| Megalobrama amblycephala has been regarded as a major species in the polyculture system of Chinese freshwater fish. Its natural distribution is limited in Liangzi Lake, Yuni Lake and Poyang Lake. In recent years, the germplasm resources of M. amblycephala protospecies were in the threat of recession and mixed due to overfishing and domestication. In present study, we described here molecular cloning, sequences, and expression of some immune-related genes to further understand their functions. Furthermore, we analyzed the differentiation of target genes expression and immune-related indexes among Liangzihu Lake population (L), Yunihu Lake population (Y) and Poyanghu Lake population (P) of M. amblycephala to identify the better population with immune property for breeding. The main results of this study were as followed below: 1. Cloning and sequence analysis of M. amblycephala target genesFull-length cDNAs of CXCR4b and CCL4 were cloned and characterized from M amblycephala in the present study. The full-length cDNA sequence of M. amblycephala CXCR4b was 1638 bp, consisting of 55-bp 5’-untranslated region (5’-UTR),521-bp 3’-UTR including a poly-A signal sequence, and 1062-bp ORF encoding 353 amino acid residues with 39.89 kDa of the calculated molecular weight and 8.59 of theoretical isoelectric point. The phylogenetic analysis indicated that M. amblycephala CXCR4b shared higher sequence identity with the homologues of common carp than other fishes. M. amblycephala CXCR4b contained typical motifs of seven transmembrane domains (TM). The putative C107-C194 (located in the EC-1 and EC-2) disulfide bridge and the nearby tyrosin Y188 of M. amblycephala CXCR4b were fully conserved in this receptor family. Additionally, it contained a conserved DRYLAIV motif at the N-terminal of the second intracellular loop.The full-length of M. amblycephala CCL4 was 913 bp, consisting of 180 bp of 5’-UTR,285 bp of ORF encoding 94 amino acid residues, and 448 bp of 3’-UTR with a poly-A signal sequence. The calculated molecular weight was 10.43 kDa and theoretical isoelectric point was 9.24. Like all other CC chemokines, M. amblycephala CCL4 harbored the four conserved cysteine residues. The analysis showed that the transmembrane domain of all CC chemokines was located in the least conserved N-terminal. The phylogenetic analysis demonstrated that CCL4 protein of M. amblycephala was clustered with the homologues of the bony fish CCL4.2. Temporal expression of M. amblycephala target genesQuantitative RT-PCR analysis indicated that the expression levels of nine target genes in all pro-hatching stages were significantly higher than most post-hatching stages (P< 0.05), and reached the first peak at 10 hpf to 28 hpf, then decreased remarkably at hatching stage (38 hpf, namely). Nevertheless, target genes mRNA rapidly reached the second peak at 62 hpf (P<0.05). These results suggested that all nine target genes may play a key role in the embryonic development of M. amblycephala.3. Effect of acute toxicity of nitrite on M. amblycephala target genesExpression analysis of target genes in kidney, spleen, intestine, gill, liver and blood at different time points after different nitrite concentration stress by relatively quantitative RT-PCR indicated that all target genes mRNA in all sampled tissues was quickly up-regulated after nitrite exposure and reached the peak level at 24 h or 48 h post-exposure. Our findings have fully confirmed that all target genes were related with non-specific immune system, supporting a potential pro-inflammatory function for some target protein or induced by inflammation.4. Estimation and comparison of immune property among three populationsAnalysis of the target gene mRNA in different tissues of different M. amblycephala populations indicated the expression levels in the population Y were obviously higher than in the other two populations (P<0.05). Furthermore, non-specific immune and haematological indexes were detected, the results showed that the activity of catalase (CAT), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in population Y were lower than other two populations. In the liver and spleen, the content of malondialdehyde (MDA) in population L was significantly lower than population Y (P <0.05), but being identical with population P. We also had known that the ordinal proportion of thrombocyte of three populations were P>Y> L, and the proportion of thrombocyte of population Y was significantly lower than the other two populations (P< 0.05). However, the proportion of lymphocytes was the opposite trend. The size of red cells of population L was significantly smaller than the other two populations (P< 0.05). Taken together of all results have demonstrated that the immune property of population Y was the worst among the three populations. |
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