Study On Key Gene Expression,Enzyme Activity And Transcriptome Analysis Of Osmotic Pressure Of Litopenaeus Vannamei During Desalination

The aquatic environment is particularly important for crustaceans.Changes in salinity can cause many changes in crustacean physiology and metabolism.This makes the adaptation mechanism between salinity and crustaceans a hot research topic in aquaculture.Based on the low-salt environment background of Litopenaeus vannamei's osmotic pressure regulation,we explored and analyzed the adaptation mechanism of Litopenaeus vannamei to salinity from three different levels of enzyme activity,gene cloning and transcriptome.The basic data obtained can provide support for the study of crustacean osmotic pressure regulation and immune regulation in the future research.1.In order to explore the ion transport,antioxidant and metabolic immunity of Penaeus vannamei under short-term low-salt aquaculture conditions.Healthy litopenaeus vannamei(length 3.5±0.5cm;body weight 0.2±0.08g)was divided into low-salt group(0.5psu)and normal salinity group(30psu)in this experiment.After 7days feeding,determined Na~+K~+-ATPase activity in gills and hepatopancreas and MDA content in hepatopancreas and serum as well as catalase(CAT),superoxide dismutase(SOD),glutathione peroxidase(GPx),?-glutamyl trans Peptidase(GGT),acid phosphatase(ACP),alkaline phosphatase(ALP).The results showed that the activity of Na~+K~+-ATPase and SOD in 0.5psu was higher than in the normal salinity group;the MDA content was higher than the normal salinity group in 0.5psu;the activity of ACP,ALP and GSH-Px in 0.5psu was significantly lower than the normal salinity group,and there was no significant change in?-GT and CAT between 0.5 psu and 30 psu.The increased enzyme activity can be considered as the positive response of Litopenaeus vannamei to the decrease of external salinity.The inhibited enzyme activity is due to the excessive change of salinity,and the damage of the body caused by the toxicity of Litopenaeus vannamei makes some antioxidant enzymes and metabolic immune enzymes all inhibited.2.Illuminated high-throughput transcriptome sequencing analysis of two groups of Litopenaeus vannamei during salt reduction and normal salinity,respectively,obtained 56598024 and 55440854 raw reads,after data filtering,the low-quality redundant bands were removed,and the two groups were respectively Get 55978038and 54859860 clean reads.A total of 23,202 genes were compared with the six databases,and 19252 genes were successfully annotated,accounting for 82.98%.Comparing the two sets of transcriptome data,a total of 12,101 genes with differential expression were found,of which 9727 genes were up-regulated and expressed A total of2373 genes were down-regulated,indicating that during the period of salinity reduction,Litopenaeus vannamei actively regulates each gene to regulate its osmotic pressure in response to changes in salinity.GO annotations show that differential genes are enriched in three major categories of functions;KEGG Patyeway analysis shows that differentially expressed genes involve 44 metabolic pathways,of which 12 pathways are significantly differentially enriched,and in-depth understanding of 12 pathways may help to understand the response mechanism of Litopenaeus vannamei in the process of seeing low salinity;by constructing a protein interaction network,we can understand the signals between gene clusters that may play an important role in the response of Litopenaeus vannamei to the reduction of salinity Conduction,but at present the interaction and function of these genes are unclear and need further study.3.Calcium-activatedchloridechannelregulator(CLCA)isa metalloproteinase-dependent family which plays an important role in animals.In order to figure out the function of CLCA1 in Litopenaeus vannamei,we cloned full-length of CLCA1 c DNA by RACE technology and analyzed relative expression by q RT-PCR.Rresults showed the full sequence of CLCA1 c DNA is 3129bp which contains 2847bp open-reading flame,175bp 5'UTR and 107bp 3'UTR.The CLCA1 gene codes 984animo acid contains 1 transmembrane region and VMA domain.The animo acid sequence analyzing showed there are low similarity of Litopenaeus vannamei and other species,the highest similarity is the CLCA2 amino acid sequence of procambarus clarkii,which is 47.83%.q RT-PCR results showed that CLCA1 gene was expressed in all tissues of Litopenaeus vannamei,expression are higher in intestine,gill and hepatopancreas.Results showed the expression of CLCA1 gene was down-regulated in four tissues as salinity decreased which means CLCA1 involved in osmotic pressure regulation of Litopenaeus vannamei.The above research laid the foundation for the study of CLCA1 gene in crustaceans.