Studies On Aquaporins Genes Of Hydatid Of Echinococcus Granulosus

Objective1.The sequences and structure properties of EgAQPs were analyzed by bioinformatics methods to provide foundation for researches of the biological functions of EgAQPs.2.The optimal reference genes for studies on expression profiles of EgAQPs genes in different developmental stages of hydatid by RT-qPCR method were selected to lay foundation for exploring the relationship between the transcript expression statuses of EgAQPs genes and the formation or accumulation of hydatid fluid.3.Eg AQPs genes were cloned and heterogeneous expressed in Xenopus laevis oocytes to identify their water channel activities,which could provide foundation for understanding the roles and mechanisms of EgAQPs genes in the formation or accumulation of hydatid fluid.Methods1.The sequences and structure properties of EgAQPs were analyzed by bioinformatics methods,and phylogenetic tree was constructed to compare the relationship between EgAQPs and AQPs from various organisms.Bioinformatics tools,such as ProtParam,MotifScan,Conserved Domain,TMHMM 2.0,ProtScale,were used to analy the physiological biochemical characters,post-translational modification sites,conservative structural domain,transmembrane regions,hydrophilicity/hydrophobicity.Clustal omega was used to perform multiple sequence alignment,and phylogenetic tree was constructed by MEGA 6.06.2.The stability of commonly used reference genes of hydatid in vitro and in vivo was evaluated by geNorm software to select the stable reference genes under specific experimental conditions.With the established method of RT-qPCR for the reference genes recommended by geNorm,the transcript expression profiles of Eg AQPs genes in different developmental stages of hydatid could be detected.Protoscoleces were cultured in vitro or inoculated in the abdominal cavities of mice to establish infection models of hydatidosis.geNorm software was used to evaluate the stability of five candidate reference genes,including ND1,ATP6,elp,actin and ActII,in samples from different developmental stages of hydatid in vitro and in vivo.With the established method of RT-qPCR for the reference genes recommended by geNorm,the transcript expression profiles of EgAQPs genes in different developmental stages of hydatid could be detected.3.EgAQPs genes were cloned by RT-PCR.The heterologous expression vectors pCS107-EgAQPs were constructed and expressed in X.laevis oocytes in order to identify their water channel activities.E.granulosus protoscoleces were harvested and total RNA was prepared.Pairs of primers were designed and restriction sites EcoRI/xhoI were added.EgAQPs genes were amplified by RT-PCR and cloned into p CS107 vector.The heterologous expression vectors pCS107-Eg AQPs were confirmed by digested with Eco RI/xhoI,and sequencing.cRNA encoding EgAQPs were injected into X.laevis oocytes,and the cell volumes of X.laevis oocytes injected at different time points were recorded.P_f value was calculated to identify these proteins are functional water channel or not.4.The whole protein,cytosolic protein and membrane protein of X.laevis oocytes were prepared.Western blotting was conducted to test the expression statuses of EgAQP4 and EgAQP9 in the cell membranes of X.laevis oocytes.Results1.The results of bioinformatics analysis suggested that the MIP superfamily signature was predicted in all nine Eg AQPs,including EgAQP,EgAQP1,three transcripts of EgAQP4(accession number:CDS21745,CDS21752 and CDS21753),two transcripts of EgAQP9(accession number:CDS21164 and CDS22736),Eg AQPAn G and EgAQPFA-CHIP,which indicated that Eg AQPs are members of MIP superfamily.NPA motifs were not conserved,especially the first NPA motif various among EgAQPs.The number of transmembrane regions of EgAQPs varies from zero to five.In the phylogenetic tree constructed by the neighbor joining method,all nine EgAQPs were divided into two branches,the aquaporins and the aquaglyceroporins.Two transcripts of EgAQP9(accession number:CDS21164 and CDS22736)are members of aquaglyceroporins;Eg AQP,EgAQP1,three transcripts of EgAQP4(accession number:CDS21745,CDS21752 and CDS21753),EgAQPAnG and EgAQPFA-CHIP are members of aquaporins.2.According to the analysis results of geNorm software,the stable reference genes were different under different experiment conditions.Gene Act II and actin were optimal for RT-qPCR detecting of EgAQPs gene transcript expressions of E.granulosus protoscoleces in vitro;Gene ATP6and ND1 were optimal for RT-qPCR detecting of Eg AQPs