Study On Transcriptional Status And Functions Of Aquaporin Genes Of Echinococcus Granulosus

Aquaporins(AQPs)are a group of transmembrane channel proteins with intact plasma membranes,which can efficiently transport water molecules and some small molecules.They are widely presented on prokaryotes and eukaryotes cell membranes.It was originally found on the erythrocyte plasma membrane.By sequencing the E.granulosus genome,it was found that there were 12 genes encoding AQPs in E.granulosus.In order to study whether E.granulosus AQPs(EgAQPs)have regulatory functions on the formation of hydatid cysts fluid,our research group studied the water permeability function of these genes in X.laevis oocytes in the early stage.But none of these six genes were found to have water permeability.Therefore,this experiment continues to study the AQPs functions of the remaining 6 genes.1.The secondary hydatids cultured in vitro and inoculated in BALB/C mice were collected respectively,and the relative transcript expression levels of 6 EgAQPs genes from hydatid cysts cultured in vitro and in mice were detected by RT-qPCR.The results of RT-qPCR showed that the relative transcript levels of EgAQPs genes in the hydatid cysts were very low or even undetectable except for EgAQP3 and EgAQP9-1 at the beginning of culture in vitro.2.Full-length primers of EgAQP3,EgAQP4-1,EgAQP4-4 and EgAQP9-1 genes were designed according to the coding region sequence of EgAQPs genes in NCBI database.These genes were amplified by PCR using cDNA of hydatid cysts as template.The EgAQP4-2 and EgAQP4-4genes were synthesized by chemical synthesis.Six EgAQPs genes were cloned successfully,and no mutation was found after sequencing the recombinant plasmid p CS107-EgAQPs.These 6 EgAQPs were injected in X.laevis oocytes to verify their water channel functions.The relative volume change rates and water permeability coefficients of X.laevis oocytes injected with EgAQPs genes were showed no significantly difference compared with the negative control(p > 0.05),and these 6 EgAQPs genes did not show water channel functions in the oocytes.Western blot assay was used to detect the 6 EgAQPs genes expression of total protein,cytoplasmic protein and membrane protein on the oocytes.The results showed that the proteins were not expressed at the expected theoretical molecular weight of aquaporins.3.The results of codon-optimized bioinformatics analysis showed that the P.pastoris expression system may be more suitable for the exogenous expression of EgAQPs genes than the X.laevis oocyte expression system.According to the analysis of transcriptome of aquaporin-encoding genes in the protoscoleces of E.granulosus,the expression level(FPKM)of EgAQP9-1 was the highest among the EgAQPs genes.This analysis result was consistent with the results of the qPCR experiment of EgAQP9-1 gene,so we choose EgAQP9-1 gene to verify.The recombinant plasmid p PIC9 KEgAQP9-1 was constructed using p PIC9 K plasmid as a vector.And then the recombinant plasmid was electrotransformed into P.pastoris GS115 competent cells.The protein expression was induced by methanol and the protein was detected by Western Blot.The recombinant plasmid pPIC9K-EgAQP9-1 was successfully constructed and electrotransformed into P.pastoris GS115 competent cells.Western Blot detection showed that there was an obvious band of EgAQP9-1 protein at the theoretical molecular weight of about 37 k D,but there were also two obvious bands at the molecular weight of about 17 kD and 20 kD,which might be caused by shearing during EgAQP9-1 protein synthesis or might be the non-specific bands after the reaction with antibodies.The transcription level of EgAQPs genes were very low during the encystation process of the protoscoleces formation of secondary echinococcus,and these genes did not show the function of water permeability in X.laevis oocytes.The EgAQP9-1 gene with the highest transcriptional expression level was selected to be expressed in P.pastoris cells,and bands were detected at the positions equal to and less than the theoretical molecular weight.In conclusion,the EgAQPs genes exist in the cyst wall of hydatid cysts.And EgAQPS genes may gradually be silent or low expression during the evolutionary process,which just prevents the outflow of hydatid cyst fluid,leading to the gradual accumulation of hydatid fluid.