Investigating The Functions Of Akirin Gene In The Resistance Of Crayfish Procambarus Clarkii To WSSV Infection Based On Transcriptome Sequencing

Procambarus clarkii has become one of the important freshwater crustaceans aquaculture species in China.With the expansion of the breeding scale,the increase of the breeding density,the deterioration of the breeding environment,and the frequent occurrence of diseases,the industrial development is seriously hindered.The high fatality rate of white spot syndrome virus（WSSV）infection of Crayfish has a huge impact on related industries.At present,most of the research on WSSV focuses on the etiology,epidemiology and detection methods,there are relatively few studies on the infection mechanism and immune mechanism of WSSV.Crayfish are primarily based on innate immune defenses against pathogens through the involvement of hemocytes.Hematopoietic tissue,as the generating organ of hemocytes of crayfish plays an important role in the immune response of P.clarkii.In order to better understand the immune response of hematopoietic tissues to WSSV,the present study established the transcriptome data of hematopoietic tissues in different time periods after P.clarkii infection with WSSV,and screened and analyzed key differentially expressed genes（DEGs）and significantly enriched pathways.Akirin,a nuclear transcription factor involved in regulating gene expression of IMD signaling pathway and NF-κB-dependent natural immune response,was screened through transcriptome database.To explore the role of Akirin in the process of WSSV infection resistance,the recombinant Akirin protein was obtained by prokaryotic expression,and the dsRNA of Akirin was synthesized in vitro.The main results of the present study are as follows:1.According to the known WSSV sequence,specific primers were designed to amplify the WSSV envelope protein VP28 sequence.The amplified sequence was codon optimized,and the VP28 recombinant plasmid was contructed.According to the quantitative relationship of plasmid concentration,a standard for WSSV copy number detection was established,and the standard curve of virus copy number with respect to CT value was drawn by RT-qPCR depending on the standard.The pET30-VP28 recombinant vector was constructed,the VP28 recombinant protein was recombinantly expressed in E.coli.The recombinant VP28 protein was purified by Ni2+affinity chromatography,and the anti-rabbit polyclonal antibody of VP28 was prepared.2.The transcriptome database of hematopoietic tissues at different time points after P.clarkii infection with WSSV was constructed by transcriptome sequencing technology.The expressions of some hematopoietic cytokines were also analyzed.After assembly,an average of 47,712,411 clean reads were obtained in the control and WSSV-infected groups.A total of 35,945 single genes were found with an N50 length of 1554 bp.Under the functional classification,enrichment and pathway analysis of different databases,about 257 differentially expressed genes（DEGs）were identified,of which 139 were up-regulated and 118 were down-regulated.GO functional analysis showed that these DEGs were mostly involved in immune response activation,complement activation,complement fixation,negative regulation of humoral immune response,and secretion of cytokines.In KEGG analysis,these DEGs were shown to be involved in extracellular matrix-receptor interaction,transcription factor HIF-1α and it signaling pathway,glycolysis/gluconeogenesis,thyroid hormone signaling pathway,and glucagon signaling pathway.RT-qPCR analysis of nine selected genes confirmed the reliability of RNA-Seq results.3.A nuclear factor Akirin was identified from the transcriptome of P.clarkii.The Akirin protein of Crayfish is composed of 204 amino acids,and the nuclear localization signal peptide motif（KRRR）is conserved in its family.PcAkirin is highly expressed in the stomach,intestine and hepatopancreas.Akirin transcript levels were significantly elevated in hemocytes and hepatopancreas following Aeromonas hydrophila infection.In addition,the prepared Akirin recombinant protein could improve the expression level of immune-related genes,and rPcAkirin could help to resist WSSV infection.Conversely,dsRNA interference with the Akirin gene increased the susceptibility of crayfish to infection with A.hydrophila and WSSV.This result was more evident in the cumulative mortality of Crayfish infected with A.hydrophila and WSSV.The above results all demonstrate that Akirin plays an important role in the innate immune response of crayfish,protecting it from WSSV and bacterial infections.