gene transcript expressions of germinal cells of cyst walls of hydatids in vivo.With the established method of RT-qPCR for the reference genes recommended by geNorm,the transcript expression profiles of Eg AQPs genes in different developmental stages of hydatid in vitro and in vivo could be detected.The transcript expressions of genes EgAQP3,EgAQP4 and EgAQP9 of protoscoleces were much higher than that of germinal cells of cyst walls of hydatids,which were hardly detected from 3 to 7 months after the protoscoleces inoculation in the abdominal cavities of mice.The transcript expressions of genes EgAQP,EgAQP1,EgAQPFA-CHIP,EgAQPAnG were very low in both protoscoleces and germinal cells of cyst walls of hydatids from different developmental stages after the protoscoleces inoculation in the abdominal cavities of mice,which were hardly detectable also.3.Genes EgAQP3,EgAQP4 and EgAQP9 were amplified successfully by RT-PCR,when total RNA isolated from protoscoleces were used as templates.The gene sequence similarity between the reverse complementary sequence of EgAQP3 and the nucleotide sequence of EgAQP9 was 99%.The protein sequence similarity between EgAQP3 and EgAQP9 was 100%,which indicated that EgAQP3 and EgAQP9 were encoded by the same gene.EgAQP3 and Eg AQP9 were designated as EgAQP9 in this study.The heterologous expression vectors pCS107-EgAQP4 and pCS107-Eg AQP9 were constructed and expressed in X.laevis oocytes in order to identify their water channel activities.Eg AQP4and EgAQP9 could not perform their water channel activities in X.laevis oocytes.4.Western blotting was conducted to test whether EgAQP4 and EgAQP9 were expressed in the cell membranes of X.laevis oocytes or not.The results of Western blotting indicated that EgAQP9 was expressed successfully in the cell membranes of X.laevis oocytes.The expression of EgAQP4 could not be detected in whole protein,cytosolic protein and membrane protein of X.laevis oocytes,which indicated that Eg AQP4 could not be expressed in X.laevis oocytes.Conclusions1.The results of bioinformatics analysis suggest that Eg AQPs are members of MIP superfamily,the number of transmembrane regions of EgAQPs are different from that of the typical AQPs.Furthermore,NPA motifs are not conserved among EgAQPs.2.The optimal reference genes were selected by geNorm in vitro and in vivo and could be used to standardize the transcript expression levels of EgAQPs genes.During the development of hydatid cysts from protoscoleces in the abdominal cavities of mice,the transcript expression levels of EgAQPs genes of germinal cells of cysts walls of hydatids decreased greatly compared with the protoscoleces.3.Genes Eg AQP3,EgAQP4 and EgAQP9 were cloned successfully in this study.Since EgAQP3 and Eg AQP9 were encoded by the same gene,EgAQP3 and Eg AQP9 were designated as EgAQP9 in this study.EgAQP4and EgAQP9 could not perform water channel activities in the trial of X.laevis oocytes express system.The expression of EgAQP9 was detected in the cell membranes of X.laevis oocytes by Western blotting analysis.The expression of EgAQP4 could not be detected in whole protein,cytosolic protein and membrane protein of X.laevis oocytes.4.Based on the above mentioned results,this study might present a hypothesis about the relationship between the formation or accumulation of hydatid fluid and Eg AQPs:the transcript expression levels of EgAQPs genes of germinal cells of cyst walls of hydatids decreased greatly compared with the protoscoleces might be explained by the reason that a protoscolx mainLy be composed of parenchymal tissue.EgAQPs play important roles in parenchymal tissue.During the development of hydatid cysts from protoscoleces in the abdominal cavities of mice,parts of cells of the parenchymal tissue may be lysised into water,the component of hydatid fluid.The dysfunctions or low expression levels of these AQPs in germinal cells of cyst walls of hydatids are beneficial to the living of germinal cells.The water outside the lumen of cyst can not enter the germinal cells on the cyst wall through these AQPs to cause cell lysis.Furthermore,the water in the lumen of cyst can neither enter the germinal cells on the cyst wall through these AQPs to cause cell lysis,nor can it be transported through these AQPs in germinal cell membranes to the outside of cyst wall,so that the accumulation of hydatid fluid can increase the volume of hydatid cyst.As a result of water reservation,the hydatid cysts can grow up and survive for a long time in the organs or tissues of intermediate hosts.