Molecular Characteristics And Functional Analysis Of Bx42 In Macrobrachium Nipponense

Macrobrachium nipponense is an important economic species cultivated in China.Due to the existence of a large number of pathogenic microorganisms in the water,the cultivation of M.nipponense is severely restricted.At present,the research on the innate immune mechanism of aquatic crustaceans has become a hot spot.Through bioinformatics research to find key genes for disease resistance,and verify through traditional experiments,so as to regulate the immunity of crustaceans and achieve the purpose of healthy breeding.SNW1 is a considerable conserved protein in the evolution process.In mammals,SNW1 is mainly involved in pre-mRNA splicing and transcriptional modulation.Bx42 is the homologue of SNW1 in mammals,and Bx42 shares similar functions with SNW1,mainly involved in the pre-mRNA splicing and transcription modulate.However,up to now,the existence and function of Bx42 are still poorly understood in invertebrates.In the current study,a new SNW domain-containing protein MnBx42 from M.nipponense was identified and its potential role in immune response was studied,the specific results are as follows:(1)The cloned MnBx42 gene has a full length of 7467 bp,including an open reading frame of 1653 bp,which encodes 550 amino acids.Amino acid multiple sequences alignment shows that the Bx42 protein is highly conserved during the evolution from invertebrates to vertebrates,and there was a few differences of SNW/SKIP domain between vertebrates and invertebrates.The phylogenetic tree was constructed by the NJ method,and the results showed that M nip-ponense Bx42 was first grouped with Bx42 in invertebrates,and then grouped with Bx42 in vertebrates.(2)The result of real-time quantitative PCR analysis showed that MnBx42 is mainly expressed in the intestine,gills,and hepatopancreas,and relatively low in the eyestalk,blood cells,muscle,stomach,heart and nerves.The result of immunocytochemistry indicated that MnBx42 protein mainly located in the nucleus.(3)With stimulated with Aeromonas hydrophila,LPS,Staphylococcus aureus and White Spot Virus(WSSV),the expression levels of MnBx42 exhibit different degrees of significant decrease,indicating that it may be involved in the innate immune response.(4)Compared with the control group(GFP protein silenced),after interference of MnBx42 gene the susceptibility of M.nipponense to bacterial infection is significantly increased.At the same time,the results of bacterial clearance experiments showed that after interference of MnBx42 the amount of bacterial load in the gills was significantly increased.(5)The results of blood cell phagocytosis experiments showed that after interference of MnBx42 gene significantly reduced the phagocytosis of A.hydrophila.The results of dual luciferase reporter assay showed that MnBx42 can significantly activation of the NF-κB promoter.In line with this,when MnBx42 is silenced in vivo,the expression level of antimicrobial peptides(AMPs)genes,including ALF2,ALF3,ALF4,ALF5,Crul and Cru2,as well as genes in the NF-κB signaling pathway,including Dorsal,Relish,TAK1,TAB1,IKKβ and IKKε were significantly reduced.In summary,MnBx42 gene can not only regulate cellular immunity,but also regulate humoral immunity to exert antibacterial function.These findings can rovide new insights about Bx42 in crustaceans and pave the way for a better understanding of the innate immune system of crustaceans.