Zhuanjian Li

Discovery and functional characterization of leptin and its receptors in Japanese quail (Coturnix japonica)

Leptin is an important endocrine regulation factor of food intake and energy homeostasis in mammals; however, the existence of a poultry leptin gene (LEP) is still debated. Here, for the first time, we report the cloning of a partial exon 3 sequence of LEP (qLEP) and four different leptin receptor splicing variants, including a long receptor (qLEPRl) and three soluble receptors (qLEPR-a, qLEPR-b and qLEPR-c) in Japanese quail (Coturnix japonica). The qLEP gene had high GC content (64%), which is similar to other reported avian leptin genes. The encoded qLEP protein possessed the conserved pair of cysteine residues that are required to form a lasso knot for full biological activity, but shared relatively low identities with LEPs of other vertebrates. The translated qLEPRl protein contained 1143 amino acids and shared high amino acid sequence identity with a chicken homolog (89% identity). qLEPRl also contained all the motifs, domains, and basic tyrosine residues that are conserved in the LEPRl proteins of other vertebrates. qRT-PCR analysis showed that LEP and the four LEPR variants were expressed extensively in all tissues examined; the expression levels of LEP were relatively high in hypothalamus, skeletal muscle, and pancreas, while the expression levels of the LEPRs were highest in the pituitary. Compared with the expression levels of juvenile qLEP and total qLEPR (including all LEPR variants), the expression levels of mature qLEP and total qLEPR were up-regulated in the hypothalamus and pituitary, and down-regulated in the ovary. The expressions of LEP/LEPR increased when fasting and decreased when refeeding in the brain and peripheral tissues of juvenile quail, which suggested that the LEP/LEPR system modulated food intake and energy expenditure, although, unlike in mammals, LEP may actually act to inhibit food intake during fasting, at least in juvenile quail. The results indicate that qLEP and qLEPR have unique expression patterns and that the encoded proteins play important roles in the regulation of reproduction and energy status in Japanese quail.

Systematic analysis of the regulatory functions of microRNAs in chicken hepatic lipid metabolism.

Laying performance is an important economic trait in hens, and this physiological process is largely influenced by the liver function. The livers of hens at 20- and 30-week-old stages were investigated using the next generation sequencing to identify the differences of microRNA expression profiles. Compared with the 20-week-old hens, 67 down- and 13 up-regulated microRNAs were verified to be significant differentially expressed (false discovery rate, FDR ≤ 0.05) (SDE) in the 30-week-old. We also identified 13 down- and 6 up-regulated novel differentially expressed (DE) microRNAs. miR-22-3p and miR-146b-5p, which exhibit critical roles in mammalian lipid metabolism, showed the most abundant expression and the highest fold-change, respectively. A total of 648 potential target genes of the SDE microRNAs were identified through an integrated analysis of microRNAs and the DE genes obtained in previous RNA-sequencing, including FADS1, FADS2, ELOVL6 and ACSL5, which are critical lipid metabolism-related regulators. Bioinformatic analyses revealed that target genes were mainly enriched in lipid-related metabolism processes. This work provides the first study of the expression patterns of hepatic microRNAs between 20- and 30-week old hens. The findings may serve as a fundamental resource for understanding the detailed functions of microRNAs in the molecular regulatory systems of lipid metabolism.

Association of estradiol on expression of melanocortin receptors and their accessory proteins in the liver of chicken (Gallus gallus)

The melanocortin receptor accessory proteins (MRAP and MRAP2) are small single-pass transmembrane proteins that regulate the biological functions of the melanocortin receptor (MCR) family. MCRs comprise five receptors (MC1R-MC5R) with diverse physiological roles in mammals. Five MCR members and two MRAPs were also predicted in the chicken (Gallus gallus) genome. However, little is known about their expression, regulation and biological functions. In this study, we cloned the MRAP and MRAP2 genes. Sequencing analysis revealed that the functional domains of MRAP and MRAP2 were conserved among species, suggesting that the physiological roles of chicken MRAP and MRAP2 could be similar to their mammalian counterparts. Tissue expression analysis demonstrated that MRAP was expressed in the adrenal gland, liver, spleen, glandular stomach and lungs, while MRAP2 is predominantly expressed in the adrenal gland. All five MCRs were present in the adrenal gland, but showed different expression patterns in other tissues. The MC5R was the only MCR member that was expressed in the chicken liver. The expression levels of MRAP in chicken liver were significantly increased at sexual maturity stage, and were significantly up-regulated (P<0.05) when chickens and chicken primary hepatocytes were treated with 17β-estradiol in vivo and in vitro, respectively; however, expression levels of PPARγ were down-regulated, and no effect on MC5R was observed. Our results suggested that estrogen could stimulate the expression of MRAP in the liver of chicken through inhibiting the expression of transcription regulation factor PPARγ, and MRAP might play its biological role in a different way rather than forming an MRAP/MC2R complex in chicken liver during the egg-laying period.

