Analysis of the Biosynthesis Pathway of Produced SW by Endophytic Fungi from Locoweed Using Nitrosoguanidine Mutation and High Throughput Sequencing Technology

HAO Baocheng;SONG Xiangdong;GAO Yan;WANG Xuehong;LIU Yu;CHEN Keyuan;LIANG Yan;HU Yuyao;XING Xiaoyong;WEN Fengqin;HU Yonghao;LIANG Jianping;Key Laboratory of New Animal Drug Project,Gansu Province,Key Laboratory of Veterinary Pharmaceutics Discovery,Ministry of Agriculture,Lanzhou Institute of Husbandry and Pharmaceutical Sciences,Chinese Academy of Agriculture Sciences;College of Veterinary Medicine,Gansu Agricultural University;

HAO Baocheng%SONG Xiangdong%GAO Yan%WANG Xuehong%LIU Yu%CHEN Keyuan%LIANG Yan%HU Yuyao%XING Xiaoyong%WEN Fengqin%HU Yonghao%LIANG Jianping%Key Laboratory of New Animal Drug Project,Gansu Province,Key Laboratory of Veterinary Pharmaceutics Discovery,Ministry of Agriculture,Lanzhou Institute of Husbandry and Pharmaceutical Sciences,Chinese Academy of Agriculture Sciences%College of Veterinary Medicine,Gansu Agricultural University

Alternaria Section Undifilum oxytropis;;nitrosoguanidine;;mutagenesis screening;;high-throughput sequencing;;biosynthetic pathway

The elucidation of the producing swainsonine biosynthesis pathway of Alternaria Section Undifilum oxytropis,an endophytic fungi isolated from locoweed,which can provide theoretical basis and technical support for the biological fermentation to obtain a large number of SW for anti-tumor applications and detoxification breeding of locoweed.Used different doses of nitrosoguanidine,reacted with spore suspension of Alternaria Section Undifilum oxytropis at 120r·min~(-1) for 5,10,15,20,25,and 30 min at 28℃,respectively,and selected good growth strains were inoculated with PDA medium.The culture was continued for 24 days,and theα-mannosidase inhibition method was used to detect SW content in the mycelia and the fermentation broth to evaluate the genetic stability of growth.The mycelium was pretreated with glass beads and liquid nitrogen disruption method.The genomic DNA extraction kit was used to extract the genomic DNA of the original strain and the mutant strain D4.The purified and endonuclease-strained DNA library was constructed and detected by fluorescent quantitative PCR.Evaluate library construction quality and use the Ion torrent PGMTMsequencing platform to complete high-throughput whole-genome sequencing of mutant and original strains.The results showed that a mutant D4 was successfully obtained by chemical mutagenesis.The high-throughput sequencing technology was spliced and assembled using MIRA software,and 19 and 21 related genomic fragments of mutant D4 and original strain were obtained respectively.The experimental results were analyzed by BLAST and GO software,and annotation to the key synthesis genes of SW in Alternaria Section Undifilum oxytropis,such as SwnK,SwnH2,SwnH1,SwnRputative amino acid transporter,etc.,and the key synthesis enzymes,such as pyrroline-5-carboxylate reductase,formate/glycerate dehydrogenase catalytic,saccharopine reductase-like protein,combined with KEGG database,the possible biosynthetic pathway of SW is predicted in Alternaria Section Undifilum oxytropis.The above results will contribute to clarify the SW biosynthesis mechanism of Alternaria Section Undifilum oxytropis.