Enhancement And Transcriptome Analysis Of SiRNA Inhibiting WSSV In Litopenaeus Vannamei With Peptidoglycan

In recent years,the rate of WSSV infection had been declined,but the WSSV virus infection was still a major threat to shrimp farmingin certain conditions,so the study of shrimp antiviral treatment and its mechanism were of great significance.In the normal state of shrimp,the immune protective rate was low-lying by immune effect of peptidoglycan.There was not effective against WSSV infection by si RNA interference and the incidence of its immune protection was low.The experimental designed that strengthen the immune conditions,changes in incidence of WSSV infection,and then using siRNA interference,observation of WSSV infected shrimp,the protection rate of change, expect to find an effective anti WSSV treatment technology and theory,and in the late virus prevention of parent to provide technical and theoretical basis.The main content of this experiment are the following three aspects.(1)Peptidoglycan was canextracted from the cell wall of Vibrio alginolyticus and dissolved the concentrations of 0.05 mg/m L,0.1mg/m L and 0.15mg/m L with ultrasonic,which were injected into Litopenaeus vannameiei infected WSSV by intramuscular injection.Then we recorded deaths of Litopenaeus vannameiei and collected hepatopancreas and serum in order to detect immune indexes in shimp at different time points in reagen box method.(2)In frist,si RNA interfered WSSV was confirmed with the optimal concentration.Then the optimal concentration of si RNA was injected into Litopenaeus vannameiei after 24 h infected WSSV in shrimp,we recorded the number of the death of Litopenaeus vannameiei at different time points with qPCR and fluorescence quantitative detection.(3)After determined the rate of immune protection injected peptidoglycan and the effection of si RNA interference,then Litopenaeus vannameiei was injected peptidoglycan and infected WSSV,vp28-siRNA was aslo injected in shrimp at different time points,we recorded the shrimp immune protection rate.Based on the experimental results,we gatered hepatopancreas of shrimp in five different groups,transcriptome sequencing and analysis.With the analysis of the specific research results are as follows:1.Litopenaeus vannameiei were injected peptidoglycan with the concentration of0.05mg/m L(the PSA group),0.1mg/m L(the PSB group) and 0.15mg/m L(the PSC group).After 24 hours,shrimps was infected WSSV,we determine the rate of the protection and common immune index with the serum and hepatopancreas.The experimental results showed that:the cryopreservation of WSSV crude extract liquid was injected in shrimp body and re-extracted,PCR analysis showed that WSSV was positive.In first day,there was a large number of deaths after infected WSSV crude extract liquid in shrimp.Then the detection of PCR was positive and the shrimp was died with WSSV infection.Compa-red with the control group,The protective rate of PSA groupwas36.67%,the PSB group was 46.67%,the PSC group was 56.67%.The rate of shrimp protection was as increasing as the concentration of peptidoglycan.The vitality of PO enzyme in the serum of Litopenaeus vannameiei was as follows:the PSA group reached the peak value at 72 h,the PSB group was at 48 h,the PSC group also was 24 h.At the same time,the experimental group in the hepatopancreas of shrimp of ACP, AKP, SOD and NOS activity were significant differences(P<0.01),compared with the control group.The ACP activity in the PSA group reached the peak value at 24 h,the AKP was 12 h,the SOD was 48 h,NOS was 24 h.The activity of ACP, AKP and SOD in PSB group reached the peak value at 48 h, the NOS was 12 h,but there was a peak in 96 h again.The AKP and ACP activity in PSC group reached the peak value at 24 h,SOD was 12 h and decreased gradually,there was a peak in 72 h again.NOS was 6h and decreased gradually.2.The optimum concentration of siRNA is 0.1μg/μL,so each tail was injected 100μL and the effect of interference is the best.The rate of protection was 40%.The virus copy number was detected by realtime-PCR in shrimp and the results showed that the best interference effection was in 48 h,continued to 120 h.3.Litopenaeus vannameiei was injected peptidoglycan and infected WSSV,vp28-siRNA was aslo injected in shrimp at different time points.The experimental results show that the rate of protection was 80% in 6h injection group,in 12 h injection group was 63.33%,in 24 h group was 56.67%.The rate of protection was higher than the single injection of siRNA or peptidoglycan group.4.We collected five groups of the hepatopancreas for transcriptome sequencing and analysis.Through the Illumina platform of Hiseq2000 sequencing,the total output was24,001,958,340 nt.In results,the total assembly Unigene was 35,274,a total length was38,807,225 nt,the average length was 1,100 nt, N50 was 2,012 nt.Genetic analysis showed differential expression in BSA group,compared with P6 A group.There were 13,344up-regulation gene and 943 gene down-regulation in the expression.The results revealed that effect of injection of peptidoglycan and vp28-si RNA is very obvious.Through thedifferential,Gene pathway enrichment analysis revealed that endocytosis lysosomal,EB virus infection pancreatic juice,bile secretion,toxoplasmosis and Amoeba diseas pathways were significant enrichment in P6 A group contrasted with the BSA,WSA,PWA and WRA groups.These pathways were existed in shrimp but existed in humans.There may be similar with human pathway in shrimp and taked a model significance.