Expression of Thyroid Hormone Responsive SPOT 14 Gene Is Regulated by Estrogen in Chicken ( Gallus gallus )

Thyroid hormone responsive spot 14 (THRSP) is a small nuclear protein that responds rapidly to thyroid hormone. It has been shown that THRSP is abundant in lipogenic tissues such as liver, fat and the mammary gland in mammals. The THRSP gene acts as a key lipogenic activator and can be activated by thyroid hormone triiodothyronine (T3), glucose, carbohydrate and insulin. Here we report that chicken THRSP is also abundant in lipogenic tissues including the liver and the abdominal fat, and its expression levels increased with sex maturation and reached the highest level at the peak of egg production. Structure analysis of the THRSP gene indicates that there is a conscious estrogen response element (ERE) located in the −2390 – −2402 range of the gene promoter region. Further studies by ChIP-qPCR proved that the ERα interacts with the putative ERE site. In addition, THRSP was significantly upregulated (P < 0.05) when chickens or chicken primary hepatocytes were treated with 17β-estradiol in both the in vivo and in vitro conditions. We therefore conclude that THRSP is directly regulated by estrogen and is involved in the estrogen regulation network in chicken.

Effect of polymorphism within miRNA-1606 gene on growth and carcass traits in chicken.

Genetic variations in microRNAs (miRNAs) including primary miRNAs, precursor miRNAs and mature miRNAs can lead to phenotypic variation by altering the biogenesis of miRNAs and/or their binding to target mRNAs. Increasing functional studies suggest that polymorphisms occurring in miRNAs can lead to phenotypic variation in farm animal. Here, we identified a single nucleotide polymorphism (SNP) located in the precursor of chicken miRNA-1606 gene. The association study on body indexes, body weight at different growth stages, and carcass traits was performed in a Gushi-Anka F2 population resource. The SNP was not only significantly associated with body weight at 10 and 12 weeks, respectively, but also with chicken shank length, chest depth and body slanting length at 8 weeks; shank length, pectoral angle, body slanting length and pelvis breadth at 12 weeks, respectively. And the polymorphism was also significantly associated with carcass traits including semi-evisceration weight, evisceration weight, breast muscle weight, leg weight and carcass weight as well. The observed values of individuals with CA genotype were significantly higher than CC genotype both in body weight at different stages and carcass traits. This SNP altered the predicted second structure of pre-mir-1606, with the altering of the free energy values. And the relative expression level of mature miRNA between CA and AA was significantly changed in leg muscle. Our data suggested that miRNA-1606 may be a candidate gene associated with chicken growth traits.

Characterization of the visfatin gene and its expression pattern and effect on 3T3-L1 adipocyte differentiation in chickens

Visfatin is a newly identified adipocytokine that plays an important role in the determination of fat traits. In this study, we investigated the characterization of visfatin and the relationship between gene expression and chicken development to provide a theoretical basis for studying visfatin functions. The main results are summarized as follows: The 1482-bp full coding sequence of the visfatin gene of silky fowl was obtained and found to encode 493 amino acids. This gene contains 26 phosphorylation sites and a conserved domain of the NAPRTase family but no signal peptide sequence. It exhibits six functional motifs, including an amidation site. In chickens, visfatin is a highly conserved protein. The highest expression of visfatin was found in breast muscle and the lowest in bone marrow. There was no difference in expression between visceral fat and subcutaneous fat. However, the expression of visfatin in the bone marrow, liver, kidneys, and subcutaneous and visceral fat of broiler chickens was significantly higher than that in silky fowl (P<0.05). Visfatin mRNA levels in the bone marrow decreased with development (P<0.05) but increased in the liver and leg muscle. Visfatin gene expression in the liver, heart and bone marrow did not differ in silky fowl according to sex. A visfatin fusion protein caused a significant increase in the expression of adipocyte differentiation markers (PPARγ, aP2, C/EBPα, and FAS) compared with the control group and a decrease compared with the insulin group. Taken together, the results of the present study contribute to a better understanding of the expression and role of the visfatin gene in chickens.

