Study On The Function Of SwnR Gene Of Swainsonine From Metarhizium Anisopliae

Current research shows that swainsonine,a toxic component of locoweed,is mainly produced by three types of fungi that including R.leguminicola,M.anisopliae,and A.oxytropis.However,the genes involved in the swainsonine biosynthesis pathway and catalytic enzymes are unclear.Therefore,in this study,we performed a gene deletion system for synthetic swainsonine suspected catalytic enzyme gene SwnR from M.anisopliae based on PEG-mediated transformation protocol.The aim of this study is mainly related to investigate the role of the SwnR gene in the biosynthesis pathway of the swainsonine of M.anisopliae and provide a theoretical basis for further studies on swainsonine biosynthesis pathway and related catalytic enzyme genes.1.The SwnR gene primers were designed according to the standard sequence on GeneBank.The SwnR gene was amplified by amplification using the cDNA of M.anisopliae as a template.The PCR sequencing result was 99% homologous to the SwnR gene sequence BLAST.2.The gene deletion vector of SwnR gene of M.anisopliae containing Benomyl resistance gene was constructed by In-Fusion Cloning using pUC19 vector as a backbone.The upstream and downstream gene sequences of SwnR were linked to Benomyl resistance.The constructed vector was successfully verified by PCR and sequencing3.The preparation conditions of the protoplasts of M.anisopliae used the hyphae of M.anisopliae with different fermentation time,under different enzymatic hydrolysis combinations and different enzymatic hydrolysis time.The results showed that M.anisopliae was cultured in CS medium for 3days,and the hyphae were treated with 1% cellulase,1% snail enzyme and 0.5% lysing enzyme and placed at 30? for 3h at 100 rpm.The protoplast preparation was the best.4.The homologous fragment is amplified by PCR.The purified fragment is transformed into the protoplast by PEG transformation.The SwnR gene mutant strain were screened by Benomyl,and its DNA was extracted and verified by PCR.The results showed that SwnR Gene deletion strain was successfully constructed.5.Fermentation culturing the mutants obtained from the above experiments and wild-type strains,and the content of swainsonine in the fermentation broth of wild-type and mutant strains was 119.652?g/mg and 11.831?g/mg by Q Exactive high-resolution mass spectrometry.The results showed that the SwnR gene expression of the mutant strain was significantly down-regulated by RT-qPCR.In this study,the SwnR gene deletion strain of M.anisopliae was successfully obtained.It was proved that the SwnR gene plays an important regulatory role in the swainsonine biosynthesis pathway of M.anisopliae provided an important theoretical basis in the swainsonine synthesis of A.oxytropis and the prevention and control of locoism in animals.