Molecular cloning, expression profiles and associations of KLF6 gene with intramuscular fat in Tibetan chicken.

The aim of this study was to clone KLF6 gene of Tibetan chicken, clarify its temporal-spatial expression characteristics, and build the correlation between the expression level of KLF6 gene and IMF content in different developmental stages. RT-PCR was used to clone Tibetan chicken KLF6 gene, qPCR was used to detect the expression level of KLF6 gene in different tissues and developmental stages. The sequence of KLF6 gene was 919 bp including a complete 852 bp CDS region. The gene was highest expression in lung tissues, which was significantly higher than in other tissues (p < 0.01). In male Tibetan chicken breast muscle the levels of KLF6 mRNA were negatively related to IMF content (r=-0.097, p > 0.05), while in females they were positively correlated (r = 0.077, p > 0.05). At the age of 119-210 days, the expression of KLF6 mRNA in the male chicken leg muscles was highly positively correlated (r = 0.506, p < 0.01), but negatively correlated in the female chicken leg muscles (r=-0.198, p > 0.05). The expression level of KLF6 in breast muscle decreased gradually with the increasing age, while in leg muscle the expression level increased firstly and then descended with the increasing age.

Transcriptome analyses reveal genes of alternative splicing associated with muscle development in chickens.

Alternative splicing (AS) of pre-mRNA is a central mode of genetic regulation in higher eukaryotes. High-throughput experimental verification of alternative splice forms, functional characterization, and regulation of alternative splicing are key directions for research. However, little information is available on the transcriptome-wide changes during different ages in different chicken breeds. In this study, the sequencing reads of chicken muscle tissues collected from White feather broiler (day 42) and Luning Chicken (day 70, 120, 150) were mapped to the chicken genome. Results showed that a total of 16,958 genes were annotated, with 2230 differentially expressed genes (DEGs) when comparing White feather broiler and Luning Chicken, and an average of 700 DEGs when comparing different ages in Luning Chicken. Functional classification by Gene Ontology (GO) and pathways analysis for selecting the genes showed most DEGs were related to muscle development and immune response. Of the 16,958 genes, a total of 6249 genes (36.85%) underwent AS events, and over 40% were specifically expressed in each library. Additionally, 6 DEGs (SRPK3, ENSGALG00000022884, CCL4, GATM, SESN1, PTTG1IP) involved in muscle development and immunity response were found to be alternatively spliced among all the four muscle tissues. In conclusion, we present a complete dataset involving the spatial and temporal transcriptome of chicken muscle tissue using RNA -seq. These data will facilitate the understanding of the effects of breed and age on the development of muscle and uncover that AS events of candidate genes may have important functional roles in regulation of muscle development in chicken.

Functional Identification of Allograft Inflammatory Factor 1-Like Gene in Luning Chicken.

Allograft inflammatory factor-1 (AIF-1) is an inflammation-related protein mainly produced by immune cells, such as monocyte/macrophages and activated T lymphocytes. It is essential for the survival and proinflammatory activity of immune cells. However, the function of AIF-1 in chicken still has not been defined. In the present study, AIF-1-like (AIF1L) gene was identified in Luning chicken. Bioinformatics analysis revealed that the molecular weight of the chicken AIF-1 protein was 16290.8 Da. AIF1L contained a Ca2+ binding EF hand and could interact with actin filament. Its transcript was found in all tested tissues including spleen, brain, heart, kidney, liver, thymus, bursa of Fabricius, lung, and a relative low-level expression was detected in leg muscle. Furthermore, AIF1L expression in peripheral blood lymphocyte was depressed in a dose-dependent manner with cadmium exposure and peripheral blood lymphocyte viability decrease displayed a similar pattern with AIF1L expression. The results indicated that newly identified chicken AIF1L might be associated with lymphocyte viability.

Cloning and Expression of SFRP5 in Tibetan Chicken and its Relationship with IMF Deposition.

Secreted frizzled related protein 5 (SFRP5), an anti-inflammatory adipokine, is relevant to the adipocyte differentiation. In order to clarify its role in regulating intramuscular fat (IMF) deposition in Tibetan chicken, the full-length sequence of the Tibetan chicken SFRP5 gene was cloned. The relative expression of SFRP5 gene was detected using quantitative RT-PCR in various tissues of 154 days old Tibetan chicken, as well as in breast muscle, thigh muscle, and adipose tissue at different growth stages. The results showed that SFRP5 gene was expressed in all examined tissues but highly enriched in adipose tissue. Temporal expression profile showed that the expression of SFRP5 was gradually decreased in breast muscle, but was fluctuated in thigh muscle and adipose tissue with the growth of Tibetan chicken. Furthermore, correlation analysis demonstrated that the expression of SFRP5 in breast muscle, thigh muscle and adipose tissue was correlated with IMF content at different levels. The results indicated that Tibetan chicken SFRP5 is involved in IMF deposition.

