Study On The Relationship Between Renin-angiotensin System And Salinity Stress In Pacific White Shrimp Litopenaeus Vannamei

Pacific white shrimp Litopenaeus vannamei is one kind of euryhaline mariculture species,which grows fast,has high survival rate and strong resistance to stress.It is widely favored by the aquaculture and becomes one of major cultured shrimps all over the world.Unfortunately,the aquaculture environment is getting worse and worse due to the excess cultured production and unscientific management.As a result,more and more farmers began to culture L.vannamei with fresh water.If so,a series of stress responses will occur under low salinity conditions,which lead to growth retardation and reduce immunity of L.vannamei.In order to solve this problem,previous studies have been conducted to investigate the effects of low salinity stress on L.vannamei.While studies on the relationship of endocrine and salinity stress are limited.Therefore,this paper aim to explore the relationship between renin-angiotensin system and salinity stress by the way of transcriptome,mRNA and protein levels to provide some references for further studies on effects of low salinity stress on endocrine of crustaceans and other aquatic animals.1.Transcriptome response of eyestalk from Pacific white shrimp L.vannamei to chronic low salinity stressThe X organ-sinus gland complex in the eyestalk of L.vannamei is the endocrine control center.Therefore,Illumina high-throughput sequencing technology is adopted to analyze the effect of long-term low salinity（3psu）on gene expression of L.vannamei eyestalk（25psu as control group）.In order to explore the relationship between the endocrine and salinity stress of L.vannamei.A total of 6.17×10⁷ reads were selected by De novo RNA sequencing,and the total length is 8.98×10¹⁰ bp.The results was spliced and compared using Trinity software,resulting that 17,222 Unigene were obtained,and the total length is 1.88×10⁷ bp.Further more,479 differentially expressed genes were obtained through functional annotation and KEGG enrichment analysis（fold change>2,P value<0.05）.There are 150 genes up-regulated and 329 are down-regulated.Ten endocrine-related genes were significantly changed.In which,the renin-angiotensin system has the most significant changes（P=0.016）.On the other hand,the angiotensin converting enzyme（ACE）and aminopeptidase N（AP-N）were significantly reduced in this system.Subsequently,we randomly select 20 samples from the significantly changed genes for qRT-PCR verification.The results proved the correctness and reliability of transcriptome data（R=0.97）.This study indicates that the renin-angiotensin system in L.vannamei plays an important role in the adaptation of long-term salinity stress and is an important point in the osmotic pressure regulation of L.vannamei.This study can not only provide new ideas for studying the osmotic pressure regulation of L.vannamei,but also provide some references for the study of endocrine system of crustaceans.2.Clone and analysis of the key genes in renin-angiotensin system of L.vannameiAngiotensin converting enzyme（ACE）,aminopeptidase-nitrogen（AP-N）,angiotensinⅡ-type 1 receptor（AT₁R）and renin receptor（RR）play an important role in RAS,so fragment clone and bioinformatics analysis are performed on these four genes.Results shows that the four genes respond to 2326bp,3100bp,593bp and 1116bp nucleotide sequence,respectively.The molecular mass of ACE is79kDa,the isoelectric point is 6.0,the aliphatic index is 77.65.Containing a signal peptide and 48 phosphorylation sites.And there is no transmembrane domain.O-glycosylation site is five,N-glycosylation site is four.The molecular mass of AP-N is 104kDa,the isoelectric point is 6.13,the aliphatic index is 85.32.Containing a signal peptide and 102 phosphorylation sites.And there is no transmembrane domain.O-glycation site is nine.N-glycosylation site is ten.The molecular mass of AT₁R is 19kDa,the isoelectric point is 4.73,the aliphatic index is 90.29.There is no signal peptide.There are 19 phosphorylation sites and two transmembrane domains.O-glycosylation site is one.N-glycosylation site is two.The molecular mass of RR is 36kDa,the isoelectric point is 4.95,the aliphatic index is 97.18.Containing a signal peptide and 34 phosphorylation sites.And there is a transmembrane domain.O-glycation site is three.N-glycosylation site is four.The phylogenetic tree was constructed and homologous comparison analysis was performed.It was found that the species which shows the high homology of the four genes of L.vannamei are the species that close to their taxonomic classes relatively.3.The mRNA expression of the key genes of L.vannamei in renin-angiotensin system under salinity stressAfter getting the four gene fragments of ACE,AP-N,AT₁R and RR.Eyestalk,hepatopancreas and muscle tissue were selected to analyze the expression level of mRNA.It was found that all four genes were expressed in those tissues.Under long-term low salinity stress,the expression levels of ACE and AP-N in the eyestalk of L.vannamei were significant lower than normal salinity group,while there was no significant differences in AT₁R and RR,which was consistent with the results of the transcriptome.The expression of other differential expressed genes in low salinity stress showed a decreased trend,which indicate that the RAS level in the eyestalk of L.vannamei was inhibited under low salinity stress.In hepatopancreas,the expression of ACE in low salinity was significantly reduced.In addition,under acute low salinity stress,the mRNA expression of ACE,AP-N,AT₁R and RR were detected in eyestalk,hepatopancreas and muscle at 0h,3h,6h,12h,24h,48h,72h and 96h,respectively.The results showed that ACE and AP-N were more sensitive than AT₁R and RR in the eyestalk,and significant differences were observed in ACE and AP-N firstly.At the same time,ACE and AP-N may be in a dynamic and intermittent regulation process to adapt to the stress caused by the low salinity.The responses of the four genes in hepatopancreas and muscle are not as strong as those in eyestalk,and ACE showed no differences throughout the96 hours.It can be seen that the ACE in hepatopancreas and muscle does not play a role in the process of adaptation to low salinity stress.In a word,the expression of ACE,AP-N,AT₁R and RR were different in different tissues in the process of adaptation to low salinity stress.4.The preparation of angiotensin converting enzyme antibodies of L.vannamei and the expression in salinity stressThe ACE gene fragment was obtained by cloning and the amino acid sequence,hydrophobicity and antigenicity were analyzed.Finally,the 463-643amino acid region was selected to synthesize antibody.Subsequently,the target gene fragment was synthesized by the whole gene synthesis method and the PCR product was obtained,and which was constructed in pET28a and pET32a to induce the target protein expression.The proteins were purified and quantified,and were used to immunize New Zealand rabbits,then the serum were collected and purified.The specificity and titer were identified by enzyme-linked immunosorbent assay（Elisa）.The antibody titer is 1:80,000 and the specificity is strong.This antibody was used to detect the changes of ACE protein expression in the eyestalk and hepatopancreas of L.vannamei under long-term low salinity stress by Western blot.The results showed that the expression of ACE protein was significantly lower than normal salinity group in the hepatopancreas.Therefore,we hypothesized that the expression of ACE protein in the eyestalk under low salinity stress may also be significantly lower than that of normal salinity group.Specific related results need to be further studied.