Jibin Zhang

A comprehensive microRNA expression profile of the backfat tissue from castrated and intact full-sib pair male pigs

BackgroundIt is widely known that castration has a significant effect on the accumulation of adipose tissue. microRNAs (miRNAs) are known to be involved in fat deposition and to be regulated by the androgen-induced androgen receptor (AR). However, there is little understanding of the relationship between miRNAs and fat deposition after castration. In this study, the high-throughput SOLiD sequencing approach was used to identify and characterize miRNA expression in backfat from intact and castrated full-sib male 23-week-old pigs. The patterns of adipogenesis and fat deposition were compared between castrated and intact male pigs.ResultsA total of 366 unique miRNA genes were identified, comprising 174 known pre-miRNAs and 192 novel pre-miRNAs. One hundred and sixty-seven pre-miRNAs were common to both castrated (F3) and intact (F4) male pig small RNA libraries. The novel pre-miRNAs encoded 153 miRNAs/miRNA*s and 141 miRNAs/miRNA*s in the F3 and F4 libraries, respectively. One hundred and seventy-seven miRNAs, including 45 up- and 132 down-regulated, had more than 2-fold differential expression between the castrated and intact male pigs (p-value < 0.001). Thirty-five miRNAs were further selected, based on the expression abundance and differentiation between the two libraries, to predict their targets in KEGG pathways. KEGG pathway analyses suggested that miRNAs differentially expressed between the castrated and intact male pigs are involved in proliferation, apoptosis, differentiation, migration, adipose tissue development and other important biological processes. The expression patterns of eight arbitrarily selected miRNAs were validated by stem-loop reverse-transcription quantitative polymerase chain reaction. These data confirmed the expression tendency observed with SOLiD sequencing. miRNA isomiRs and mirtrons were also investigated in this study. Mirtrons are a recently described category of miRNA relying on splicing rather than processing by the microprocessor complex to generate the RNAi pathway. The functions of miRNAs important for regulating fat deposition were also investigated in this study.ConclusionsThis study expands the number of fat-deposition-related miRNAs in pig. The results also indicate that castration can significantly affect the expression patterns of fat-related miRNAs. The differentially expressed miRNAs may play important roles in fat deposition after castration.

Polymorphism identification, RH mapping and association of placental lactogen gene with milk production traits of dairy cows.

Bovine placental lactogen (bPL) is structurally related to prolactin (PRL) and growth hormone (GH). In synergism with steroid and thyroid hormones, bPL is crucial in stimulating the development of the mammary gland, mammary cell differentiation and function. To further explore whether bPL gene is associated with milk production traits, we herein analyzed single-nucleotide polymorphisms (SNPs) within eight regions of bPL gene, which are potentially associated with five milk production traits on 1028 Chinese Holstein cows. Among these, two SNPs, NT7409(T-C) and Nt11246(G-A), were identified. The former is within exon 2; it induces an alteration of amino acid from Val to Ala. The later is within exon 4. It is a synonymous mutation. We found that there were significant associations between NT7409(T-C) and milk and protein yield. Cows of the AA genotype yielded less milk (P = 0.001) and less protein (P = 0.003) than those of genotypes AB and BB. However, on the NT11246(G-A) locus, no significant association was observed in the five milk production traits studied. In addition, bPL has been localized near markers RM185 and CC549051 with a distance of 23.2 cR on BTA 23. It is at the same position as the region including quantitative trait loci (QTLs) affecting milk and protein yields by previous linkage analysis. In summary, our findings demonstrated that the SNP within exon 2 of bPL (NT7409(T-C)) is associated with two milk production traits, and this provided further evidence that bPL could be a major gene-controlling milk production trait in Holstein dairy cattle.

Differential expression of CYB5A in Chinese and European pig breeds due to genetic variations in the promoter region

Cytochrome b5 (CYB5A) is an important electron transfer protein with homologues in a number of different organisms. In pigs, CYB5A is related to boar taint because of its role in androstenone biosynthesis. To determine the variety of CYB5A expression in pig breeds, genetic variations in the porcine CYB5A promoter region in both Chinese and European pig breeds were examined. Three single nucleotide polymorphisms (NC_010443.4:g.165901487delG, g.165901767T>C and g.165902078C>T) were identified in the porcine CYB5A promoter region. These SNPs occurred in different frequencies in Chinese and European pigs. Chinese pigs were primarily haplotype B (denoted as delG-C-T: the position of nt 165901487 of the CYB5 gene is a G deletion, nt 165901767 is C and nt 165902078 is T), except for Licha black pigs, which were primarily haplotype A (denoted as G-T-C: nt 165901487 is G, nt 165901767 is T and nt 165902078 is C), similar to European pigs. Quantitative PCR data from liver tissues demonstrated that haplotype B individuals had higher CYB5A expression than did those with haplotype A. This was confirmed by in vitro cell transfection assays, in which haplotype B individuals had higher reporter activity than did those with haplotype A. In silico analysis predicted that Myc-associated zinc-finger protein (MAZ) is a potential transcription factor at position 165901767. Electrophoretic mobility shift assays showed this polymorphism affects the stable binding of transcription factors to the CYB5A promoter, which in turn affects the expression levels of this gene. Therefore, this variation of the porcine CYB5A promoter region may explain the differences in androstenone accumulation between Chinese and European pig breeds and may also prove useful as a genetic marker to distinguish the origin of different pig breeds.

