Identification And Analysis Of Salinity Stress Response Genes In Channel Catfish(Ictalurus Punctatus)

Salt water resources are an important part of water resources in China.In recent years,with the rapid development of domestic aquaculture industry and the improvement of living standards,the demand for aquatic products is increasing.Rational utilization of salt water resources and the cultivation of salt-tolerant varieties have become one of the development directions of aquaculture industry in the future.Globally,channel catfish（Ictalurus punctatus）is an important aquaculture species,first introduced into China from the United States in the 1980s.Due to its strong environmental adaptability and excellent muscle quality,the fish has become a very important freshwater aquaculture species in China.Meanwhile,previous studies have shown that channel catfish has a certain tolerance to salinity,and low salt culture can improve its survival rate.Therefore,channel catfish has the potential to cultivate new salt tolerant varieties.In this study,channel catfish juvenile were taken as the test object,the culture environment with different salinities were taken as the trentment group,and the freshwater aquaculture was used to control group.The effects of salinity on acute toxicity,growth performance and tissue antioxidant capacity of channel catfish were investigated by different salinity tests.In the meantime,comparative transcriptome analysis was used to reveal the response mechanism of channel catfish under salinity stress and the role of MAPK（mitogen activated protein kinase）gene family under salinity stress.This study consists of the following four parts:Experiment one:Acute salinity stress experiments in 24 h,48 h and 96 h were carried out on channel catfish fry at different development stages,including the yolk sac fry,10 dah fry（10th day after hatching）and 30 dah fry（30th day after hatching）.The effects of salinity on the half-lethal concentration(LC₅₀)and safe concentration（SC）of the fry at three development stages were studied,and the limit salinity tolerance of the fry at each development stage also was compared.The results showed that the effect LC₅₀ of salinity both in 24 h and 48 h were 30 dah fry>10 dah fry>yolk sac fry,while LC₅₀in 96 h was 30 dah fry>yolk sac fry>10 dah fry.The limiting salinity tolerance of fry at three development stages were 7,4 and 9,respectively.Since the fry are weak after the yolk sac disappears and still need to go through the key period of 10 dah to 30 dah.The salinity of the stocking water body of the fry in the three stages should be controlled below 4.0,4.0,9.0,respectively.Our results have important theoretical and practical significance,which could provide guidance for the scientific stocking of channel catfish fry in coastal beach areas of Jiangsu Province.Experiment two:In order to explore the effects of salinity stress on the growth performance and tissue antioxidant capacity of channel catfish fry,the salinity of 3.0 without stress response in the above stages of this experiment is the lowest salinity,and the ultimate tolerance salinity of 30 dah fry is 9.0 is the highest salinity.The test group is set up by the equal logarithm spacing method,and the growth indexes and four physical and chemical indexes of each group are measured,evaluate its tolerance to different salinity.The results of various growth indexes showed that there was no significant difference in the growth performance and survival rate of fry when the salinity was 3.0,3.8 and4.7（P>0.05）.When the salinity was 5.9,7.3 and 9.0,the growth rate of fry slowed down and the survival rate decreased with the increase of salinity.The determination results of three antioxidant enzyme activities（Superoxide dismutase,Catalase and Glutathione peroxidase）and Malondialdehyde content showed that the activity salinity of three antioxidant enzymes in liver increased first and then decreased with the increase of salinity.When the salinity was 4.7,the activities of several antioxidant enzymes were significantly increased（P<0.05）.The activities of three antioxidant enzymes in gill and kidney tissues had no significant difference in each salinity group（P>0.05）.While the content of malondialdehyde in each tissue increased significantly with the increase of salinity（P<0.05）,indicating that the fish body would be damaged in varying degrees regardless of low salt and high salt stress.The results show that the liver tissue is more sensitive to salinity stress,which is of great significance to the saline culture of channel catfish,which lays a foundation for the follow-up experiment.Experiment three:In order to investigate the changes of gene expression level of channel catfish under salinity stress,liver samples of channel catfish were selected under salinity conditions of 0,3 and 7.3.The results showed that 927 differentially expressed genes（DEGs）were detected in the low salt group and the control group,of which 564 genes were up-regulated and 363 genes were down-regulated.There were 1356 DEGs in the high salt group and control group,of which 691 genes were significantly up-regulated and 665 genes were down-regulated.KEGG enrichment showed that low/high salt stress would affect the metabolism related pathways such as Amino acid metabolism,Fatty acid metabolism,Glucose metabolism and Steroid biosynthesis.Most of the enriched genes in these pathways were up-regulated under salinity stress.Go enrichment showed that DEGs significantly enriched in pathways related to oxygen transport,such as Oxygen carrier activity,Gas transport,Molecular carrier activity,Oxygen transport and Hemoglobin complex,and most of them were down-regulated.Scd,fas,fads2,acat,gale tm7sf2,sqle,dbt,hbb,hba and other genes related to energy consumption were screened from the liver transcriptome of channel catfish under salinity stress.Primers were designed and verified by q RT-PCR（Quantitative Reverse Transcription PCR）experiment.The results showed that the gene expression trend was consistent with the RNA-Seq results,which could be used as a candidate gene for channel catfish to deal with salinity stress.The results lay a foundation for the study of the steady-state regulation mechanism of channel catfish under salinity stress,and help to prevent the impact of extreme salinity changes on channel catfish in aquaculture practice.Experiment four:The mitogen-activated protein kinase（MAPK）gene family has been systematically described in several fish species,but less so in channel catfish,which is an important global aquaculture species.In this study,16 MAPK genes were identified in the channel catfish genome and classified into three subfamilies based on phylogenetic analysis,including six extracellular signal regulated kinase（ERK）genes,six p38-MAPK genes,and four C-Jun N-terminal kinase（JNK）genes.All MAPK genes were distributed unevenly across 10 chromosomes,of which three（MAPK8,MAPK12,and MAPK14）underwent teleost-specific whole genome duplication during evolution.Gene expression profiles in channel catfish during salinity stress were analyzed using transcriptome sequencing and q RT-PCR.Results from Reads Per Kilobase Million（RPKM）analysis showed MAPK13,MAPK14a,and MAPK14b genes were differentially expressed when compared with other genes between treatment and control groups.Furthermore,three of these genes were validated by q RT-PCR,of which MAPK14a expression levels were significantly up-regulated in treatment groups（high and low salinity）when compared with the control group,with the highest expression levels in the low salinity group（P<0.05）.Therefore,MAPK14a may have important response roles to salinity stress in channel catfish.