform(Lon PGM),high-throughput sequencing analysis was performed on the original strains and D4 mutants with large changes in SW production.Firstly,DNA was extracted from U.oxytropis and D4 mutants.The extracted DNA was purified using Beckmann C1 magnetic beads nucleic acid purification reagents.The Thermo Scientific kit was used to construct a gene library and fluorescence quantitative PCR was performed on the library.Quantitative calculations,high-throughput second-generation sequencing,and sequencing results were used to obtain the complete genome sequence of the original strain and the mutant strain D4.The key genes SwnK,SwH2,and SwH1 of U.oxytropis biosynthesis SW were annotated by BLAST and GO analysis software.,Swn R,putative amino acid transporter and key enzymes pyrroline-5-carboxylate reductase,5’-piperidenine-6-L-carboxylate,Formate/glycerate dehydrogenase catalytic,saccharopine reductase-like protein,combined with KEGG pathway database for U.oxytropis organisms The synthetic SW pathway was predicted,which provided further theoretical basis for the discovery of the biosynthesis mechanism of U.oxytropis.