Study On The Function Of VEGFR Gene Of Eriocheir Sinensis In The Process Of Spiroplasma Infection

Eriocheir sinensis is an important economic species in China.In recent years,with the continuous expansion of its breeding scale and the improvement of intensive degree,as well as the worsening of the breeding environment,diseases caused by pathogens are becoming more and more serious,which has caused huge losses,serious threat and restricting the healthy development to the aquaculture of E.sinensis in China.Among them,tremor disease（TD）is one of the most serious diseases of E.sinensis,which causing serious economic losses.A spiroplasma was identified as the pathogen of TD and named Spiroplasma eriocheiris.Previous studies have shown that the S.eriocheiris entered into the crab through the gill or surface and invaded its target cells,hemocytes.Then,through blood circulation,S.eriocheiris invaded connective,nerve and other tissues and caused the crab tremor.On the basis of this study,the full-length of two vascular endothelial growth factor receptor（EsVEGFR）gene was amplified by RACE technique,and its sequence characteristics and expression patterns were analyzed.Then,the function of gene interference was studied.This paper lays a good foundation for the in-depth study on the infection mechanism of S.eriocheiris and the host immune defense mechanism.This study is mainly carried out from the following three aspects:1.Identify different proteins of hemocytes in E.sinensis during S.eriocheiris infectionTMT technology was used to screen the differentially expressed proteins in the hemocytes of E.sinensis infected by S.eriocheiris,and a total of 212 differentially expressed proteins were screened out,which were divided into immune-related proteins,physiology-related proteins and cytoskeletal related proteins according to their functions.Of these,127 were up-regulated,85 were down-regulated,and about 42 were immune-related.Up-regulated proteins include cathepsin L,glutathione s-transferase（GST）,clotting factor B/B2,melanization protease 1,serine protease inhibitor and Peroxiredoxin1（Prx1）,etc.Down-regulated proteins include ferritin2/3（fer2/3）,lectin,lipopolysaccharide and-1,3-glucan binding protein（LGBP）,vascular endothelial growth factor receptor 1,NADPH oxidase 5（NOX5）,lysozyme C,etc.In this study,8proteins were selected,including four up-regulated proteins（cathepsin L,hemocytin,peroxiredoxin 1 and NADPH oxidase 5）and four down-regulated proteins（LGBP,ferritin 2,kazal-type protease inhibitor,VEGFR1）,to study their expression at the gene level by real-time quantitative PCR.The experimental results were consistent with the TMT screening results.2.The sequence and the expression analysis of EsVEGFR1 and EsVEGFR2Two subtypes of the VEGFR gene,EsVEGFR1 and EsVEGFR2,were amplified by RACE using the c DNA of the hemocytes of E.sinensis.The characteristics of the EsVEGFR1 c DNA sequence are as follows:the EsVEGFR1 c DNA sequence is 6470bp long and contains a 4380 bp open reading frame,encoding 1459 amino acid polypeptide.SMART site predicts that the 802nd to 1261 amino acid residues of EsVEGFR1 are a Tyr Kc domain and an ig-like domain.EsVEGFR2 c DNA sequence is5548 bp and contains a 4704 bp open reading frame encoding 1567 amino acid polypeptide,its 1 to 34 amino acid residues as a signal peptide,and four immune globulin structure domains（IG-like domain）,SMART site prediction of the 787th to809th amino acid residues as a transmembrane domain structure,864-1259 amino acid residues is a Tyr Kc structure domain.MEGA5.0 was used to analyze the sequence of EsVEGFR,and the similarity of the protein to the Rab of some aquatic crustaceans was up to 80%,and the similarity to the VEGFR sequence of insects and other invertebrates was about 60%.The technique of qRT-PCR was used to detect the distribution of two genes in the tissues of E.sinensis.EsVEGFR1 was the most expressed gene in the nerve tissues,while EsVEGFR2 was the most expressed gene in hemocytes,followed by the gill and other tissues.Since the target cells of E.sinensis infected by S.eriocheiris,were hemocytes,we selected hemocytes for the next experiment.The expression of EsVEGFR1 in hemocytes increased on the 3rd and 5th day,the rest of the time showed a downward trend.The decrease of EsVEGFR2 expression at day 3 and the upregulation of EsVEGFR2 at the rest of the time.This kind of phenomenon indicated that EsVEGFR1 and EsVEGFR2 played an important regulatory role in the infection of S.eriocheiris,which laid the foundation for further studies on the immune function of EsVEGFR.3.RNA interference of EsVEGFR1EsVEGFR1 gene was silenced by artificial transcriptional dsRNA,and qRT-PCR was used to detect the interference efficiency after dsRNA injection.The results showed that the transcription level of ESVEGFR1 specific dsRNA was significantly lower than that of the control group（PBS,ds GFP）.This demonstrated that EsVEGFR1 specific dsRNA significantly reduced the level of EsVEGFR1 transcription in hemocytes.After dsRNA was successfully silenced EsVEGFR1 gene,E.sinensis were infected with S.eriocheiris,the results showed the number of copies of S.eriocheiris in hemocytes increased significantly compared with control group,at the same time,compared with the control group（PBS+S.eriocheiris）,E.sinensisin the group ds EsVEGFR1+S.eriocheiris,began to death on the 7^th dary and the mortality increased.This further indicates that silence of EsVEGFR1 reduces the immunity of E.sinensis and promotes the infection of S.eriocheiris.In a word,these results lay a theoretical foundation for further understanding of the mechanism of E.sinensis infection by S.eriocheiris.And it is of certain reference value to produce the drugs to control the disease in practical application.