Construction Of Pyrroline-5-carboxylate Reductase Gene Knockout Strains From Undifilum Oxytropis In Locoweeds

Locoweed is one of the mainly poisonous weeds from Western grassland of China.Livestock appear classical toxic symptoms like head tremor and dyskinesia after ingesting these plants contained swainsonine,which could seriously result in the death of licestock,and caused tremendous economic losses to the pasture animal husbandry in China.The research showed that the main toxic components of locoweed is swainsonine,and Undifilum oxytropis is the primary cause of swainsonine biosynthesis in locoweed.It is now known that swainsonine can be produced by three fungi,that is Rhizoctonia leguminicola,Metarhizium anisopliae and Undifilum oxytropis.The pathway for swainsonine biosynthesis has been partially characterized in Rhizoctonia leguminicola and Metarhizium anisopliae,but the research on biosynthesis pathway and key enzymes of swainsonine in Undifilum oxytropis is unclear.In the early stage of this study,the whole genome was sequenced by high-throughput sequencing technology,and some key enzymes involved in the synthesis of swainsonine were analysed and screened out through gene function and annotation.Pyrroline-5-carboxylate reductase(P5CR)is one of thekey regulatory enzymes.Therefore,Undifilum oxytropis from locoweed was used as a research object in this study.The gene deletion vector was developed by using gene engineering techniques,specific research results are as follows:1.Primers designed from the P5 CR gene sequences according to Undifilum oxytropis genome sequencing results annotation,P5 CR gene was amplified with Undifilum oxytropis cDNA as template.BLAST comparisons revealed 99% of sequence similarity between the PCR product sequencing results and annotated P5 CR gene sequences.2.P5 CR gene deletion vector of Undifilum oxytropis was constructed with pUC19 as start plasmid,which contained the hygromycin resistance gene as a selectable marker gene.P5 CR upstream gene sequence and downstream target gene sequence were attached to both ends of the hygromycin resistance gene.The vectors were constructed successfully verified by PCR,sequencing and double enzyme digestion.3.After the constructed vector plasmid was linearized,the linearized vector was transformed into protoplasts by REMI transformation method to remove the protoplast P5 CR gene of Undifilum oxytropis.Genomic DNA of the transformed strain screened by hygromycin was extracted and verified by PCR,the results showed that P5 CR knockout strain was successfully constructed.In conclusion,the P5 CR gene knockout strain was successfully obtained in this study,and it will subsequently lay a theoretical foundation for the role of P5 CR gene in swainsonine biosynthesis of Undifilum oxytropis and new variety breeding of locoweed without swainsonine.