Molecular Cloning Of Mel Receptors Gene And Its Expressional Examanition Of Immune Organs By In-situ Hybridization In Chicken

In this study,we changed the content of melatonin in chicken by controlling illumination (long illumination(18h),short illumination(6h)and the control(12h)) and injecting exogenous melatonin(low level(10mg/kg) , high level(20mg/kg) , the control(0mg/kg),all under 24h illumination). Made counting statistics of the leukocyte,erythrocyte in peripheral blood ,the T,B lymphocyte and its sub-population by Flow cytometry.Then cloned the three sub types of melatonin receptors by RT-PCR and made sequence analysis. Then detected the mRNA expression in chicken'spleen,thymus and cloacal bursa by in situ hybridization. The results as followed:The level of the melatonin was increased with the illumination decreased,and the content of leukocyte(P<0.01),erythrocyte(P<0.01),hemoglobin(P<0.01) in peripheral blood were increased very significantly; with illumination increased,the content of melatonin decreased,and the content of leukocyte(P<0.01),erythrocyte(P<0.01),hemoglobin(P<0.01) in peripheral blood were decreased very significantly.The results of T,B lymphocyte rise and fall regularity and its sub-population by Flow cytometry showed that both the content of CD3~+CD4~+,CD3~+CD8~+ T cell and Bu-1a~+B cell were declined notably with the content of Mel decreased under long illumination(18h),compared with the control group,the differences were significant(P<0.01); in short illumination group(6h),both the content of CD3~+CD4~+,CD3~+CD8~+ T cell and Bu-1a~+Bcell were growth obviously with the content of Mel increased,and compared with the control,the differences were significant(P<0.01). After injected exogenous melatonin,the content of CD3~+CD4~+,CD3~+CD8~+ T cell and Bu-1a~+B cell were raised remarkably,and compared with control group,the low level group was increased significantly(P<0.05),the high level was increased very significantly(P<0.01) (except Bu-1a~+B cell).According to the cDNA sequence of chicken melatonin three sub types receptor (MR)chickenU 31820(Mel1a),chickenU 30609(Mel1b),chickenU 31821(Mel1c)submitted in GenBank,we designed 3 pairs of primers,amplified the partial cDNA fragment of the three receptors from chicken brain by RT-PCR,the lengths of the fragment were 603bp,463bp and 504bp respectively.Then made analysis of these sequences and the results indicated that the identity of the three melatonin receptors sequences we acquired in this study were high compared with chickenU 31820(Mel1a),chickenU 30609(Mel1b), chickenU 31821(Mel1c)in GeneBank,which were 99.0%,97.2% and 99.4%respectively.We prepared digoxigenin-labeled MR cDNA probes and detected the Mel1a,Mel1b,Mel1c mRNA in chicken immune organs(spleen,thymus and cloacal bursa) by in situ hybridization. The results showed that the three subtypes MR all expressed in chicken spleen,thymus and cloacal bursa. And the MR positive expressional signal centralized in nucleus and endochylema of T and B lymphocyte in these organs mentioned above. The positive signals distribution rule was 18h>12h > 6h and Mel1a>Mel1c >Mel1b.