Study Of Prebiotics Effect On Disease Resistance Performance And Toll-like Receptor Gene Polymorphism Of Litopenaeus Vannamei

Litopenaeus vannamei is important economic shrimp in China. But with the breeding intensity increased and aquaculture environment worsening, especially the outbreak of the disease, led to the breeding fail. So urgently needed to study these lower factors, comprehensive understanding of prawn immune system regulation mechanism, and get through improving the breeding environment, using prebiotics to increase the feed utilization efficiency, screening and establishing an effective resistance molecular genetic markers to improve Litopenaeus vannamei germplasm and reduce disease incidence. This study first introduced the expression of Litopenaeus vannamei Toll-like receptor in different tissues after challenges white spot syndrome and Vibrio alginnolyficus; Secondly, using SNPs markers to analyze Toll1 gene polymorphism of three experimental groups, such as control, WSSV and Vibrio alginnolyficus resistance, and analyze the SNPs loci associated with resistance properties; In addition, study the effects of inulin, oligosaccharide on Toll-like receptor gene m RNA expression and the influence of disease resistance to further understanding of the Toll-like receptor mediated immune response and filtering the effective feed combination; This experiment also discusses other related immune gene in order to fully understand the immune mechanism of Litopenaeus vannamei. Specific research contents are as follows:(1) The expression of Litopenaeus vannamei Toll-like receptor in different tissues after white spot syndrome and Vibrio alginnolyficus challengeThe expression of three types of Toll-like receptors m RNA expression in different tissues of Litopenaeus vannamei were detected with Real-time PCR. The results indicated that Toll1 was high expressed in muscle, heart, haemocyte and gill,while the highest expression was in the heart. Toll2 was high expressed in heart, haemocyte and gill and the highest expression was in the gill. Toll3 highest expression was in gill. All the expressions of three types of Tolls enhanced significantly after the White Spot Syndrome Virus(WSSV) challenge(P<0.05). The significant enhancements of three types of Toll m RNAs were observed in haemocyte(P<0.05), after the V.alginolyticus challenge and the Toll1 and Toll3 expression rose remarkably in gill after 3 hours and 12 hours post-V.alginolyticus challenge(P<0.05). Additionally, the three types of Toll-like receptors expression significantly enhanced after two weeks post WSSV or V.alginolyticus challenges(P<0.05).(2) Correlation analysis of Litopenaeus vannamei Toll1 like receptor gene SNPs with disease resistance performanceUsing SNPs molecular markers to dissolve Toll1 like receptor gene polymorphism of three experimental groups of Control, WSSV resistance and V.alginolyticus resistance, and SNPs loci association with resistance properties. This analysis shows that the loci c. 1430 c > T, c. 1877 c > T genotype frequency and allele frequency distribution between Control group and WSSV resistance groups were significantly different(P<0.05), and resistance allele of two locus was T, C respectively, resistance genetype was CT, CC respectively; loci c. 1877 c > T genotype frequency and allele frequency distribution between Control group and V.alginolyticus resistance group were significant differences(P < 0.05), initially genotype CC may be associated with V.alginolyticus resistance properties.(3) The effect of Inulin, Mannan Oligosaccharide(MOS) on Toll-like receptor gene expression and disease resistance of Litopenaeus vannameiFeeding with basal diet supplemented with different doses of Inulin, Mannan Oligosaccharide four weeks respectively, according to the results, the Inulin(5mg/g)、Inulin(10mg/g)、MOS(5mg/g)、MOS(10mg/g)、Inulin(5mg/g)+ MOS(5mg/g) have significantly increase on relative weight gain rate and the specific growth rate(P < 0.05); before challenge, three types of Toll-like receptors m RNA expression of Inulin(5mg/g)、Inulin(10mg/g)、MOS(5mg/g)、MOS(10mg/g)、Inulin(5mg/g)+ MOS(5mg/g)increased significantly in 14 d、28 d respectively; After 5 d post-WSSV or 10 d post-V.alginolyticus challenge, the three types of Toll-like receptors m RNA expression of Inulin(5mg/g)、Inulin(10mg/g)、MOS(5mg/g)、MOS(10mg/g)、Inulin(5mg/g)+ MOS(5mg/g)group significantly increased compared with the Control group, the best feed effect is Inulin(5mg/g)+ MOS(5mg/g) combination group.(4) The effect of Inulin, Mannan Oligosaccharide(MOS) on four related immune gene of Litopenaeus vannameiIn order to fully understand the immune mechanism of Litopenaeus vannamei, by feeding experiment, the STAT、 pro PO、Crustin、ERK gene m RNA expression of Inulin(5mg/g)、Inulin(10mg/g)、MOS(5mg/g)、MOS(10mg/g)、Inulin(5mg/g)+ MOS(5mg/g)combination were significantly higher than that of Control group(P < 0.05); After 5 d post-WSSV or 10 d post-V.alginolyticus challenge, pro PO, Crustin gene m RNA expression of Inulin(5mg/g)、MOS(5mg/g)、Inulin(5mg/g)+ MOS(5mg/g) group were significantly higher than the Control group(P < 0.05), STAT, ERK gene m RNA expression does not change significantly compared with Control group(P >0.05),, at the same time get the best feed effect is Inulin(5mg/g)+ MOS(5mg/g) combination group.