Construction Of Pipecolate Oxidase Gene Mutant Strains From Undifilum Oxytropis In Locoweeds

Locoweeds are leguminous plants,which are widely distributed in the grasslands of northwest China,the United States,Russia,Australia and Canada.Its main toxic ingredient,indolizidine alkaloid swainsonine?SW?,is the main poisonous substance of locoweeds.However,SW also has significant anti-cancer and anti-tumor function.It can directly inhibit tumor cells by altering the glycosylation of tumor cells.It can also promote the killing effect of cytokines or chemotherapeutic drugs on tumor cells.Therefore,it has received widely attention by the medical community.According to current studies,SW can be obtained from the fermentation medium of Slafractonia leguminicola and Metarhizium anisopliae.In recent years,some researchers have isolated an endophytic fungus Undifilum oxytropis from locoweed plants,and it plays a leading role in SW synthesis.It is the resource of SW in locoweeds.However,the SW biosynthetic pathway and the key enzymes of regulation in U.oxytropis is still unclear.In view of this,in the early stage of the research group,the whole genome sequencing of U.oxytropis was performed and some key enzymes that may be involved in the regulation of SW biosynthesis were screened and obtained.Pipecolate oxidase?PIPOX?is one of them.In this study,CRISPR/Cas 9 gene editing technology was used to knock out the PIPOX gene,and the PIPOX gene mutant strain of U.oxytropis was successfully constructed.The research results are as follows:1.U.oxytropis preserved in our group was activated and screened for hygromycin-resistant concentrations.The results showed that the inhibitory concentration of hygromycin for U.oxytropis was 1.5?g/mL.2.The preparation,regeneration and the vitality of protoplasts under different enzyme combinations and different enzymolysis time were investigated.The results showed that U.oxytropis was cultured in PDA medium for 15 days,and then transferred to PDB medium in shake flask for 10 days.The mycelium was composed of complex enzyme?1%snail enzyme+1%cellulose+1%lysine?treatment and 25°C,80 r/min shaker for 10 h.Protoplasts are prepared well,with a number of up to 10⁸-10⁹/mL.The prepared protoplasts were poured into double-layer regeneration medium and cultured at 25°C for 5 days.Thin white regenerated colonies appeared,indicating that the protoplasts were successfully regenerated.3.CRISPR/Cas9 gene editing technology was used to edit the PIPOX gene in U.oxytropis,the gene knockout vector was successfully constructed by using pFC332 and pFC334 plasmids.Then the knockout vector was transformed to prepared protoplasts by PEG-Ca²⁺mediated method.The transformants were selected by hygromycin.The PIPOX gene was amplified by PCR using specific primers and compared with the original sequence.The results showed that 4bp were deleted and U.oxytropis PIPOX gene mutant strain was successfully constructed.In this study,CRISPR/Cas 9 gene editing technology was successfully used to obtain the PIPOX gene mutant strain of U.oxytropis,which laid the foundation for the subsequent detection of its key role in SW biosynthesis and SW industrial production.