Characterization of the NLRC5 promoter in chicken: SNPs, regulatory elements and CpG islands.

NLRC5 plays an important role in the innate immunity and cellular immunity in many species, but the regulatory mechanism of NLRC5 expression in chickens remains unclear. In this study, a series of deletion fragments of the NLRC5 promoter region were constructed and dual-luciferase assay was performed. Then, we detected the SNP in the core region and its function. Important transcriptional regulatory elements were predicted and identified. Methylation of CpG islands was measured. The results revealed that the two core regions of -4372 to -3756 and -2925 to -2265 in the NLRC5 promoter were essential for NLRC5 mRNA expression in which a SNP (A/G), located at -2470, was found to have an effect on the transcriptional activity. Also, the STAT1 element in the second core region of the NLRC5 promoter was identified to bind with the STAT1 transcription factor, which was necessary for the transcriptional activity. In addition, many other elements in the NLRC5 promoter, including YY1 and CEBP, may contribute significantly to the expression activity of NLRC5. Moreover, two CpG islands were searched. Part of one was located in the first core region, which suggests that epigenetic modification may regulate the activity of the first promoter region, and the other was mostly in an unmethylated state. Collectively, these results suggest the complex regulation of NLRC5 expression includes SNPs, transcription factors and methylation.

Gene expression changes in chicken NLRC5 signal pathway associated with in vitro avian leukosis virus subgroup J infection.

Nucleotide-binding oligomerization domain-like receptors (NLRs) play a key role in the innate immune response as pattern-recognition receptors. However, the role of NLRC5, which is a member of the NLR family, in NF-κB activation and MHC-I expression remains debatable. Infection with the J group avian leukosis virus (ALV-J) can result in immunosuppression and a subsequent increase in susceptibility to secondary infection. This results in huge economic losses to the poultry industry worldwide. Using quantitative real-time polymerase chain reaction (qRT-PCR), we investigated the mRNA expression levels of NLRC5 signal pathway-related genes in secondary chicken embryo fibroblasts 7 days after infection with ALV-J. The results indicated that, compared with the control groups, the expression levels of TLR7, MHC-I, and IL-18 increased significantly in the infected groups at 7 days post-infection (d.p.i.). The expression levels of NLRC5 and IL-6 were conspicuously downregulated at 7 d.p.i., but the expression levels of NF-κB, STAT1, and STAT3 were not significantly altered. These results suggest that NLRC5 and some genes involved in the NLRC5 pathway play a key role in antiviral immunity, typically the response to ALV-J infection. Moreover, MHC-I expression levels vary between different cell types.

Single nucleotide polymorphism screening, molecular characterization, and evolutionary aspects of chicken Piwi genes.

The P-element-induced wimpy testis (Piwi) gene is involved in germline stem cell self-renewal, meiosis, RNA silencing, and transcriptional regulation. Piwi genes are relatively well conserved in many species, but their function in poultry species is unclear. In this study, Piwi genes were sequenced using a target-sequence capture assay in quail and 28 breeds of chicken. Single nucleotide polymorphisms (SNPs) and evolutionary aspects of these chicken breeds were then analyzed. We found that SNP sites existed mainly in the introns of a few chicken breeds, and we selected an SNP on intron 4 for further verification by Sanger sequencing, the results of which were similar to those obtained by the target-capture sequencing assay. The evolutionary analysis revealed that there were more mutations in the Chahua and Leghorn breeds than in the other breeds, and that the phylogenetic tree was divided into four main categories that suggested that Piwi is evolutionarily conserved, and mutations in the introns might be associated with gametogenesis. The screened SNPs can be used as candidate markers for Piwi, and our results provide basic information for the further study of Piwi function in poultry.

Analysis of Gene Expression Responses to a Salmonella Infection in Rugao Chicken Intestine Using GeneChips.

Poultry products are an important source of Salmonella enterica. An effective way to reduce food poisoning due to Salmonella would be to breed chickens more resistant to infection. Unfortunately host responses to Salmonella are complex with many factors involved. To learn more about responses to Salmonella in young chickens of 2 wk old, a cDNA Microarray containing 13,319 probes was performed to compare gene expression profiles between two chicken groups under control and Salmonella infected conditions. Newly hatched chickens were orally infected with S. enterica serovar Enteritidis. Since the intestine is one of the important barriers the bacteria encounter after oral inoculation, intestine gene expression was investigated at 2 wk old. There were 588 differentially expressed genes detected, of which 276 were known genes, and of the total number 266 were up-regulated and 322 were down-regulated. Differences in gene expression between the two chicken groups were found in control as well as Salmonella infected conditions indicating a difference in the intestine development between the two chicken groups which might be linked to the difference in Salmonella susceptibility. The differential expressions of 4 genes were confirmed by quantitative real-time PCR and the results indicated that the expression changes of these genes were generally consistent with the results of GeneChips. The findings in this study have lead to the identification of novel genes and possible cellular pathways, which are host dependent.

Behavior differentiation between wild Japanese quail, domestic quail, and their first filial generation.

The number of wild quail has dramatically reduced in China and reached a state of endangerment with the deterioration of the environment in recent years. In this study, we examined the ecological behaviors of quails in the cage to determine the differentiation level between wild Japanese quail and domestic quail, to detect the relationship between quail behavior and evolutionary differentiation and to analyze the possibility of restoring effective size of wild population. With the on-the-spot observations and measurements, the behaviors of 3 categories of quail, namely wild Japanese quail from the Weishan Lake area in China, domestic quail, and their first filial generation (F(1)) were studied. Domestic quail differed from wild Japanese quail in morphological pattern and ecological behaviors, including some indexes of figure type and egg, vocalization, aggression and fighting, and mating, but wild Japanese quail and domestic quail could succeed in mating and reproducing fertile hybrid offspring. There were significant differences between domestic quail and wild Japanese quail in reproductive traits, involved mating times, fertility rate, hatching rate, and hatching rate of fertilized eggs (P < 0.05). The first filial generation presented significant difference from the wild Japanese quail in vocalization, aggression and fighting, mating, hatching rate, hatching rate of fertilized eggs, and some egg indexes (P < 0.05) and significantly differ from the domestic quail in vocalization, hatching rate, and hatching rate of fertilized eggs (P < 0.05). Evolutionary differentiation between wild quail and domestic quail was still at a relatively low level because no reproductive isolation existed. The advantages of the F(1) hybrids in reproductive capacity, fertilization, and hatching recommend that releasing hybrids instead of domestic quails to the wild would be a more effective way to restore the effective size of wild quail population if necessary.