Study On The Genetic Editing And Function Of Plasmodium Falciparum Actin-related Proteins(Arps)

Background:Malaria is still one of the major infectious diseases in the world,especially Plasmodium falciparum,which seriously endangers human health.Research on the pathogenic genes of Plasmodium falciparum still needs to be deepened.In eukaryotes,the actin-related proteins?Arps?family is relatively conservative,and studies have shown that its members are a major component of gene regulation complexes.In Plasmodium falciparum,bioinformatics genomic analysis predicts Arp orthologs with similar functions,including PfArp1,PfArp4 and PfArp6.The biological function of P.falciparum Arp4 and Arp6 and the regulatory mechanism of pathogenic genes have not been systematically reported.Objective:Investigate the target genes regulated by PfArp4 and PfArp6 proteins and their mechanism of regulating gene expression.Methods: 1.The editing of Gene :Download the PfArp4?PF3D7₁422800?and PfArp6?PF3D7₀719300?sequences from the Plasmodium genomic data library PlasmoDB?https://plasmodb.org/plasmo/?,design with SnapGene software,and conduct the knocking under the guidance of CRISPR / Cas9 technical principles Out?KO?,knock in?KI?and knock down?KD?editing.Construct plasmids,polymerase chain reaction?PCR?,including modified primer PCR technology,long fragment PCR technology and composite PCR technology.Using Plasmodium falciparum 3D7_G7?existing in the laboratory?as a template,the DNA sequence to be designed was amplified,and the homologous modified DNA sequence and sgRNA sequence were ligated on the Pl6 plasmid using DNA ligase.2.Verification and screening of transgenic insect strains:After the constructed plasmid was verified by sequencing,it was mixed with the Cas9 plasmid and introduced into the wild type 3D7_G7 by electrotransfection.The gene editing and modification of Plasmodium falciparum Arp4 and Arp6 were carried out,and the drug resistance gene was screened through the constructed drug resistance gene.The genetically modified Plasmodium falciparum genes and proteins screened by drugs were first subjected to PCR to verify the modified genes,and then Western blot experiments were conducted to verify the modified gene protein expression.Finally,clone to obtain monoclonal transgenic insect strains.3.Protein expression and growth status of PfArp4 and PfArp6 genes at different stages:The R,T and S phase proteins of Plasmodium falciparum were collected respectively,and the expression of PfArp4 and PfArp6 proteins in different phases was identified by western blot experiments.Under different concentrations of GlcN drugs,protein expression down-regulation experiments were performed on the integrated Plasmodium falciparum with Ty1-Ribo tag inserted,and the degree of protein down-regulation was identified through western blot experiments.And through the effect of different drug concentrations GlcN,the number of late merozoites after two life cycles was counted,and whether the growth and development of integrated Plasmodium was affected.Co-IFA experiments were used to identify the localization of PfArp4 and PfArp6 in Plasmodium falciparum cells,and the relationship between the two.And further observe the interaction of PfArp4 and PfArp6 protein by co-IP experiment.4.The expression of PfArp4 and PfArp6 genes at the RNA and DNA levels:RNA-seq experiments were used to observe the expression of transcripts after PfArp4?+/-GlcN?.Combining ChIP with second-generation sequencing technology to enrich DNA fragments that specifically bind to PfArp4 or PfArp6 protein,and establish corresponding gene libraries.Through high-throughput sequencing,analyze the enrichment of Arp4 or Arp6 genes in Plasmodium falciparum.Results: 1.Plasmid construction and transgenic insect strains:Ten recombinant plasmids modified by KO,KI and KD were constructed.The integrated Plasmodium falciparum with Ty1-HA and Ty1-Ribo tags inserted into Arp4 and Arp6 genes,and Ty1 tag inserted into Arp4 and HA tag inserted into Arp6 was obtained.Each strain holds two monoclonal strains.2.Plasmodium falciparum Arp4 and Arp6 gene protein expression and growth status:Expression of Arp4 and Arp6 proteins of Plasmodium falciparum in different periods: Arp4 protein is mainly expressed in R phase,followed by T / S phase,and Arp6 is mainly expressed in T / S phase,followed by R phase.GlcN at a concentration of 5 mmol can down-regulate the expression of PfArp4 and PfArp6 proteins of the integrated Plasmodium falciparum inserted with the Ty1-Ribo tag.PfArp4 is most significantly down-regulated during the T phase.And in the third growth cycle,the protozoa rate was obviously reduced.However,the growth of PfArp6 integrated Plasmodium has not been affected.At a GlcN concentration of 5 mmol,PfArp4 integrated Plasmodium decreased significantly in the late merozoite number in the second growth cycle?P<0.0001?.3.Location of Arp4 and Arp6 genes in Plasmodium falciparum:Through co-IFA experiments,it was found that PfArp4 and PfArp6 proteins were located on the nucleus of Plasmodium falciparum,and the two partially overlapped,indicating an interaction.And the co-IP experiment found that the protein was down through the HA tag on PfArp6,and the pulled protein had Ty1 tagged PfArp4 protein,further proving the interaction between the two.4.The expression of PfArp4 and PfArp6 genes at the RNA and DNA levels:RNA-seq experiments found that PfArp4 protein was down-regulated after GlcN drug action,which widely affected transcript expression.The ChIP-seq experiment found that the PfArp4 and PfArp6 proteins of R.falciparum were enriched in 60 var genes,and the two proteins were enriched at the same site.Among them,PfArp4 was significantly enriched on the flanking sites of the centromere of chromosomes.The down-regulation of PfArp4 protein caused the deposition of H2 A.Z and H3K9 ac at the gene start codon,especially the deposition of H2 A.Z on the centromere of chromosome.Conclusion: 1.KI and KD strains of PfArp4 and PfArp6 were successfully obtained,and the two labeled strains with different tags were added.PfArp4 and PfArp6 proteins were located on the nucleus of Plasmodium falciparum,both parts Overlapping,experiments show that the two interact with each other;2.The induced down-regulation of PfArp4 leads to restricted growth of Plasmodium falciparum;3.PfArp4 is positively correlated with the dynamic expression of genes.The induced down-regulation of PfArp4 led to the reduction of H2 A.Z and H3K9 ac levels in the upstream region of eukaryotic genes,thereby suppressing the transcription of H2A.Z-dependent genes;4.PfArp4 is located at the flanking site of all centromeres.PfArp4 affects the centromeric function by controlling H2 A.Z deposition.