| Background: Galectins, which consiste of 15 members, are a family of lectin that bind to β-galactosides through carbohydrate-recognition domain(CRD) and play a crucial role on the regulation of cell proliferation and growth, immune defense and anti-inflammatory reaction. In recent years, Andrias davidianus have been suffered by pathogenic disease seriously. The current research mainly concentrated on the breeding and germplasm resources, while seldom on immune system.Objective: To explore the relationship between the functional mechanism and cytology of galectins, and discuss the funtion and regulating mechanism in the immune system of the Andrias davidianus, we study the spatio-temporal expression, cell location,and the temporal expression of galectins after the pathogenic infected.Methods: We filtrated the high expression gene of Gal-1, 3, 8, 9 from the RNA-Seq database, and analyzed the characteristic and molecular evolution of the galectin family.Using the qRT- PCR detect the spatial and temporal expression of galectin, and their expression after the pathogeny infection. In addition, we detect the cellular localization of Gal3 throμgh the in situ hybridization technique.Results:1. By evaluation of five candidate reference(18SrRNA, EF1-α, GAPDH,β-actin, SDHA)through the geNorm, NormFinder and BestKeeper, we found that EF1-α and SDHA were the optimum reference for the study of tissue expression, while the expression research of different development stage should choose EF1-α and GAPDH as reference. Intriguingly, EF1-α and GAPDH were recommended as the ideal reference for the expression study of Andrias davidianus after immune stimulation. 2. The open reading frame(ORF) of AdGal1 was 405 bp, coding 134 AA. AdGal8 consisted of 1008 bp ORF, which coded 335 AA, while AdGal9 contained 960 bp and 319 AA of ORF. Homology comparison showed galectin was conservative, and the statu of galectins in the evolutionary tree were in accordance with the classification status of Andrias davidianus. The temporal and spatial expression patterns showed that AdGal1, AdGal8, and AdGal9 was highest expressed in gonad, spleen, and intestines, respectively. Meanwhile, AdGal3 expression showed no significant difference in different stages. Nevertheless, AdGal1 expression was significantly higher at the age of 3 years than the age of 1 year and 2 years(P < 0.05). AdGal8 expression at the age of 1 year in spleen was significantly lower than the age of 2 and 3 years, while AdGal9 expression displayed significant difference in different growth stages.3. Raf1, which involved in the signal path of AdGal1, was in accordance with AdGal1 and increasingly expressed after the infection of Aeromonas hydrophila. While Hras appeared significantly upregulated on the 7th day. Gal3 and KRAS expression were rise significantly on the third day. Then declined on the 5th day, though still higher than the control group, and significant increased on the 7th day. AKT2, Caspase3 and ASK1 showed contrary development trend to the Gal3, which significantly lower on the third day, then rose on the 5th day, while decreased on the 7th day. The expression of Gal8, AKT2 and Erk1 were consistently rising in different range, all first appeared significantly upregulated on the 5th day. The Gal9 began to significantly upregulated expression on the 5th day, while the expression of IL8 showed no obvious correlation with Gal9.4. After virus infection, Gal1, Hras, and Raf1 displayed downward trend after rising on the 3th day. Gal1 and Raf1 significantly upregulated on the third day, then falling to the original level. Wihle the change of the Hras showed no significant. Gal3 and KRAS peaked signify rose on the 3th day, decline but no significant on the 5th day, on the 7th day fall to the level before the infection. While Caspase3 and ASK1 displayed the opposite trend, and the AKT2 showed no significant change. Both Gal8 and Erk1 up-regulated after infection, AKT2 also showed no change in the spleen. Besides, Gal9 up-regulated on the 5th day.5. By detected the cellular localization of Gal3 through the in situ hybridization technique, we found that the Gal3 mainly distributed in the cytoplasm, while the infected cell showed significantly stronger signals than the healthy cells, the distribution of Gal3 in the nucleus significantly increased than the healthy one. Additionally, the nuclear/cytoplasmic ratio was increased.Conclusions: This study sμggests that the galectin plays a promoting role in the natural immune systems of different tissues of Andrias davidianus. Morever, the regulation of galectin induced by pathogeny in Andrias davidianus may involve in MAPK/ERK, PI3/Akt, and the Caspase signaling pathways. |
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