Cloning And Functional Research Of Swnk Gene In The Endophytic Fungus Alternaria Oxytropis OW7.8 SwnK

Oxytropis glabra is a plant containing a toxic alkaloid swainsonine(SW).Its endophytic fungus produced SW,which led to the accumulation of SW in the host plant and showed toxicity.Our research group isolated its endophytic fungus Alternaria Oxytropis.SW producing endophytic fungi also included slafractonia leguminicola,Metarhizium robertsii,Metarhizium anisopliae and so on.SW synthesis pathway in fungi contains a common SWN gene cluster,swn K is a key gene which speculated encodes a multifunctional protein contains seven domains:A domain for adenosylation,T domain for thioalcoholation,KS domain for keto synthase,AT domain for acyltransferase,KR domain for polyketo reductase,ACP domain for acyl carrier protein and R domain for reductase,1-hydroxy-indolizidine from pipecolic acid was catalyzed by multi-step reaction.In this study,swn K gene(SW synthesized key gene)and its promoter in A.oxytropis OW7.8 was firstly cloned by PCR,and its bioinformatics analysis was carried out.The knockout vectors of ACP domain and R domain were constructed,and the vectors were transformed into protoplasts of A.oxytropis OW7.8.The gene knockout transformants were screened,and their SW levels were detected.The reserch provide basic data for the molecular mechanism and metabolic pathway of SW synthesis in A.oxytropis OW7.8.The results are as follows:1.The swn K gene of A.oxytropis OW7.8 was cloned including7515bp from ATG to TAA,with no intron,and it encodes 2504 amino acids and contains 7 domains:A domain 168bp-1578bp(adenosylation);T domain 1647bp-1833bp(phosphopantetheine attachment site);AT/KS domain 1899bp-4557bp(polyketide synthase);KR domain4599bp-5961bp(?-Ketoyl reductase);ACP domain 6024bp-6270bp(phosphopantetheine attachment site);R domain 6453bp-7338bp(?-aminoadipate reductase lys2 and thioester reductase domain of NRPSs).2.The upstream regulatory region of swn K gene was cloned,including promoter,enhancer,light response element,hypoxia specific inducing element,-52bp to-57bp was TATA box,-81bp to-85bp was CAAT box,-158bp to-163bp was G-box,-186bp to-191bp was CCAAT box,including 1 TATA box,4 CAAT boxes and 1 G-box.3.The ACP domain knockout vector(5568bp)and R domain knockout vector(5224bp)of swn K gene were constructed.Their contain upstream DNA homologous region sequence+hph+downstream DNA homologous region sequence and amp^r,It is the shuttle vector of Escherichia coli and filamentous fungi.A R domain of swn K gene editing vector(16510bp)was also constructed,which contains target,amp^r,hph,gpd A promoter and trp C terminator,It is also a shuttle vector between Escherichia coli and filamentous fungi.4.The swn K gene knockout vectors were transformed to protoplasts of A.oxytropis OW7.8 mediated by PEG,?ACP1 and?ACP2 knockout mutants of swn K gene were screened,cultured for 20 days no SW was detected in?ACP1 and?ACP2 mycelia,while the level of SW in wild-type strain OW7.8 mycelia was 5.072mg/g.Considered losed swn K gene ACP domain function,the substrate Malonyl-COA cannot bind and condense with pipecolic acid to produce 1-hydroxy-indolizidine,so SW cannot be synthesized,the knockout strains?ACP1 and?ACP2 do not contain SW.5.The colonies morphology of?ACP1 and?ACP2 are different from wild type A.oxytropis OW7.8,which showed irregular colony shape,irregular edge,milky white,radial growth,and no pigment accumulation on the back.