Cloning And Expression Analysis Of NLRPs Inflammasome Members From Chinese Giant Salamander(Andrias Davidianus)

Innate immunity is the body’s first line of defense against pathogen microbial invasion and is activated by a series of highly conserved pattern recognition receptors(PRRs)that recognize the pathogen associated molecular patterns(PAMPs)of certain pathogens or their products.NLRs(Nucleotide binding domain and oligomerization domain-like receptors),as a class of intracellular pattern recognition receptors,play a role in inflammatory response and clearance of pathogenic microorganisms.NLRPs(NACHT,LRR and PYD domains-containing proteins),a member of the NLRs superfamily,is a newly discovered class of intracellular PRRs,which has attracted wide attention because of its ability to participate in the formation of inflammasome.NLRPs inflammasome complex is composed of three structures: NLRPs family protein,apoptosis-associated speck-like protein containing CARD(ASC)and caspase-1.Chinese giant salamander(Andrias davidianus),commonly known as "giant salamander",is a second-class aquatic wildlife protected by the state.However,due to the infection of pathogenic microorganisms,especially the bacterial infection in the process of breeding,the reports of salamander disease gradually increase,which has become an important factor restricting the healthy development of salamander breeding industry.It is of great practical significance to study the antibacterial defense mechanism of salamander in order to promote the sustainable and healthy development of salamander breeding industry.In this study,the cDNA sequences of Chinese giant salamander NLRPs family members(AdNLRP1,AdNLRP3 and AdNLRP-like),AdASC and AdCaspase-1 were cloned by using the methods of reverse transcription polymerase chain reaction(RT-PCR)and rapid amplification of cDNA ends(RACE).The sequences were analyzed by bioinformatics and the expression patterns of the above genes in different tissues after normal and bacterial infection were studied by using realtime qPCR(qPCR).The results are as follows:(1)This paper firstly cloned the genes of NLRPs inflammasome members AdNLRP1,AdNLRP3,AdNLRP-like,AdASC and AdCaspase-1,and analyzed the sequence characteristics of the above-mentioned genes.The cloned sequences of AdNLRP1,AdNLRP3,AdNLRP-like,AdASC and AdCaspase-1 were 558 bp,2816 bp,564 bp,2034 bp and 403 bp,respectively.The number of nucleotides encoded by an open reading frame(ORF)was 185 aa(amino acids),871 aa,187 aa,444 aa,and 122 aa,respectively.The results of SMART prediction and multi-sequence analysis prove that AdNLRP1,AdNLRP3,AdASC and AdCaspase-1 contain typical and conservative domains,while AdNLRP-like does not.Phylogenetic tree analysis showed that AdNLRP1,AdASC and AdCaspase-1 are evolutionarily close to reptiles,while AdNLRP3 is close to mammals.(2)The expression of AdNLRP1,AdNLRP3,AdNLRP-like,AdASC and AdCaspase-1 in different tissues of healthy giant salamander was detected by fluorescence quantitative PCR.The results showed that AdNLRP1,AdNLRP3,AdNLRP-like,AdASC and AdCaspase-1 were constitutively expressed in the tested tissues.Furthermore,the expression of Aeromonas hydrophila after abdominal infection was analyzed.The results showed that the expression levels of AdNLRP1,AdNLRP3,AdNLRP-like,AdASC and AdCaspase-1 were significantly up-regulated in different tissues after Aeromonas hydrophila infection.In this study,giant salamander was taken as the experimental subject,and for the first time,NLRP1,NLRP3,NLRP-like,ASC and Caspase-1 of NLRPs inflammasome members were cloned and their expression was analyzed,which clarified the expression change rule of NLRPs inflammasome in giant salamander during bacterial infection.The results will lay a solid foundation for an in-depth understanding of the immune function and mechanism of NLRPs inflammasome in giant salamanders.