The Interaction Of Peroxinectin With Integrin,CuZn-SOD And The Effect Of Cd²⁺ Stress On Immune System:Based On The Transcriptome Analysis In Chinese Mitten Crab ?Eriocheir Sinensis?

In this study,transcriptome analysis of Eriocheir sinensis in normal physiological and Aeromonas hydrophila infected state was done with high throughput sequencing technology,and some immune related gene sequences were screened.On this basis,the interaction of peroxinectin with integrin,CuZnSOD was analyzed by using bimolecular fluorescence complementation?BiFC?,the changes of the expression of immune related genes in Eriocheir sinensis under Cd²⁺stress were detected by real time fluorescence quantitative PCR technology,and the morphological and structural differences of the gill tissue of Eriocheir sinensis after Cd²⁺stress were analyzed by tissue slicing technology.The main results were as follows:?1?Two cDNA?Control and Infected group?libraries were successfully constructed and sequenced on Illumina HiSeq 4000 platform.More than 5 Gb database output was obtained from each transcriptome.Respectivelly,63.85×10⁶ and64.91×10⁶ raw reads were generated by sequencing,while 47.85×10⁶ and 48.36×10⁶clean reads remained after screening.These clean reads were then assembled into93,328 and 78,731unigenes,with N50 of 1014 bp.?2?Approximately 15.79%?5.10%?20.08%?15.53%and 28.29%non-redundant unigenes of the control transcriptomes and 13.19%?4.37%?17.90%?14.0%8 and25.92%were annotated to homology sequences of Pfam?String?Swissprot?KEGG and NR databases,Respectivelly.?3?According to GO analysis,6,233 and 5,068 unigenes were assigned to three main categories including biological process,cellular component and molecular function,with the dominant subcategories to be cellular process,cell and cell part,binding.Then,6,233 and 5,068 unigenes were classified to 26 COG groups.We also found that the function groups related to immune signaling included signal transduction and defense mechanism.KEGG pathway analysis showed that 11,625and 9,120 unigenes were claried to five kinds of pathways.Among them,709 and 519unigenes were relevant to immune system,signal transtruction,immune molecules and interaction.We also found that 158 unigenes were related to immune system.These sequences will provide valuable data for immune study and disease control of the crab.?4?A total of 1,683 differentially expressed genes?DEGs?were detected after RPKM normalization and comparison between the two transcriptomes.1,450 genes were up-regulated while 233 were down-regulated in infected transcriptome compared with control transcriptome.38,370 and 36,127 putative SNPs from control and infected transcriptomes,respectivelly.They may offer important resources of candidate markers for future selective breeding of E.Sinensis.?5?BiFC system testing found that the cell adhesion protein?Peroxinectin?binds to integrin and CuZn-SOD in eukaryotic cells,and the three genes of PXN,Integrin and CuZn-SOD were significantly down-regulated after Aeromonas hydrophila infection,.?6?The expression of seven immune-related genes?LGBP?PPAF?Dorsal?Integrin?GPx?GST?PXN?were quantitatively analyzed by means of quantitative real-time PCR in haemocyte under Cd²⁺stress.The results showed that Cd²⁺had a significant effect on immune-related genes and the expression patterns of seven genes in this study were different.?7?Tissue slicing technology analysis found that the complete degree of gill structure decreased and the thickness of gill is increased under Cd²⁺Stress.