Construction Of RNA Interference Strain Of PksCT Gene Of Monascus Ruber And Analysis Of Its Effect On Regulation Of Citrinin

Some strains can produce Citrinin(CIT).Previous studies have found that the precursor m RNA of citrinin polyketide synthase gene pksCT can produce two kinds of m RNA(pksCT? and pksCT?)through alternative splicing(AS).To regulate the amount of citrinin synthesis,but the regulatory mechanism is not yet clear.In this paper,Monascus ruber M7 was taken as the research object,and appropriate concentration of cycloheximide was added to the culture medium of Monascus ruber to study the nonsense-mediated m RNA decay(NMD)on pksCT? and pksCT? degradation level.Using RNA interference technology to construct an RNA interference vector with a hairpin structure and a reverse double promoter structure,with the help of Agrobacterium tumefaciens-mediated red yeast transformation,interference strains that interfere with pksCT? and both pksCT? and pksCT? are obtained.Detection and analysis of transcriptome the main findings are as follows: 1.Degradation of pksCT? and pksCT? by cycloheximide.The cycloheximide obviously inhibits the expression of the citrinin gene.From the 4th to the 12 th day of Monascus growth,the ratio of pksCT? and pksCT? expression increased first and then decreased.The amount of synthesis has a certain correlation.2.Interfere with pksCT? and pksCT? and pksCT? Monascus mutant strains simultaneously.Using RNA interference technology,gene cloning technology was used to construct hairpin RNA(hp RNA)interference expression vector,and the vector was transferred into Monascus M7 to achieve heterologous expression by means of the Agrobacterium tumefaciens transformation method table.Screening successfully obtained mutant strains of Monascus that interfered with pksCT? alone and pksCT? and pksCT? simultaneously.3.Transcriptome analysis of Monascus mutant strains that interfere with pksCT? alone and pksCT? and pksCT?The interference of pksCT? alone and pksCT? and pksCT? at the same time reduced the expression of M7 alternative splicing to varying degrees,indicating that RNA interference can significantly reduce the transcription of the two m RNAs pksCT? and pksCT?,and the resulting mutant strain citrinin yields significantly cut back.The analysis of differentially expressed genes revealed that the interference of the pksCT? sample group alone with the M7 control group affected glyoxalase-like domains,short-chain dehydrogenase,glucose-methanol-choline oxidoreductase,and MFS(Major Facilitator Superfamily)The gene expression level of protein expression is significantly increased,and the gene expression level of enoyl-(acyl carrier protein)reductase is significantly reduced.While simultaneously interfering with the pksCT? and pksCT? sample groups compared with the M7 control group,it affected the gene expression of glyoxalase-like domains,short-chain dehydrogenase,carbonic anhydrase,and enoyl-(acyl carrier protein)reductase A significant increase.Changes in the expression of these genes also affect the biosynthesis of citrinin.Through the analysis of GO and KEGG pathways,the two pathways of Seroid biosynthesis and Oxidative phosphorylation may be the influencing factors for the interference of pksCT? alone and the synthesis of pksCT? and pksCT? red yeast mutant citrinin.4.Successful construction of M.ruber M7 strain expressing green fluorescent protein EGFPUsing gene cloning method,the EGFP gene was transferred into the hygromycin resistance gene Escherichia coli and Agrobacterium shuttle plasmid pCAMBIA3300,using the Agrobacterium tumefaciens transformation method table to transfer the vector into Monascus M7 to achieve heterologous expression,After genome verification,the M.ruber M7 strain expressing green fluorescent protein EGFP was screened,and the difference between the content of citrinin in the fermentation broth and M7 citrinin by the ultra high-performance liquid chromatography was within 3%,indicating successful construction The M7 strain expressing green fluorescent protein EGFP provides a good starting strain for the study of the construction of reverse dual promoter system RNA interference with the synthesis of Monascus citrinin.5.Successful construction of the interference green fluorescent protein expression vectors pCAMBIA3300-neo-PtrpC-EGFP-Intron2-Pgpd A and pCAMBIA3300 –neo-PtrpC-EGFP Exon3-Pgpd AUsing the method of gene cloning,clone intron 2 or exon 3 as a spacer sequence into the plasmid p Silent-Dual1(p SD1)containing the reverse dual promoter system.The gene of the spacer sequence was cloned into the shuttle plasmid pCAMBIA3300 containing the neomycin resistance gene Escherichia coli and Agrobacterium.After plasmid verification and sequence comparison,the interference green fluorescent protein expression vector pCAMBIA3300-neo-PtrpC-EGFP-Intron2-Pgpd A and pCAMBIA3300-neo-PtrpCEGFPExon3-Pgpd A.With the help of the Agrobacterium tumefaciens transformation method,the vector was transformed into the M7 strain that successfully expressed green fluorescent protein EGFP to achieve heterologous expression.After genomic verification,two mutant strains that interfered with green fluorescent protein expression of Aspergillus were obtained.