Data in support of the discovery of alternative splicing variants of quail LEPR and the evolutionary conservation of qLEPRl by nucleotide and amino acid sequences alignment

Leptin receptor (LEPR) belongs to the class I cytokine receptor superfamily which share common structural features and signal transduction pathways. Although multiple LEPR isoforms, which are derived from one gene, were identified in mammals, they were rarely found in avian except the long LEPR. Four alternative splicing variants of quail LEPR (qLEPR) had been cloned and sequenced for the first time (Wang et al., 2015 [1]). To define patterns of the four splicing variants (qLEPRl, qLEPR-a, qLEPR-b and qLEPR-c) and locate the conserved regions of qLEPRl, this data article provides nucleotide sequence alignment of qLEPR and amino acid sequence alignment of representative vertebrate LEPR. The detailed analysis was shown in [1].

Genetic diversity of mtDNA D-loop sequences in four native Chinese chicken breeds

ABSTRACT 1. To explore the genetic diversity of Chinese indigenous chicken breeds, a 585 bp fragment of the mitochondrial DNA (mtDNA) region was sequenced in 102 birds from the Xichuan black-bone chicken, Yunyang black-bone chicken and Lushi chicken. In addition, 30 mtDNA D-loop sequences of Silkie fowls were downloaded from NCBI. The mtDNA D-loop sequence polymorphism and maternal origin of 4 chicken breeds were analysed in this study. 2. The results showed that a total of 33 mutation sites and 28 haplotypes were detected in the 4 chicken breeds. The haplotype diversity and nucleotide diversity of these 4 native breeds were 0.916 ± 0.014 and 0.012 ± 0.002, respectively. Three clusters were formed in 4 Chinese native chickens and 12 reference breeds. Both the Xichuan black-bone chicken and Yunyang black-bone chicken were grouped into one cluster. Four haplogroups (A, B, C and E) emerged in the median-joining network in these breeds. 3. It was concluded that these 4 Chinese chicken breeds had high genetic diversity. The phylogenetic tree and median network profiles showed that Chinese native chickens and its neighbouring countries had at least two maternal origins, one from Yunnan, China and another from Southeast Asia or its surrounding area.

Promoter analysis and tissue expression of the chicken ASB15 gene

ABSTRACT 1. This study was conducted to explore the promoter region of the chicken ASB15 gene by detecting the activities of the dual luciferase reporter gene and to assess expression profiles of the ASB15 gene in 10 different tissues from Gushi chickens. 2. Five dual luciferase reporter gene vectors were constructed and transfected into DF1 cells. The activities of recombined plasmids were measured and the core promoter was confirmed by bioinformatic analysis. Total RNA was extracted and the relative expression of the ASB15 gene was examined. 3. Data analysis indicated that the promoter was located from −955 to −212 bp. Results showed that the chicken ASB15 gene was expressed in heart, breast muscle and leg muscle. 4. This study has confirmed the promoter region and the expression profile of the chicken ASB15 gene, which provides a foundation for further exploring its transcriptional regulation and function.

SNP in pre-miR-1666 decreases mature miRNA expression and is associated with chicken performance.

Polymorphisms in miRNA genes could potentially alter various biological processes by influencing the processing and (or) target selection of miRNAs. The rs14120863 (C > G) mutation, which we characterized in a Gushi-Anka F2 resource population, resides in the precursor region of miR-1666. Association analysis with chicken carcass and growth traits showed that the SNP was significantly associated with carcass weight, evisceration weight, breast muscle weight, leg muscle weight, and body weight at 8 weeks of age, as well as some body size indexes including shank girth, chest breadth, breast bone length, and body slanting length, in the Gushi-Anka F2 resource population. Quantitative RT-PCR results showed that miR-1666 expression levels in muscle tissues differed within various genotypes. Experiment in DF1 cells further confirmed that the SNP in miR-1666 could significantly alter mature miRNA production. Subsequently, using dual-luciferase report assay, we verified that miR-1666 could perform its function through targeting of the CBFB gene. In conclusion, the SNP in the precursor of miR-1666 could significantly reduce mature miR-1666 production. It may further affect the function of miR-1666 through the target gene CBFB, hence it is associated with chicken growth traits.