Cloning and polymorphisms of yak lactate dehydrogenase B gene.

The main objective of this work was to study the unique polymorphisms of the lactate dehydrogenase-1 (LDH1) gene in yak (Bos grunniens). Native polyacrylamide gel electrophoresis revealed three phenotypes of LDH1 (a tetramer of H subunit) in yak heart and longissimus muscle extracts. The corresponding gene, ldhb, encoding H subunits of three LDH1 phenotypes was obtained by RT-PCR. A total of six nucleotide differences were detected in yak ldhb compared with that of cattle, of which five mutations cause amino acid substitutions. Sequence analysis shows that the G896A and C689A, mutations of ldhb gene, result in alterations of differently charged amino acids, and create the three phenotypes (F, M, and S) of yak LDH1. Molecular modeling of the H subunit of LDH indicates that the substituted amino acids are not located within NAD+ or substrate binding sites. PCR-RFLP examination of G896A mutation demonstrated that most LDH1-F samples are actually heterozygote at this site. These results help to elucidate the molecular basis and genetic characteristic of the three unique LDH1 phenotypes in yak.

Prokaryotic expression and immunogenicity analysis of yak recombinant myostatin.

Myostatin (MSTN) is a negative regulator of skeletal muscle growth. The objective of the present study was to express yak (Bos grunniens) recombinant MSTN protein in E. coli and study its characteristics of immunogenicity. cDNA encoding yak MSTN mature peptide was amplified by reverse-transcription PCR, and cloned into pET28a(+) vector and expressed in E. coli. The expressed recombinant MSTN was purified by affinity chromatography and used to prepare rabbit anti yak MSTN antibody. The results showed that yak MSTN mature peptide gene contained 330 bp nucleotides coding 109 amino acids. Content of the target protein accounted for 21% of the total expression products when MSTN-pET28a(+)-BL21(DE3) bacterium was incubated in LB medium with 0.1 mM IPTG for 6 hours. The molecular weight of the purified yak MSTN recombinant protein was 16.5 kDa, exhibiting excellent immunogenicity as shown by ELISA. The obtained recombinant MSTN of yak is suitable for further analysis of yak MSTN functions.

Alternative splicing of testis-specific lactate dehydrogenase C gene in mammals and pigeon.

The objective of the present study was to confirm the widespread existence of alternative splicing of lactate dehydrogenase c (ldhc) gene in mammals. RT-PCR was employed to amplify cDNAs of ldhc from testes of mammals including pig, dog, rabbit, cat, rat, and mouse, as well as pigeon. Two to six kinds of splice variants of ldhc were observed in the seven species as a result of deletion of one or more exons or insertion of partial sequence of an intron in the mature mRNA. The deleted exons occur mostly in exons 5, 4, 6, and 3. The insertion of a partial sequence of introns, which resulted in an abnormal stop codon in the inserted intron sequence, was observed only in dog and rat. The deletion of exons also resulted in a reading frame shift and formation of a stop codon in some variants. No alternative splicing was observed for ldha and ldhb genes in testis of yak. Native polyacrylamide gel electrophoresis and Western blot analysis revealed no obvious LDH-C4 activity derived from expressed ldhc variants. Our results demonstrated the widespread and unique existence of alternative splicing of ldhc genes in mammals.

High-altitude adaptation of yak based on genetic variants and activity of lactate dehydrogenase-1.

This study investigates the molecular mechanism by which yaks (Bos grunniens) adapt to hypoxia based on lactate dehydrogenase (LDH). Three LDH1 variants of the yak were revealed in tissue extracts by electrophoresis, including LDH1-F, LDH1-M, and LDH1-S. Kinetic analysis using purified LDH1 variants showed that the yak LDH1-M variant exhibited a similar K (m) (NADH) and the same mobility on a gel as bovine LDH1, and the LDH1-F variant showed significant differences in K (m) values for NADH or pyruvate from the other two variants of yak LDH1 and bovine LDH1. Among the three muscles assayed, yak longissimus dorsi showed the highest LDH activity and the lowest malate dehydrogenase (MDH) activity; heart muscle was exactly the opposite. Our results suggest that the three LDH1 variants might play an important role in the adaptation to hypoxia.