Screening Of Lipid Raft Protein LvFlotillin Interacting Protein And Study On Its Gene Function In Litopenaeus Vannamei

Litopenaeus vannamei,as one of the important species in shrimp farming,has brought enormous economic value to China’s aquaculture industry.White spot syndrome virus(WSSV)is the deadliest pathogen of L.vannamei,which restricts the healthy development of shrimp farming.Conducting research on the pathogenic mechanism of WSSV and the immune defense mechanism of shrimp will help to deeply explore the molecular mechanisms of host virus interactions and provide theoretical basis for the prevention and control of shrimp diseases.Flottilin is a lipid raft marker,and its two protein molecules,Flottilin-1 and Flottilin-2,form micro-regions on the cell membrane and participate in such vital processes as cell endocytosis,cytoskeletal regulation,and protein sorting and transportation.The previous research group has confirmed that L.vannamei LvFlotillin1A and LvFlotillin2 are related to WSSV infection,and they are co-located at the cell membrane and may be involved in the endocytosis of WSSV.To further understand the molecular mechanism of LvFlotillin-mediated WSSV endocytosis.In this study,LvFlotillin was used as the research object,and the possible regulation of downstream genes and signaling pathways as well as interaction proteins related to WSSV endocytosis were explored through the methods of transcriptomics and protein omics.Transcriptomics analysis showed that LvFlotillin was closely related to the regulation of cytoskeletal proteins and membrane proteins.Its over-expression might promote the formation of endocytosis and phagosomes,and participate in the regulation of apoptosis,cell phagocytosis,autophagy,RNA degradation and other processes.LvFlotillin1A mainly promotes the fusion of vesicles,while LvFlotillin2 promotes the transport of vesicles and protein sorting in the endometrium.Flotillin usually performs its biological functions in cells as oligomers.We found the interaction between LvFlotillin1A and LvFlotillin2 in shrimp by immunoprecipitation experiment.Further mass spectrometric identification of the immunoprecipitates in WSSV-infected shrimp tissues revealed that 59 potential interacting proteins of LvFlotillin1A and LvFlotillin2 were obtained.Through in vitro immunoprecipitation verification,seven proteins interacting with LvFlotillin were finally obtained,which are ATP synthase(Lvβ-ATPase),heat shock protein(LvHSC70),actin(Lvβ-actin),lectins(LvLectin2,LvLectin3,LvFCN1),serine protease(LvCSP).These interacting proteins might participate in the infection process of WSSV through a complex with LvFlotillin.In summary,in this study,we initially analyzed the biological functions and molecular regulatory mechanisms that LvFlotillin may be involved in and screened for LvFlotillin interacting proteins associated with WSSV infection.The results will help to further reveal the role of LvFlotillin during WSSV infection and provide a theoretical basis for disease control in penaeid shrimp.