Cloning And Expression Analysis Of Three HSP And CuZnSOD Genes In Cherax Quadricarinatus

Cherax quadricarinatu belongs to the arthropod phylum,crustacea,decapoda,Parastacidae family,Cherax genus,native to Australia,has been widely cultivated in China,has produced huge economic value.Cherax quadricarinatus suffered from aquatic disease outbreaks and bacterial infections during the breeding process,which caused significant losses to the red crawfish aquacu Lture industry.However,it is unclear how the innate immune regu Latory network of Cherax quadricarinatus is under the stress of pathogenic microorganisms.Therefore,this study conducted fu Ll-length transcriptome sequencing of Cherax quadricarinatus and cloned Cherax quadricarinatus HSP60,HSP 70,HSP 90 Waiting for the fu Ll length of the cDNA sequences of three different HSPs genes and CuZnSOD genes,they were analyzed informatively,and the fluorescent quantitative PCR was used to study the four genes in different tissues in normal shrimp and in Vibrio alginolyticus,Vibrio parahaemolyticus and Aeromonas hydrophila after infection Expression changes in gills.The main findings are as follows :(1)Extract the total RNA of normal Cherax quadricarinatus tissues separately,perform Pac Bio Sequel sequencing,and analyze by bioinformatics method.Using SMRT,a total of 423407 polymerase reads(21.24 Gb)can be obtained,with an average read length of 50165 bp,N50 of 83833 bp,366570 CCS,and fu Ll-length non-chimeric reads(FLNC)of 289232,the average length of FLNC 3717 bp;13449 transcripts were annotated in total,12631 transcripts were mapped to 354 KEGG pathways,coding sequences were identified by ANGEL software,14220 coding sequences were obtained and 8319 had complete ORF,all predictions were made using BLASTN The lnc RNA(4333)was identified as a novel lnc RNA with an average length of 665.39 bp,and21108 SSRs were identified in all tested transcripts.It enriched and supplemented the genomic information of the Cherax quadricarinatus.(2)Cherax quadricarinatus HSP 60 gene cDNA is 1731 bp in total length,encodes 576 amino acids,theoretical molecu Lar weight is 61.27 kda,isoelectric point is 5.23,contains a hexapeptide and octapeptide repeat sequence,C-terminal contains ATP binding motif and typical GGM repeat motif;HSP70gene c NDA is 1932 bp fu Ll length,encodes 643 amino acids,theoretical molecu Lar weight is 70.29 k Da,isoelectric point is 5.27,contains ATP / GTP binding site,NLS-BP and EEVD motif;HSP90 gene c NDA The fu Ll length is2199 bp,encoding 732 amino acids,the theoretical molecu Lar weight is 84.47 k Da,the isoelectric point is 4.89,and includes the Gxx Gx G motif,NLS-BP and a MEEVD motif;Amino acids,the theoretical molecu Lar weight is 17.05 kda,and the isoelectric point is 5.16.Contains four highly conserved copper-zinc binding sites.The evolutionary tree comparison found that the cloned Cherax quadricarinatus HSPs and CuZnSOD genes were highly conserved in different animals.(3)Take Cherax quadricarinatus separately,extract the total RNA of each tissue,synthesize cDNA,and carry out fluorescence quantitative expression analysis,QRT-PCR resu Lts show that the Cherax quadricarinatus HSP 60,HSP70,HSP 90 and CuZnSOD gene heart,hepatopancreas,gill,Intestine,gonad,eye stem,muscle,abdominal nerve and other 8 kinds of tissues are all expressed,but the expression patterns in male and female shrimp tissues are different.HSP60 gene is most expressed in male hepatopancreas,heart and gonads,and is highest in female gonads and heart;HSP 70 gene is most expressed in male and female hepatopancreas and gonads;HSP 90 is expressed in male gonads and intestines The highest expression was found in the hepatopancreas and gonads in females;The CuZnSOD gene is most highly expressed in the male hepatopancreas and intestine,but the highest in the female stem and intestine.(4)Use Vibrio alginolyticus,Vibrio parahaemolyticus and Aeromonas hydrophila to stress the Cherax quadricarinatus,collect the gill tissues of the shrimp 3,6,9,12,24,48 h after stress and extract total RNA After reverse transcription to cDNA,fluorescence quantitative PCR showed that after Vibrio alginolyticus stress,HSP 60 had the highest expression at 12 h,HSP 70 had the highest expression at 9 h,and HSP 90 had the highest expression at 6 h.CuZnSOD had the highest expression in the gills of dead shrimp at 3 h,and the highest expression in the gills of surviving shrimp at 24 h;after infection with Vibrio parahaemolyticus,HSP 60 had the highest expression at 9 h and HSP 70 had the highest expression at 12 h.,HSP 90 had the highest expression in the gills of the surviving shrimp at 3 h,the highest expression in the gills of the dead shrimp at 9 h,and the highest expression of CuZnSOD at 6 h;after infection by Aeromonas hydrophila,HSP 60,HSP 70 The expression level of HSP 90 was the highest at 24 h,and CuZnSOD was the highest in the gills of the dead shrimp at3 h,and the highest in the gills of the surviving shrimp at 6 h.The resu Lts of QRT-PCR suggest that both HSPs gene and CuZnSOD gene are involved in the stress response of Cherax quadricarinatus to pathogenic microorganisms,and play an important role in the immune response of Cherax quadricarinatus.In conclusion,this study completed the fu Ll-length transcriptome sequencing of Cherax quadricarinatus,enriching and supplementing the transcript information of Cherax quadricarinatus.Three different HSPs genes HSP 60,HSP 70,HSP 90 and CuZnSOD genes of Cherax quadricarinatus were cloned,and their expression changes in different tissues and after being infected by different bacteria were analyzed by fluorescence quantitative PCR.This experiment is only cloning and preliminary Has studied the expression of HSPs and CuZnSOD genes of Cherax quadricarinatus,and its specific functions have yet to be further studied using RNAi and overexpression techniques.