| Microsporidium is a typical obligate eukaryotic microorganism.Enterocytozoon hepatopenaei(EHP),as a microsporidia that mainly infects the hepatopancreas of Litopenaeus vannamei,can cause hepatopancreatic microsporidiosis(HPM).There are no obvious pathological symptoms in the early stage of this disease infection,and Litopenaeus vannamei will not show significant slow growth until 30-45 days after infection.Due to the long infection cycle and no effective prevention and control measures,the spread of EHP has brought huge economic losses to the global shrimp aquaculture industry.The infection mechanism of microsporidia is quite special,that is,under appropriate environmental stimulation,microsporidia germinates,the inner polar filaments of spores are quickly released to the outside and form hollow polar tubes,through which the infectious sporoplasm is transported to the host cell to complete the infection.It was found that aquaporin was involved in activating the germination process of microsporidia.Therefore,the identification and functional study of EHP aquaporin will help us better analyze the germination mechanism of EHP,and provide ideas and references for the prevention and control of EHP.In this study,sequence characteristics and expression localization characteristics of EHP hypothesized aquaporin EHP00＿492 were analyzed,and it was found that EHP00＿492 was a potential aquaporin with conserved characteristics of aquaporin family members.In order to further explore the function of EHP00＿492,the germination rate of EHP,the changes of the endometrium structure of EHP and the blocking experiment of EHP00＿492 antibody were comprehensively analyzed under the condition of high sucrose solution,and it was confirmed that EHP00＿492 has the function of regulating the osmotic pressure in EHP spores.The main research results are as follows:1.Bioinformatics analysisAccording to the conserved analysis of gene locus of different microsporidia,EHP hypothetical aquaporin EHP00＿492 was screened.There is a conserved aquaporin superfamily domain(pfam:MIP)in the whole gene sequence region,and the TMHMM software was used to analyze the amino acid sequence and found that there are 6transmembrane domains.At the positions of amino acids 12-31,41-60,91-113,113-155,176-198,218-240,the protein sequence also contains two conserved structure motifs of aquaporins,NPA(Asn-Pro-Ala).The two moths were symmetrical in the position of 69-71 aa and 201-203 aa,respectively.According to the results of homologous sequence alignment,it was found that aquaporins had very high homology in microsporidia,and the highest homology with Enterocytozoon bieneusi postulant aquaporins(Gen Bank No.EBI＿27080)was as high as 73%.The sequence similarity with Encephalitozoon cuniculi(ECU07＿0740)was 47%,the sequence similarity with Encephalitozoon hellem(EHEL＿060160)was 48%,and the sequence similarity with Encephalitozoon intestinalis(Eint＿070680)was 46%.EHP00＿492 phylogenetic tree was drawed by MEGA software,also observed that the closest evolutionary relationship with the protein is the aquaporin of Enterocytozoon bieneusi.In addition,phylogenetic tree results showed that the aquaporins of microsporidium were more closely related to those of yeast than those of human and prawn.The three-dimensional structure model of EHP00＿492,Ec AQP and Nb AQP was constructed by Alpha Fold and the results were compared and analyzed.The results showed that the three-dimensional structure of these three proteins overlaps highly at 10-32 aa,42-126 aa,129-160 aa and 172-242 aa.It suggests that its function is conserved in microsporidia.2.Expression and localization of EHP00＿492EHP spores were isolated and purified from EHP-infected hepatopancreas of penaeus shrimp by differential centrifugation combined with sucrose density gradient centrifugation.Total RNA of EHP spore was extracted and purified,converted into c DNA template for RT-PCR amplification,and a single band of about 750 bp was obtained,indicating that EHP00＿492 coding gene was normally transcribed in EHP spore.The recombinant plasmid p Cold-TF-EHP00＿492 was induced by Isopropyl-1-thio-β-D-galactoside.The recombinant plasmid Pcold-Tf-EHP00＿492 was heterologous expressed in Escherichia coli.The recombinant protein EHP00＿492 was obtained,and the recombinant protein EHP00＿492 was immunized with rabbits to prepare polyclonal antibody.The specificity of EHP00＿492 rabbit antibody was detected by Western blot.The results showed that EHP00＿492 rabbit antibody could specifically recognize a natural protein of about 21 k Da in the total protein of EHP spore.The molecular weight of the protein was slightly smaller than the predicted molecular weight.Subcellular localization of EHP00492 was analyzed by Indirect immunofluorescence analysis(IFA)and Immunoelectron microscopy(IEM).It was found that the antibody could locate specifically to the wall and plasma membrane of EHP mature spores,and TF tag rabbit antibody could not label EHP mature spores.Therefore,based on the sequence,gene cloning,protein expression and localization characteristics of EHP00＿492,we confirmed that EHP00＿492 basically conforms to the conserved characteristics of aquaporin family and is a potential aquaporin of EHP.3.The function of EHP00＿492In 1990,it was proved that high sucrose solution could inhibit N.algerae(Na)germination.In order to study the effect of high sucrose solution on EHP germination,2M sucrose and 0.1 M KOH were used to treat spores respectively and the germination rate was calculated.The results showed that the spore germination rate of EHP was significantly inhibited after 2 M sucrose treatment,while the spore treated with 0.1 M KOH as the control group could still germinate normally.Moreover,after sucrose dialysis,EHP was found to recover the germination ability,indicating that sucrose could inhibit the germination of EHP.Since the germination of microsporidia is closely related to the expansion of anterior polaroplast and vacuole,and it has been shown that lipophilic membrane dye Di I can label the endospore of microsporidia,in this study,Di I was used to stain EHP,and it was observed that it can be labeled.Note one end of the mature spore of EHP,showing intense orange-red fluorescence.EHP was then treated with sucrose and the changes of endospore were observed by Di I staining.IFA results showed that EHP membrane structure was significantly reduced in sucrose treatment group compared with control group.The results showed that sucrose prevented the endosporal structure of EHP from expanding,and therefore from ejecting the polar tube.In order to clarify the function of EHP00＿492 in EHP germination,we found that the endospore of EHP spore blocked by EHP00＿492 antibody was not affected by sucrose,suggesting that EHP00＿492 played an important role in EHP germination.We constructed a recombinant vector p IZT-EHP00＿492-GFP and expressed EHP00＿492protein on the cell membrane of Sf9,which provided the basis for a better study of the function of EHP00＿492. |
