Yaqiu Lin

Selection of reference genes for gene expression studies related to intramuscular fat deposition in Capra hircus skeletal muscle.

The identification of suitable reference genes is critical for obtaining reliable results from gene expression studies using quantitative real-time PCR (qPCR) because the expression of reference genes may vary considerably under different experimental conditions. In most cases, however, commonly used reference genes are employed in data normalization without proper validation, which may lead to incorrect data interpretation. Here, we aim to select a set of optimal reference genes for the accurate normalization of gene expression associated with intramuscular fat (IMF) deposition during development. In the present study, eight reference genes (PPIB, HMBS, RPLP0, B2M, YWHAZ, 18S, GAPDH and ACTB) were evaluated by three different algorithms (geNorm, NormFinder and BestKeeper) in two types of muscle tissues (longissimus dorsi muscle and biceps femoris muscle) across different developmental stages. All three algorithms gave similar results. PPIB and HMBS were identified as the most stable reference genes, while the commonly used reference genes 18S and GAPDH were the most variably expressed, with expression varying dramatically across different developmental stages. Furthermore, to reveal the crucial role of appropriate reference genes in obtaining a reliable result, analysis of PPARG expression was performed by normalization to the most and the least stable reference genes. The relative expression levels of PPARG normalized to the most stable reference genes greatly differed from those normalized to the least stable one. Therefore, evaluation of reference genes must be performed for a given experimental condition before the reference genes are used. PPIB and HMBS are the optimal reference genes for analysis of gene expression associated with IMF deposition in skeletal muscle during development.

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 Km (NADH) and the same mobility on a gel as bovine LDH1, and the LDH1-F variant showed significant differences in Km 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.

Expression profiles and associations of adiponectin and adiponectin receptors with intramuscular fat in Tibetan chicken

ABSTRACT 1. Adiponectin and its receptors (ADIPOR1 and ADIPOR2) are novel endocrine systems that act at various levels to modulate glucose and lipid metabolism. This study was designed to investigate the spatial expression of adiponectin, ADIPOR1 and ADIPOR2 genes in various tissues in Tibetan chicken. The temporal expression of adiponectin and its receptor mRNAs were also studied in adipose tissue, breast muscle and thigh muscle and the correlations of the levels of adiponectin, ADIPOR1 and ADIPOR2 mRNA with the contents of intramuscular fat in breast muscle and thigh muscle of Tibetan chicken were determined. 2. Quantitative real-time PCR detected chicken adiponectin, ADIPOR1 and ADIPOR2 mRNA transcripts in heart, liver, spleen, lung, kidney, skeletal muscle and adipose tissue. 3. Adipose tissue contained the highest amount of adiponectin mRNA followed by the kidney and liver. The expression levels of ADIPOR1 mRNA were significantly higher in adipose tissue, lung and spleen, and adipose tissue exhibited significantly higher levels of ADIPOR2 mRNA followed by the spleen and lung compared with other tissues. 4. Temporal expression profiles of adiponectin, ADIPOR1 and ADIPOR2 mRNA showed gender differences in adipose tissue and skeletal muscle at certain ages. In adipose tissue, adiponectin mRNA was higher in 154-d-old females and ADIPOR1 mRNA was higher in 154-d-old males: Adiponectin and ADIPOR2 mRNA were higher, and ADIPOR1 mRNA was lower, in thigh muscle in female compared with male chickens. 5. The correlation data showed that, except for adiponectin mRNA, the levels of ADIPOR1 and ADIPOR2 mRNA in thigh muscle of males were significantly positively correlated with IMF (r = 0.206 for the ADIPOR1 gene and r = 0.676 for the ADIPOR2 gene). 6. Taken together, it was concluded that adiponectin and the ADIPOR1 and ADIPOR2 genes are ubiquitously expressed in various tissues of Tibetan chicken and the expression of the adiponectin system is gender-dependant at certain ages in adipose tissue and skeletal muscle.

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

ABSTRACT 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.

Quantitation of alternative splicing variants of lactate dehydrogenase C gene in testes of adult yak, sexually immature yak calf and sterile male hybrid of yak

Huang, L., Jin, S-Y., Xu, Y-O., Li, Y-P., Lin, Y-Q. and Zheng, Y-C. 2012. Quantitation of alternative splicing variants of lactate dehydrogenase C gene in testes of adult yak, sexually immature yak calf and sterile male hybrid of yak. Can. J. Anim. Sci. 92: 291-296. The main objective of the present study was to analyze quantitatively the alternative splicing of the lactate dehydrogenase C (ldhc) gene in the testes of yak (Bos grunniens) and male sterile yak hybrid. RT-PCR amplification of ldhc cDNA in the testes revealed eight splice variants formed by the deletion of one or more exons in the mRNA transcripts. The deleted exons occur mostly in exons 7 and 4. The deletion of exons caused reading frame shift and formation of stop codon in some variants. Quantitative real-time RT-PCR analysis using ldhc variant-specific primers showed that the mRNA level of full length ldhc decreased dramatically in the testes of sexually immature yak calf (n=6) and male sterile hybrid cattle-yak (n=4) compared with that of adult yak (n=14). The proportions of the ldhc variants assayed differed significantly among adult yak, yak calf and cattle-yak; more ldhc transcripts were spliced in immature or sterile testes. Our results suggest that the alternative splicing could play a role in the regulation of ldhc expression in testes, and could be one factor that plays a role in infertility of yak hybrids.

