Study On The Role Of Anopheles MiR-2944a-3p And Its Predicted Target Gene Torso-like In The Process Of Plasmodium Infecting Anopheles

Objective:To explored the regulatory effects of Anopheles miR-2944a-3p and its predicted target gene torso-like(tsl)on Plasmodium infection in Anopheles.The miR-2944a-3p in Anopheles mosquitoes is significantly down-regulated after Plasmodium infection,and the tsl gene plays an important role in controlling the development of the Drosophila cellular immune system.To verify the distribution of Anopheles miR-2944a-3p and its predicted target gene tsl in Anopheles mosquito tissues and the targeting relationship between the two through experiments.Then to further explore its role in the process of malaria parasite infection in Anopheles and understand the relationship between the two.The interaction of molecular mechanisms and the development of new malaria control strategies provide a theoretical research basis for advancing my country’s malaria elimination plan and the global vision of malaria-free.Methods:According to the coding sequences of Drosophila melanogaster and An.gambiae tsl genes,the complete genome of An.dirus was retrieved and the An.dirus tsl gene was characterized.Specific primers were designed and the target gene was amplified using PCR and reverse-transcription PCR assays.The physicochemical properties,signal peptide,transmembrane structure,secondary structure and tertiary structure of the encoded protein TSL were analyzed using bioinformatics tools.Use the Clustal W method in the MEGA 6 software to align the selected protein sequences,and construct a phylogenetic evolution analysis based on the maximum likelihood method(ML),maximum parsimony(MP)and neighbor joining method(NJ)(Bootstrap detection value is 1000 times).In addition,the specific expression of the tsl gene was detected in various tissues of An.dirus using a quantitative real-time PCR assay.Using bioinformatics methods to predict that miR-2944a-3p will target the 3’UTR region of the tsl gene,first carry out oligo transfection and plasmid transfection verification,and use 293T cell genomic DNA as a template to design wild-type and mutations vector sequences of the tsl gene.And set up the control group PC:the two repeats of adi-miR-2944a-3p inhibitor were built into the pmirGLO vector group;pmirGlO:the empty vector group.And clone them into pmirGLO vector to construct a dual luciferase gene reporter vector.The miR-2944a-3p mimics and mimics NC were transfected into 293T cells with the wild-type vector,mutant vector and PC vector,respectively,and the relative fluorescence value was further detected.The qRT-PCR method was used to understand the existence and expression of miR-2944a-3p and tsl,and to detect the expression level of female Anopheles mosquitoes in the head,chest,abdomen and remaining parts.On the one hand,to identify the effect of miR-2944a-3p on Plasmodium infection:According to the base sequence of miR-2944a-3p,the company synthesized miRNA mimics(mimics)and miRNA negative control mimics(NC).The female Anopheles 3-5 days old after emergence were selected for microinjection of two simulants,and the overexpression efficiency was tested two days after injection.At the same time,BALB/c mice infected with Plasmodium berghei(infection rate 4%～6%)were infected with two groups of mosquito vectors.The midgut was dissected 8 days after infection,and the intestinal plasmodium in the two groups was calculated by fluorescence microscopy.The number of oocysts was statistically tested with the Mann-Whitney test.On the other hand,to identify the effect of tsl gene on Plasmodium infection:using RNAi technology,according to the sequence of Anopheles stephensi tsl gene and GFP gene,amplify and purify,and then connect with T vector and transform with DH5a competent cells,after colony PCR After verification,the plasmid was extracted and the in vitro expression vector was constructed,and the experimental group dstsl and the control group dsGFP were prepared respectively.The female Anopheles 3-5 days old after emergence were selected for microinjection of two kinds of dsRNA,and knock-down efficiency was tested two days after injection.Four days after injection,BALB/c mice infected with Plasmodium berghei(infection rate of 4%～6%)were infected with two groups of mosquito vectors.The midgut was dissected 8 days after infection,and the intestinal malaria in the two groups was calculated by fluorescence microscope The number of protozoan oocysts was statistically tested by Mann-Whitney test.In this way,to verify the effect of miR-2944a-3p and predict the target gene tsl on Plasmodium infection.Results:The An.dirus tsl gene was 16751 bp in length with a CDS region of 1134 bp,encoding 377 amino acids,and the encoded TSL protein was a stably hydrophilic protein.The TSL protein was predicted to be a secretory protein that was located in extra-mem-brane regions containing signal peptides.The secondary structure of the TSL protein contained α-helix(51.72%),extended strand(12.20%),β-bridge(4.78%)and random coil(31.30%)in the secondary structure,and a 3D homology model was generat-ed using 5cj9.1.A as a template.Phylogenetic analysis revealed a close genetic relationship in the TSL protein between An.dirus and An.farauti.In addition,quantitative real-time PCR assay detected the tsl gene expression in the head,chest,abdomen and foot of An.dirus,with the highest expression in the head and low expression in the feet.In the experimental group,adi-miR-2944a-3p mimics was co-transformed with pmirGLO-ADIR006646-RA wild-type plasmid for 48 hours.Compared with the co-transformed mimic NC group,the expression of luciferase was statistically different(P=0.0365);When the binding site of adi-miR-2944a-3p in the 3’UTR sequence was mutated and co-transformed with adi-miR-2944a-3p mimics,there was no significant difference in luciferase expression compared with the co-transformed mimic NC group(P=0.7360).This result indicates that after 48 hours of transfection,the ADIR006646-RA 3’UTR sequence constructed into the vector in this experiment can bind to adi-miR-2944a-3p,but the binding effect is weak.The miR-2944a-3p and tsl genes are expressed in the head,chest,abdomen,and feet of An.stephensi.And the expression of miR-2944a-3p is higher in the breast,and the expression of tsl gene is higher in the head.To identify the effect of miR-2944a-3p on Plasmodium infection:Compared with the mimics NC group,the expression of miR-2944a-3p increased by about 80%in the mimics group.Next,the number of oocysts of Plasmodium midgut of Anopheles mosquitoes 8 days after infection was calculated by Mann-Whitney test,and there was no significant difference in oocyst counts between mimics and NC groups(P=0.8).To identify the effect of tsl gene on Plasmodium infection:the concentration of prepared dstsl was 3.70 ug/ul,and the concentration of dsGFP in the control group was 3.77 ug/ul.Two groups of Anopheles mosquitoes were injected with dstsl and dsGFP from the chest.Two days after the injection,qPCR was used for knockdown detection.Compared with the control group dsGFP,the difference of dstsl was statistically significant(P=0.0005),and the expression of tsl was knocked down by about 61%.Next,the number of oocysts of Plasmodium midgut of Anopheles mosquitoes 8 days after infection was calculated by Mann-Whitney test,and there was no significant difference in oocyst counts between the dstsl and dsGFP groups(P=0.61)·Therefore,miR-2944a-3p and tsl genes did not affect Anopheles infection by malaria parasites.Conclusion:This study not only identified the tsl gene of An.dirus at the genomic level,analyzed and predicted its protein structure,but also obtained its tissue-specific expression characteristics.Subsequently,the dual luciferase reporter experiment was used to verify the targeting relationship between miR-2944a-3p and tsl genes.In order to further verify the function of miRNA and its target genes,the Anopheles mosquitoes were subjected to in vivo RNAi(knock down the tsl gene)through the overexpression of miR-2944a-3p followed by the in vitro construction of expression vectors,challenged by malaria parasite infection.The result showed that both of miRNA and tsl had no obvious effect on the malaria parasite infection status.