Transcriptome Construction Of Gills Of Cherax Quadricarinatus Under Aeromonas Hydrophila Stress And Cloning And Expression Analysis Of Theww SHSP Genes

Cherax quadricarinatus have been widely introduced and cultured around the world and are severely affected by pathogens such as viruses and bacteria.Small heat shock proteins(s HSPs)have been shown to have a positive effect on the immune response of animals,but their function in resistance to bacterial stress in Cherax quadricarinatus remains to be further clarified.Therefore,in this study,transcriptome sequencing was carried out in the gill tissue of the second generation,and three s Hsps genes(Cqs Hsps,named CqHsp24.06,CqHsp41.97 and CqHsp40.17)with significant expression differences were detected by transcriptome,cloning,informatics analysis and expression changes were studied.The main results are as follows:(1)The second generation transcript database of gills tissue of Cherax quadricarinatus was constructed.A total of 133322601 fragments were read from six experimental samples,and 39776631602 bases were detected,with GC content ranging from 37.41% to 40.79%.A total of 51,298 transcripts were annotated after comparison with existing databases.Analysis of differentially expressed transcripts showed that there were 1917 differentially expressed transcripts in the stress death group compared to the control group.Compared with the control group,there were 3872 differentially expressed transcripts in the stress survival group.Compared with the stress survival group,there were1184 differentially expressed transcripts in the stress death group.(2)To GO enrichment analysis of differentially expressed transcript,stress survival group and stress the transcription of the function of enrichment of differentially expressed between death group and control group function in the transcription of the differential expression of stress concentration in biological processes,and different aspects of cellular components,in the aspect of molecular function the same,both on antioxidation and the activity of electron carrier function expression differences.The KEGG database was used for further functional enrichment analysis of these differentially expressed transcripts.Compared with the control group,the differentially expressed transcripts in the stress group were more reflected in metabolization-related functional pathways,among which carbon metabolism pathway,lysine degradation pathway and oxidative phosphorylation pathway were the most correlated.The differential expression of transcriptional functions between the stress survival group and the stress death group was also enriched in metabolic related pathways,among which carbon metabolism pathway and oxidative phosphorylation pathway were the most correlated.Analysis of differential expression enrichment of transcripts of the same functional pathway showed that there were more differential expression enrichment of transcripts in carbon metabolism pathway,oxidative phosphorylation pathway,lysosomal pathway and phagosome pathway.(3)The encoding sequences of CqHsp24.06,CqHsp41.97 and CqHsp40.17 were 654 bp,1128 bp and 1074 bp,and the molecular weights were24.06,41.97 and 40.17 k Da,respectively.Their Hsp21 sequences are similar to those of other crustaceans and have similar motifs and domains.CqHsp24.06 contains alpha-Crystin-HSPS＿P23-like superfamily domain,while CqHsp41.97 and CqHsp40.17 contain metazoan＿ACD domain.The amino acid sequences of CqHsp 24.06,CqHsp 41.97 and CqHsp 40.17 all contained multiple protease sites.The CqHsp 21 marker sites of the three small molecule heat shock proteins were 5,6,3 and 1.The HSP21 sites were similar to those of Macrophthalmus japonicus,Penaeus vannamei and Macrobrachium nipponense.CqHsp41.97 and CqHsp40.17 have the same Motif 8 as macrobrachium nipponense.CqHsp24.06 does not have Motif 8,which is similar to Hsp21 motif of Japanese bigeye crab and South American White shrimp.(4)In normal tissues,the expression of CqHsp24.06 was highest in intestinal tract,CqHsp41.97 in gills and CqHsp40.17 in nerves.Under bacterial stress,the relative expression levels of CqHsp24.06,CqHsp41.97 and CqHsp40.17 in gills of Cherax quadricarinatus firstly increased and then decreased.Under the stress of Aeromonas hydrophila,CqHsp24.06 and CqHsp41.97 had the highest expression levels at 3 h and 12 h.The results showed that Cqs Hsps were involved in the immune response of red chelambarus,and the sensitivity of Cqs Hsps to Vibrio algolytica was higher than that of Aeromonas hydrophila.In conclusion,this study completed the full-length transcriptome sequencing of C.quadricarinatus,and enriched and supplemented the transcriptome information of C.quadricarinatus.Through functional annotation and enrichment analysis of differentially expressed transcripts,it was found that carbon metabolism pathway,oxidative phosphorylation pathway,lysosomal pathway and phagosomal pathway were involved in the immune response of red catfish.Three different s HSP genes related to the four pathways were also cloned: CqHsp24.06,CqHsp41.97 and CqHsp40.17.The changes of their expression in different tissues and infected by different bacteria were analyzed by fluorescence quantitative PCR.In this study,the bioinformatics analysis of cloned s HSP was carried out,and the relative expression of s HSPs gene in C.quadricarinatus was preliminarilly studied.However,the mechanism of s HSP protein in C.quadricarinatus and its role in immune response need to be further clarified.