Expression profiles and associations of muscle regulatory factor (MRF) genes with growth traits in Tibetan chickens

ABSTRACT 1. Muscle regulatory factors (MRFs), including Myf5, Myf6 (MRF4/herculin), MyoD and MyoG (myogenin), play pivotal roles in muscle growth and development. Therefore, they are considered as candidate genes for meat production traits in livestock and poultry. 2. The objective of this study was to investigate the expression profiles of these genes in skeletal muscles (breast muscle and thigh muscle) at 5 developmental stages (0, 81, 119, 154 and 210 d old) of Tibetan chickens. Relationships between expressions of these genes and growth and carcass traits in these chickens were also estimated. 3. The expression profiles showed that in the breast muscle of both genders the mRNA levels of MRF genes were highest on the day of hatching, then declined significantly from d 0 to d 81, and fluctuated in a certain range from d 81 to d 210. However, the expression of Myf5, Myf6 and MyoG reached peaks in the thigh muscle in 118-d-old females and for MyoD in 154-d-old females, whereas the mRNA amounts of MRF genes in the male thigh muscle were in a narrow range from d 0 to d 210. 4. Correlation analysis suggested that gender had an influence on the relationships of MRF gene expression with growth traits. The RNA levels of MyoD, Myf5 genes in male breast muscle were positively related with several growth traits of Tibetan chickens (P < 0.05). No correlation was found between expressions of MRF genes and carcass traits of the chickens. 5. These results will provide a base for functional studies of MRF genes on growth and development of Tibetan chickens, as well as selective breeding and resource exploration.

Identification of differentially expressed genes through RNA sequencing in goats (Capra hircus) at different postnatal stages.

Intramuscular fat (IMF) content and fatty acid composition of longissimus dorsi muscle (LM) change with growth, which partially determines the flavor and nutritional value of goat (Capra hircus) meat. However, unlike cattle, little information is available on the transcriptome-wide changes during different postnatal stages in small ruminants, especially goats. In this study, the sequencing reads of goat LM tissues collected from kid, youth, and adult period were mapped to the goat genome. Results showed that out of total 24 689 Unigenes, 20 435 Unigenes were annotated. Based on expected number of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM), 111 annotated differentially expressed genes (DEGs) were identified among different postnatal stages, which were subsequently assigned to 16 possible expression patterns by series-cluster analysis. Functional classification by Gene Ontology (GO) analysis was used for selecting the genes showing highest expression related to lipid metabolism. Finally, we identified the node genes for lipid metabolism regulation using co-expression analysis. In conclusion, these data may uncover candidate genes having functional roles in regulation of goat muscle development and lipid metabolism during the various growth stages in goats.

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.

Expression profiles and associations of FGF1 and FGF10 with intramuscular fat in Tibetan chicken

ABSTRACT 1. FGF1 and FGF10, two paracrine members of the fibroblast growth factor (FGF) gene family, play critical roles in the development, structural and metabolic remodelling of adipose tissue. 2. The objective of this study was to investigate the expression profiles of FGF1 and FGF10 genes in breast muscle and thigh muscle in 5 developmental stages (1, 81, 119, 154 and 210 d old) in Tibetan chickens. The possible relationships between expression of these genes and intramuscular fat (IMF) content were analysed in Tibetan chickens. 3. Expression profiles showed that FGF1 and FGF10 mRNA were ubiquitously expressed in various tissues of 154-d-old Tibetan chickens. Lung tissue contained the highest amount of FGF1 and FGF10 mRNA while breast muscle and thigh muscle exhibited lower levels of FGF1 and FGF10 mRNA in both males and females compared with other tissues (P < 0.05). Temporal expression of FGF1 and FGF10 in breast and thigh muscle showed similar tendencies in males and females, respectively, with peaks in thigh muscle at 119-d-old and breast muscle in 1-d-old males and females, respectively. 4. Correlation analysis suggested that gender had an influence on the relationships of FGF1 and FGF10 expression with IMF content in thigh muscle. The RNA levels of FGF1 and FGF10 genes in male thigh muscle were positively related to IMF content of Tibetan chickens (P < 0.01), while the correlations were shown to be negative in female thigh muscle (P > 0.05). 5. These results provide a basis for functional elucidation of FGF1 and FGF10 genes on adipocyte development and intramuscular fat deposition, as well as selective breeding and resource exploration of local poultry breeds.

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

ABSTRACT 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.