Function Of FREP And SRB-Croquemort On Immune Response In Litopenaeus Vannamei

Litopeneaus vannamei(L.vannamei), also known as pacific white shrimp, Due to its rapid growth rate, the larger volume of individual adult, the longer duration of the breeding period, the demand for protein relatively low, but the meat quality relatively high and in high density and low salinity waters farming, etc., Quickly became the number one shrimp farming of our country and the world, however, with the continuous expansion of the scale of farming,shrimp farming issues are becoming increasingly serious disease in which the most serious hazards are viruses and bacteria, therefore, the research on shrimp immune defense mechanism, the immune basis for understanding shrimp has great economic value for shrimp farming.In this study, gene specific primers were designed according to the homologous species sequences with RACE technology,which obtained L.vannamei two fibrinogen-related protein gene(LvFREP1 and LvFREP2) and a class B scavenger receptor Croquemort gene(LvSRBCroquemort), the structural characteristics of these genes and their homology was analyzed by Bioinformatics, and then by semi-quantitative PCR(RT-PCR) to detect the differential expression of ten different organizations, including gill, hepatopancreas, hemolymph, heart,intestine, muscle, nerve tissue. by the real-time fluorescence quantitative PCR(qRT-PCR), the expression of these three genes were tested after injecting V. Anguillarum, M.lysodeikticus and WSSV at different points. In addition, after SRB-Croquemort was silenced by dsRNA interfering and then injection of V. harveyi, the expression of related genes were detected at different time points, These genes have been made a preliminary study and obtained the following results:(1) There have been obtained three kinds of LvFREP1 sequences, defined as LvFREP1-1,LvFREP1-2 and LvFREP1-3. LvFREP1-1 gene length is 802 bp with an open reading frame of783 bp, encoding 260 amino acids, LvFREP1-2 gene length is 1714 bp with an open reading frame of 1689 bp, encoding 562 amino acids, LvFREP1-3 gene length is 1593 bp with an open reading frame of 1569 bp, encoding 522 amino acids; LvFREP2 gene length is 942 bp with the open reading frame of 939 bp, encoding 312 amino acids; The coding region length of LvSRB-Croquemort gene is 1602 bp, encoding 533 amino acids.(2) Phylogenetic analysis showed that: the three genes with Marsupenaeus japonicas corresponding gene had the highest homology, however, the homology with other species were relatively low.(3) Tissue expression distribution showed that: LvFREP1 gene was only detected in thegills, hemolymph, intestinal, heart and nerve tissue in which the highest expression was in the gills; LvFREP2 expressed higher levels in hemolymph and gill, other organizations little or no expression; LvSRB-Croquemort gene was expressed in all detected tissues, the highest expression level was in the hemolymph and testis.(4) Temporal expression of pathogen stimulation showed that, in V. anguillarum group,the expression of LvFREP1 gene gradually increased, decreased at 48 h, in M. lysodeikticus group, the expression level gradually rised high, and then the difference reached significant and extremely significant, in WSSV group the expression increased at 24h; In V.anguillarum group, LvFREP2 gene whose expression levels were significantly higher at 6h, the expression level began to decrease at 12 h, In M. lysodeikticus and WSSV stimulation group,there had two peak period of expressions at 6h and 48h; LvSRB-Croquemort gene, in V.anguillarum group, after 6h it reached the maximum, then decreased gradually, in M.lysodeikticus group, its expression had a obvious rise in 48h; in WSSV group, its expression reduced first and then increased again.(5) After silencing LvSRB-Croquemort gene using dsRNA interference, the expression level of shrimp three antimicrobial peptides ALF, Crustin, Pen3 generally had a significant downward trend after silencing the gene, while the two transcription factors, Dorsal had no significant change in the expression and Relish decreased slightly first and then increased again.These results suggested that: LvFREP1, LvFREP2, LvSRB-Croquemort genes might be involved in shrimp innate immune responses against pathogen stimulation.