Effect Of Cadmium Stress On Immune Response Of Two C-Type Lectins Of The Freshwater Crab Sinopotamon Henanense

In order to study the effect of cadmium stress on the immune response of C-type lectins in the freshwater crab Sinopotamon honanense,we firstly used the stress method of cadmium followed by bacteria to select two C-type lectin genes ShLec21 and ShLec23 sensitive to cadmium stress from the transcriptome sequencing results of S.honanense by semi-quantitative RTPCR and real-time fluorescence quantitative technique.Then,the full-length cDNAs of ShLec21 and ShLec23 were cloned by RACE technology and their homology and phylogenetic analysis were performed.At the same time,the tissue distribution of ShLec21 and ShLec23 mRNA was detected by RT-PCR.The expression of two C-type lectin mRNAs in the hepatopancreas and hemolymph tissues was further determined by absolute quantitative method after the treatment of cadmium followed by Aeromonas hydrophila insults.Finally,recombinant prokaryotic expression plasmids of the two C-type lectins,ShLec21 and ShLec23,in fusion with GST were constructed.Recombinant fusion proteins were expressed and purified.Polyclonal antiserum against rShLec21 and rShLec23 were collected from the rabbits.Moreover,the antibacterial activity of rShLec21 and rShLec23 against Escherichia coli,A.hydrophila,Staphylococcus aureus,and Pichia pastoris were detemined by plate inhibition experiments.The results shown that:1.Screening C-type lectin genes of S.henanense in response to cadmium stressBased on the transcriptome information of S.honanense hepatopancreas,semi-quantitative RT-PCR and real-time fluorescence quantitative methods were used to detect the expression of all the available C-type lectin after the crab were treated by cadmium with different concentrations or time followed by A.hydrophila.Two C-type lectin genes,namely ShLec21 and ShLec23,were selected.2.Cloning and expression patterns of ShLec21 and ShLec23 in S.henanenseTwo full-length cDNAs of the ShLec21 and ShLec23 from S.honanense were obtained.The results showed that the full-length cDNAs of ShLec21 and ShLec23 are 863 bp and 681 bp in length,respectively.The ShLec21 cDNA contained a 459 bp open reading frame that encoded a putative 152-amino-acid protein,and ShLec23 cDNA contained a 495 bp open reading frame that encoded a putative 164-amino-acid protein.The similarity of ShLec21 amino acid sequence to EsLecG of Eriocheir sinensis was up to 65% and the similarity of ShLec23 amino acid sequence to PtLP of Portunus trituberculatus reached 48%.However,the similarity of the amino acid sequence encoded by ShLec21 and ShLec23 is only 32%.ShLec21 and ShLec23 were widely expressed in seven selected tissues,namely blood,hepatopancreas,gills,intestine,muscle,ovaries and spermarium.ShLec21 was highly expressed in hepatopancreas,intestine and ovaries,and ShLec23 was highly expressed in hepatopancreas.After cadmium stress at different time points(0,1,4 and 7d),there was no significant change in the expression of ShLec21 and ShLec23 mRNA in hepatopancreas or hemolymph.After A.hydrophila infection,the expression levels of ShLec21 and ShLec23 mRNA in the hepatopancreas were highly significantly down-regulated(p<0.01),and the expression of ShLec23 mRNA was down-regulated in the hemolymph(p<0.05)as well.However,after the Cd-stressed crabs were reinfected with A.hydrophila,the expression of ShLec21 and ShLec23 mRNA in the hepatopancreas and hemolymph was significantly(p<0.05)or highyly significantly(p<0.01)up-regulated in the cadmium-treated group at certain time points and concentration(except for the different concentrations of cadmium treatment of ShLec21).3.Prokaryotic expression,polyclonal antibody preparation and antibacterial activity of ShLec21 and ShLec23 in S.henanenseThe recombinant fusion proteins rShLec21 and rShLec23 with GST tags were efficiently expressed as inclusion bodies at 0.2 mmol/L IPTG,28℃,150 rpm for 10 h.Purified rShLec21 and rShLec23 were obtained by renaturation and purification of inclusion bodies.Using the purified recombinant fusion protein as antigen,rabbit anti-rShLec21 and rShLec23 polyclonal antibodies were prepared,and the titer was more than 1:100000 by ELISA analysis.However,the purified and refolded rShLec21 and rShLec23 showed no antibacterial activity against E.coli,A.hydrophila,S.aureus,and P.pastoris through plate inhibition circles experiments,and the reason for this awaits further investigation.Conclutions:First,ShLec21 and ShLec23 are C-type lectin genes which are sensitive to cadmium stress and may play a role in the innate immunity of Sinopotamon henanense in response to cadmium stress.This study lays a foundation for the further studies of innate immunity of C-type lectins in Sinopotamon henanense under cadmium stress.Second,through systematic bioinformatics analysis and tissue distribution results,it was hypothesized that ShLec21 and ShLec23 play different roles in innate immunity of Sinopotamon henanense.Absolute quantification results showed that cadmium can alter the response patterns of ShLec21 and ShLec23 in response to A.hydrophila infection,and can regulate the innate immunity of Sinopotamon henanense.Third,high titer rShLec21 and rShLec23 polyclonal antibodies were obtained.The results of plate inhibition assays do not indicate that ShLec21 and ShLec23 have no antibacterial activity against E.coli,A.hydrophila,S.aureus,and P.pastoris.An alternative explaination is that GST tags carried by rShLec21 and rShLec23 affect the structure and function of the two C-type lectins,which resulted in the negative results.Whether ShLec21 and ShLec23 have antibacterial activity were not proved in this study and it awaits to be approved by further research.In total,this study provides theoretical basis and practical tools for further study of the function of ShLec21 and ShLec23 genes in innate immunity of Sinopotamon henanense.