| Coccidiosis is the most frequent and harmful parasitic disease in intensive poultry industry. In our country, the incidence rate of it is high enough to cause immense economic loss. The Eimeria tenella is the most important species among the genus.Although there were more and more immunological researches on coccidiosis in recent years, molecular mechanisms of parasite invasion and host defense is still not complete clear due to its complex life history and huge genome size. In order to reveal the interaction between the parasite and the host at molecular level, the differentially expressed genes in the spleen of chickens infected with E. tenella were analyzed by c DNA-SRAP technique at the transcriptional level in this study. The main contents and results are as follows:(1) Establishment of c DNA-SRAP system for chicken spleen RNA sample. The total RNA was extracted from the spleen of 14 day-old chickens, c DNA-SRAP system was optimized by means of single factor experiment and orthogonal tests. The optimized c DNA-SRAP reaction system was as follows: d NTPs 0.25 m M, Mg2+ 2.0m M,Taq DNA polymerase 2.25 U, primer 0.55μM, c DNA templete 20 ng. Cycling parameters were: 5 cycles of 5-minute-long 94 ℃ pre-denaturation, 1-minute-long 94 ℃denaturation, 1-minute-long 36℃ annealing and 1-minute-long 72℃ extension; and 35 cycles of 1-minute-long 94 ℃ denaturation, 1-minute-long 55 ℃ annealing,1-minute-long 72℃ extension, and 10-minute-long 72℃ extension at last. Stable and clear polymorphic bands could be obtained with above c DNA-SRAP system and cycling parameters.(2) c DNA-SRAP analysis of spleen infected with E. tenella. c DNA samples of infected and control chicken was used as template for c DNA-SRAP reaction with 256 arbitary primer pairs, and differential bands were analyzed by 1.6% agarose gel electrophoresis. After the initial round screening, 12 up-regulated genes were obtained.9 up-regulated genes were verified by the biological repeat and c DNA-SRAP of these12 genes. The result of bioinformatic analysis showed that 6 of them are known functional genes: MLL5, P2RY8, PLCD1, LAP3, IL-17 RA and WHSC1L1 gene respectively.(3) Real Time RT-PCR analysis of differentially expressed genes in spleen of chickens infected with E. tenella. 6 differentially expressed genes were quantitatively analyzed by means of Real time RT-PCR technology. The results showed that MLL5 was up-regulated 1.52 folds after infection with E. tenenlla, P2RY8 gene up-regulated2.33 folds, PLCD1 up-regulated 1.48 folds, LAP3 up-regulated 1.57 folds, IL-17 RA up-regulated 1.41 folds, WHSC1L1 up-regulated folds. The data were analyzed with the independent samples T-test for significance analysis, which showed the expression level of WHSC1L1 and P2RY8 were significantly different(P<0.01), and the expression level of MLL5, PLCD1, LAP3 and IL-17 RA were significant differences(P<0.05).The up-regulation of MLL5, P2RY8, PLCD1, LAP3, IL-17 RA and WHSC1L1 genes indicates that these genes play important roles in the invasion and host defense in the early stage of infection of Eimeria tenella. PLCD1 can amplify the signal regulatory pathway of Ca2+ so as to play its role in the intrinsic immune reaction. LAP3 can help antigen peptide to form MHCI molecule compound on cell surface. IL-17 RA, as the cytokine receptor, participates the process of inflamation. The immunological functions of these three genes indicate that they play important roles in immune reactions against coccidium. MLL5 gene has the promotive function for cell proliferation, WHSC1L1 gene may be involved in the development of cancer, and the function of P2RY8 is still uncertain, as a result, the biological meaning of the up-regulation of them in E. tenenlla infection needs to be calrified through further researches. |
PDF Full Text Request