Cloning And Functional Study Of SwnH1 Gene From Endophytic Fungus Alternaria Oxytropis OW7.8 In Oxytropis Glabra

Our previous study found that saccharopine reductase（Sac）plays an important role in the SW synthesis of A.oxytropis,however,other key enzyme genes involved in each step of SW synthesis have not been further studied.In some SW producing Metarhizium anisopliae,it is speculated that the 2-oxyglutarate/Fe（Ⅱ）-dependent dioxygenase encoded by swnH1 catalyzes（1S,2S/R,8a S）1,2-dihydroxyindolicidine to produce SW.In this study,swnH1 and cDNA were cloned from A.oxytropis OW7.8 for the first time and analyzed by bioinformatics.Gene knockout vector,overexpression vector and gene editing vector were constructed and transformed into A.oxytropis OW7.8 protoplasts,the transformants were screened by regeneration culture,and verified by PCR and DNA sequencing.Analysis the effect of swnH1 gene on SW synthesisin in A.oxytropis OW7.8 provides basic data for exploring the molecular mechanism and metabolic pathway of SW synthesis in this fungus.The main results are as follows:1.The primers were designed based on the genome sequence of A.oxytropis OW7.8,and swnH1 and cDNA were cloned by PCR.The ORF of the gene was 921 bp and predicted to encode 306 amino acids.The length of the gene sequence from the start codon ATG to the end codon TAG is 987 bp,including a 66 bp intron.The intron boundary sequence conforms to the GT-AG rule.Bioinformatics prediction of SwnH1 amino acid sequence showed that its molecular weight was36377.78 Da,isoelectric point was 6.61,and its molecular formula was C₁₆₂₄H₂₅₆₈N₄₄₄O₄₇₆S₁₄.It encoded hydrophilic protein,no signal peptide and transmembrane domain,and was probably located in the cytoplasm.2.A.oxytropis OW7.8 was inoculated on PDA media containing different concentrations of Hyg B（0.4μg/m L,0.5μg/m L,0.6μg/m L,0.7μg/m L,0.8μg/m L,0.9μg/m L,1μg/m L,2μg/m L,3μg/m L,4μg/m L,5μg/m L,10μg/m L,15μg/m L,20μg/m L）.it was found that the concentration of Hyg B≥1μg/m L can completely inhibit fungal growth.Therefore,the transformants can be screened according to this.3.The upstream homologous arm（618 bp）and downstream homologous arm（322 bp）of swnH1 and the 5’-end（764 bp）and 3’-end（931 bp）of Hygromycin Phosphotransferase（hph）were amplified by PCR.The swnH1 knockout fragment was ligated by Split-marker method and connected to the p MD^TM19-T vector to construct the swnH1knockout vector（5008 bp）.4.The cDNA sequence of swnH1 with Eco RⅠand Bam HⅠrestriction sites at both ends was amplified.After double enzyme digestion of p BARGPE1-Hygro plasmid vector with the above two restriction enzymes,the two were connected to construct the overexpression vector of swnH1 gene（7087 bp）with Pgpd A as promoter and Ttrp C as terminator,and the vector was transformed into OW7.8protoplasts.An overexpression transformant swnH1-T1（Hyg B 2μg/m L）was screened.Compared with the wild-type OW7.8,the colony of the transformant morphology is different,which is manifested in that the colony is black,loose and flaky,and the growth rate is slow.5.The sg RNA expression cassette of swnH1 gene was amplified by overlapping PCR,and connected with p FC332 vector by seamless cloning.The CRISPR-Cas9 gene editing vector（16510 bp）of swnH1gene was constructed.The vector contains g RNA scaffold,Amp^r,hph,promoter Pgpd A and terminator Ttrpc.