Construction And Functional Analysis Of The Rab6 And Rab17 Gene Disruption Mutants In Monascus Aurantiacus AS3.4384

Monascus,as a traditional medicine-food homologous microorganism,can produce a variety of beneficial metabolites,such as pigment,lovastatin,γ-aminobutyric acid and so on.However,Monascus also produce citrinin with renal toxicity.Therefore,it is very important to study the metabolic pathway of Monascus with appropriate methods to promote its application.The growth of filamentous fungi is mainly achieved by adding cell walls and membranes at the apex of mycelia,which is closely related to exocytosis occurring in the Spitzenk(?)rper and endocytosis occurring in mycelia.In eukaryotic cells,the transport of material is mediated by vesicles.Rab protein is a molecular transport switch,which plays an important role in vesicle formation,transport and fusion.In this paper,the efficient gene knockout system of Monascus was established by knocking out Ku70 gene of Monascus aurantiacus AS3.4384,and then on this basis,Rab6 and Rab17 genes that may be related to Monascus pigment and citrinin synthesis were knocked out to study the functions of Rab6 and Rab17 in the growth and metabolism of Monascus.Then,the co-localization and interaction between the Rab17 protein and the citrinin-related protein were studied by constructing strains containing both m Cherry labeled citrinin biosynthesis related proteins and e GFP labeled Rab17 protein.This study provides theoretical basis for studying the metabolic process of Monascus,and lays the foundation for the safety application of Monascus.The main research contents are as follows:1.The replacement Ku70 gene targeting vector p CAMBIA-△Ku70(hph)with hygromycin selective marker was constructed by seamless cloning technique.Then,the target vector was transferred into M.aurantiacus AS3.4384 by agrobacterium-mediated transformation.After hygromycin resistance plate screening and PCR verification,the Ku70 gene deletion strain AS△Ku70 was obtained.The colony morphology,sporulation capacity,pigment and citrinin production and homologous recombination efficiency of AS△Ku70 and AS3.4384 strains were analyzed.The results showed that the loss of Ku70 did not affect the growth,reproduction and sporulation capacity of Monascus and the biosynthesis of pigment and citrinin,and the efficiency of homologous recombination of AS△Ku70 strain was increased more than 6 times.AS△Ku70 strain can be used as a model strain for efficient gene knockout of M.aurantiacus AS3.4384,providing research materials for subsequent gene knockout.2.A replacement Rab6 gene targeting vector p CAMBIA-△Rab6(G418)with G418 resistance gene as selective marker was constructed by seamless cloning technique.Then,the target vector was transferred into Monascus AS△Ku70 by agrobacterium-mediated transformation.After G418 resistance plate screening and PCR verification,△Rab6 heterokaryotic strain with impaired mycelium growth was obtained,but no homozygous was obtained after single spore isolation,and △Rab6heterokaryotic strain was lost.The results showed that the deletion of Rab6 gene might lead to severe damage of mycelia extension and death of Monascus.Rab6 protein may play an important role in material transport from endosome to golgi or golgi to endoplasmic,and regulate mycelial extension and polar growth of Monascus.3.A replacement Rab17 gene targeting vector p CAMBIA-△Rab17(G418)with G418 resistance gene as selective marker was constructed by seamless cloning technique.Then,the target vector was transferred into Monascus AS△Ku70 by agrobacterium-mediated transformation.After G418 resistance plate screening and PCR verification,△Rab17 heterokaryotic strain with with floccular mycelia was obtained.The results showed that Rab17 protein may be closely related to the endosome maturation of Monascus,and its absence may lead to the failure of Monascus mycelia to grow normally,leading to death.However,because Monascus mycelia and spores have multiple nuclei,Rab17 can be partially missing,through the complementary role of the nucleus,and are passed in the form of △ Rab17 heteronuclear culture.4.The fusion expression vector of m Cherry and CtnE/ CtnH/ Orf1/ Orf5 was transferred into the Monascus AS3.4384::Rab17 by agrobacterium-mediated transformation.After G418 resistance plate screening and PCR verification,AS3.4384::Rab17::ctnE,AS3.4384::Rab17::ctnH,AS3.4384::Rab17:: orf1,and AS3.4384::Rab17::orf5 strains overexpressing both citrinin-related protein with m Cherry label and Rab17 protein with e GFP label were obtained.About 40 h of culture,Rab17 and CtnE proteins were co-located on partial mycelium and cleistothecium,Rab17 and CtnH proteins were co-located on cleistothecium,Rab17 and Orf1 proteins were co-located on vacuole-like structure at mycelium branch,cell membrane and cleistothecium,Rab17 and Orf5 proteins were co-located on mycelium cell membrane and cleistothecium.The results showed that Rab17 protein may interacted with citrinin biosynthesis related proteins,and involved in the growth and reproduction process of Monascus and the biosynthesis of pigment and citrinin.