Study On The Structure And Function Of Autophagy-related Genes In Ovine Babesia

Ovine babesiosis is a tick-borne blood protozoan disease characterized by fever,hemoglobinuria and hemolytic anemia.At present,studies have shown that ovine Babesia is widespread in China,and high infection rate of ovine Babesia seriously affects the health and sustainable development of small ruminant industry.Autophagy is a process,which relied on the lysosomal pathway,to degrade longevity proteins or damaged organelles under the control of autophagy-related genes(ATG).In recent years,researches have revealed that ATGs are not only involved in regulating the occurrence of Plasmodium and Toxoplasma autophagy,but also play key roles in maintaining the homeostasis of apicoplast,mitochondria and other organelles and the growth and replication of parasites.Further more,there is a potential relationship with the production of resistant strains(antimalarial drugs such as dihydroartemisinin,chloroquine and piperazine).Babesia,Plasmodium and Toxoplasma belong to apicomplexan protozoa,however,there is no research report on whether there is an autophagy system and the structure and function of ATGs in Babesia.Therefore,this study took Babesia sp.Xinjiang(BspXJ)as the research object,through the bioinformatics analysis of ATGs of piroplasm,determination of apicoplast and mitochondrial genome of ovine Babesia,evaluation the effects of autophagy inhibitors and inducers on the expression abundance of ATGs,the establishment of gene editing technology of ovine Babesia,analysis of ATGs subcellular localization and identification of interacting proteins,to clarify the function of ATGs.It provided basic data for in-depth research on the function of ATGs of Babesia,and also established a technical platform for the use of gene editing technology to carry out research on gene function of Babesia.The main research results are summarized as following:1.The bioinformatics analysis of the ATG of piroplasm showed that only part of the core ATGs existed in the Babesia genome,including ATG3,ATG4,ATG7,ATG8,ATG18 and Vps34.Among them,the catalytic or binding sites of the ATGs protein of different species are highly conserved,and have also been shown to be suitable as a molecular target for taxonomic research on apicomplexan protozoa.Secondly,the sequence anaysis of apicoplast and mitochondrial genomes of ovine Babesia showed that their genome sizes were 29916–30846 bp and 5767–5946 bp,respectively.The phylogenetic analysis indicated that ovine Babesia were divided into Babesia sp.and Babesia motasi,and B.motasi was further divided into two subspecies.The results provided organelle genome information for the subsequent study of ATGs gene function.2.The optimal transfection conditions were screened by transient transfection.The results showed that the optimal transfection buffer,program and plasmid dosage were Human T Cell nucleofector solution,V-024,20 ?g,respectively,and the promoter activity were Actin IG > ef1?AIG > ef1?BIG > Rap IG.On the basis of the successful transient transfection,the e GFP-BSD fusion protein was inserted into the ef1?locus of the BspXJ genome by using homologous recombination technology.The results of PCR,WB and IFA showed that a monoclonal strain of Babesia stably expressing the e GFP-BSD protein was obtained,which indicated that BspXJ transient transfection and stable transfection system were successfully established.It provided technical support for subsequent research on gene function of Babesia.3.The ATGs polyclonal antibody of BspXJ were prepared and analyzed,which showed that the expression of natural ATG protein were low.The BspXJ strains overexpressing ATGs were constructed by using the established stable transfection system,and identified by q PCR,PCR,WB and IFA.And the results showed that the BspXJ strains overexpressing ATG3 and ATG8 were successfully obtained.Then subcellular localization of ATG3 and ATG8 proteins and the interacting proteins were analyzed and identified.The results showed that ATG3 and ATG8 are partially co-localized with apicoplast and mitochondria,and the proteins interacting with them are mainly involved in the regulation of biological processes such as autophagy,ubiquitin degradation,invasion and the main functions of mitochondria.4.QPCR was used to detect the expression abundance of ATGs at different time points after treatment with the optimal concentration of BspXJ autophagy inhibitor and inducer.The results showed that the optimal concentration of 3-methyladenine,bafilomycin A1,chloroquine and rapamycin were 2 m M,12.5n M,12.5 ?M and 5 ?M,respectively.Both HBSS and rapamycin autophagy inducers could increase the expression abundance of ATGs after treating BspXJ,in which the expression of ATG8 increased by 3–26.5 times,while after treatment with autophagy inhibitors,the expression abundance of ATGs decreased/increased less than one,which indicated that the ATGs of BspXJ were involved in regulating its autophagy process.5.The stable transfected gene knockout strains were not obtained after multiple rounds of screening by using homologous recombination technology to knock out ATG3 and ATG8,which indicated that ATG3 and ATG8 were key genes in the growth and replication process of BspXJ.In order to further study its gene function,the p BS-m Cherry-AID-3HA plasmid was transfected into the strain stably expressing TIR1-3Flag,and the strain stably expressing m Cherry-AID-3HA was successfully screened.Four hours after 400 ?M IAA was added,it was found that the expression level of m Cherry-AID-3HA protein was significantly reduced,which indicated that the conditional gene knockout system of BspXJ was successfully established.This provided a technical platform for future research on the mechanism of ATG3 and ATG8.In summary,BspXJ was successfully established the transient,stable transfection and conditional gene knock-out technology,which preliminarily clarified that there is an autophagy pathway in the BspXJ,and the ATGs of BspXJ were involved in the regulation of autophagy pathways.It provided basic data for the in-depth development of the mechanism of ATGs of Babesia,and also provided a technical platform for the study of invasion,reproduction,pathogenicity,metabolism and other mechanisms of Babesia.