CRISPR/Cas9 Technology To Explore The Knockout Method Of Monascus Aurantiacus AS3.4384 PksCT Gene

Monascus is a kind of filamentous fungus which can be used as medicine and food.It can produce pigment,Monacolin K and other secondary metabolites in the process of fermentation.They have anti-cancer,lipid-lowering,antibacterial and other effects.However,at the same time,Monascus can also produce a mycotoxin citrinin,which has teratogenic,carcinogenic and mutagenic effects,thus limiting the wider application of Monascus.It has important practical significance to explore a safe,reliable and simple method to reduce citrinin and solve the problem of citrinin production by Monascus.CRISPR/cas9 gene editing technology can be used for precise and fixed-point editing,with the advantages of simple operation and high efficiency,which provides a new direction for gene editing.Therefore,the main purpose of this paper is to explore the application of CRISPR/Cas9 gene editing technology in Monascus.We try to solve the problem of citrinin production by Monascus from the perspective of molecular biology,and seek a new method for gene editing of Monascus genome.Firstly,the effect of sgRNA and spCas9 on the target gene in vitro was discussed in this paper;then,according to the results of in vitro validation test,a sgRNA was selected to construct the vector;finally,the effect of the vector on the growth of Monascus and whether the target gene was edited were studied.The main results are as follows:1.Adding flavonoids in the fermentation culture of Monascus rice,adding 1%genistein to 30 g rice can reduce citrinin by 31.3%,adding 1%luteolin can reduce citrinin production by 37.7%.2.Three sgRNAs were digested in vitro,The first target located in the first exon of pksCT gene has the best digestion effect in vitro,The efficiency of enzyme digestion can reach 75%.3.108 ?/ml Monascus spore suspension was cultured on hygromycin plate for 7 d,and 80 ?g/ml hygromycin could completely inhibit the growth of Monascus.4.PBARGPE-Hygro-EGFP plasmid is a fungal expression plasmid.The gpdA promoter and trpC terminator of EGFP gene can play a role in Monascus by fluorescent protein analysis.5.The results of sequencing confirmed that Ps6C and Ps knock out vectors were successfully constructed.6.After the Ps plasmid was transferred into Monascus,it could grow on the resistant plate,but the Monascus without Ps plasmid could not grow,which proved that the plasmid had been successfully transferred into Monascus.