Study On Response Mechanism To The Infection Of Decapod Iridescent Virus 1 In Litopenaeus Vannamei

As a new virus of shrimp,Decapod iridescent virus 1（DIV1）which could cause mortality up to 100%in crustaceans has caused huge losses to the shrimp breeding industry in recent years.However,most of the studies on DIV1 focused on the virus itself,and paid less attention to the changes of shrimp post infection.On this basis,Litopenaeus vannamei was selected in this study to study the effect of DIV1 infection on shrimp in order to explore the mechanism of response to DIV1 infection in L.vannamei.The results can provide theoretical basis for disease control of shrimp.The specific research contents and results are as follows:1.According to the toxicity experiment of DIV1 on L.vannamei,the survival of L.vannamei was recorded.By calculation,the lethal concentration(LC₅₀)of DIV1infecting L.vannamei at 48,72,96,and 156 hours post infection（hpi）were 4.86×10⁶,5.07×10⁵,2.13×10⁵,and 2.38×10⁴ copies/μg DNA,respectively.2.Transcriptome sequencing on the hemocytes was used to study the effect of DIV1 infection on gene expression in L.vannamei.A total of 168,854 unigenes were obtained,with the average length of 601 nt and the N50 length of 807 bp.The function annotation results showed that 48,135,28,835,53,506,and 43,824 unigenes were annotated in the Nr,KEGG,Swissport,and KOG databases,respectively.A total of 1,112 differentially expressed genes were obtained（DEGs）,including 889up-regulated and 223 down-regulated.KEGG signaling pathway enrichment analysis showed that most of these DEGs were mainly annotated to signaling pathways related to metabolic functions.Fructose and mannose metabolism,biosynthesis of amino acids,glycolysis/gluconeogenesis,inositol phosphate metabolism,and carbon metabolism were members of top 20 signaling pathways with the most enriched DEGs.Triosephosphate isomerase（TPI）gene participates in these signaling pathways,suggesting that Lv TPI gene plays an important role in DIV1 infection of L.vannamei.3.Triosephosphate isomerase is an enzyme in the glycolytic pathway,which catalyzes the reversible interconversion of the triose phosphate glyceraldehyde3-phosphate（GAP）and dihydroxyacetone phosphate（DHAP）.Based on the results of transcriptome sequencing,three kinds of sequences annotated as TPI gene were found,named Lv TPI-Like,Lv TPI-Blike and Lv TPI-Blike1.The c DNA sequences of Lv TPI-Like,Lv TPI-Blike and Lv TPI-Blike1 were cloned using RACE-PCR.It was showed that Lv TPI-Like,Lv TPI-Blike and Lv TPI-Blike1 genes encode 284,267,and266 amino acids,respectively.The results of protein homology analysis showed that the similarity of Lv TPI-Like compared with other species ranged from 44.13%to51.50%,with the highest similarity of Penaeus monodon.The similarity of Lv TPI-Blike compared with other species ranged from 59.59%to 73.44%,with the highest similarity of Palaemon carinicauda.The similarity of Lv TPI-Blike1compared with other species ranged from 58.06%to 87.22%,with the highest similarity of P.monodon.q PCR was used to detect the expression of Lv TPI in different tissues of L.vannamei.The results showed that Lv TPI-Like,Lv TPI-Blike,and Lv TPI-Blike1 genes were constitutively expressed in L.vannamei,with the highest level in the stomach,gill,and hepatopancreas,respectively.4.In order to identify the function of Lv TPI during the infection of DIV1,RNA interference（RNAi）technology was used to silence the Lv TPI-Like,Lv TPI-Blike and Lv TPI-Blike1.It was found that the silence of Lv TPI-Like and Lv TPI-Blike1 caused a large number of deaths of L.vannamei,with the survival rate was only 50%and 7.5%at 48 hpi.L.vannamei was infected by DIV1 at 48 hours after the silence of Lv TPI-Like and Lv TPI-Blike.There was no significant difference in the survival rate of L.vannamei among the three groups.However,the numbers of DIV1 in haemocytes,hepatopancreas,intestine,gill and muscle of Lv TPI-like and Lv TPI-Blike silenced L.vannamei were reduced at different degrees when compared the control group,which indicated that Lv TPI-Like and Lv TPI-Blike maybe beneficial for the replication of DIV1 in L.vannamei.In this study,transcriptome sequencing was performed on hemocytes of L.vannamei infected with DIV1,and related candidate genes for L.vannamei response to DIV1 were screened.The Lv TPI-Like,Lv TPI-Blike and Lv TPI-Blike1 gene were cloned and functionally studied.The results prove that Lv TPI-Like and Lv TPI-Blike can effectively inhibit DIV1 replication in L.vannamei.This further proves that Lv TPI gene plays an important role in the process of DIV1 infecting the host.