Differential expression of cyclin G2, cyclin-dependent kinase inhibitor 2C and peripheral myelin protein 22 genes during adipogenesis.

Increase of fat cells (FCs) in adipose tissue is attributed to proliferation of preadipocytes or immature adipocytes in the early stage, as well as adipogenic differentiation in the later stage of adipose development. Although both events are involved in the FC increase, they are contrary to each other, because the former requires cell cycle activity, whereas the latter requires cell cycle withdrawal. Therefore, appropriate regulation of cell cycle inhibition is critical to adipogenesis. In order to explore the important cell cycle inhibitors and study their expression in adipogenesis, we adopted a strategy combining the Gene Expression Omnibus (GEO) database available on the NCBI website and the results of quantitative real-time PCR (qPCR) data in porcine adipose tissue. Three cell cycle inhibitors - cyclin G2 (CCNG2), cyclin-dependent kinase inhibitor 2C (CDKN2C) and peripheral myelin protein (PMP22) - were selected for study because they are relatively highly expressed in adipose tissue compared with muscle, heart, lung, liver and kidney in humans and mice based on two GEO DataSets (GDS596 and GDS3142). In the latter analysis, they were found to be more highly expressed in differentiating/ed preadipocytes than in undifferentiated preadipocytes in human and mice as shown respectively by GDS2366 and GDS2743. In addition, GDS2659 also suggested increasing expression of the three cell cycle inhibitors during differentiation of 3T3-L1 cells. Further study with qPCR in Landrace pigs did not confirm the high expression of these genes in adipose tissue compared with other tissues in market-age pigs, but confirmed higher expression of these genes in FCs than in the stromal vascular fraction, as well as increasing expression of these genes during in vitro adipogenic differentiation and in vivo development of adipose tissue. Moreover, the relatively high expression of CCNG2 in adipose tissue of market-age pigs and increasing expression during development of adipose tissue was also confirmed at the protein level by western blot analysis. Based on the analysis of the GEO DataSets and results of qPCR and Western blotting we conclude that all three cell cycle inhibitors may inhibit adipocyte proliferation, but promote adipocyte differentiation and hold a differentiated state by inducing and maintaining cell cycle inhibition. Therefore, their expression in adipose tissue is positively correlated with age and mature FC number. By regulating the expression of these genes, we may be able to control FC number, and, thus, reduce excessive fat tissue in animals and humans.

Identification of CTLA2A, DEFB29, WFDC15B, SERPINA1F and MUP19 as Novel Tissue-Specific Secretory Factors in Mouse

Secretory factors in animals play an important role in communication between different cells, tissues and organs. Especially, the secretory factors with specific expression in one tissue may reflect important functions and unique status of that tissue in an organism. In this study, we identified potential tissue-specific secretory factors in the fat, muscle, heart, lung, kidney and liver in the mouse by analyzing microarray data from NCBI’s Gene Expression Omnibus (GEO) public repository and searching and predicting their subcellular location in GeneCards and WoLF PSORT, and then confirmed tissue-specific expression of the genes using semi-quantitative PCR reactions. With this approach, we confirmed 11 lung, 7 liver, 2 heart, 1 heart and muscle, 7 kidney and 2 adipose and liver-specific secretory factors. Among these genes, 1 lung-specific gene - CTLA2A (cytotoxic T lymphocyte-associated protein 2 alpha), 3 kidney-specific genes - SERPINA1F (serpin peptidase inhibitor, Clade A, member 1F), WFDC15B (WAP four-disulfide core domain 15B) and DEFB29 (defensin beta 29) and 1 liver-specific gene - MUP19 (major urinary protein 19) have not been reported as secretory factors. These genes were tagged with hemagglutinin at the 3’end and then transiently transfected to HEK293 cells. Through protein detection in cell lysate and media using Western blotting, we verified secretion of the 5 genes and predicted the potential pathways in which they may participate in the specific tissue through data analysis of GEO profiles. In addition, alternative splicing was detected in transcripts of CTLA2A and SERPINA1F and the corresponding proteins were found not to be secreted in cell culture media. Identification of novel secretory factors through the current study provides a new platform to explore novel secretory factors and a general direction for further study of these genes in the future.

Mild heat stress enhances differentiation and proliferation of Japanese quail myoblasts and enhances slow muscle fiber characteristics

The objective of this study was to investigate the effect of mild heat stress on muscle fiber hyperplastic and hypertrophic growth in quail primary myogenic cells to better understand the mechanisms leading to increased skeletal muscle development in avian embryos incubated at a higher temperature. Compared to control cultures maintained at 37°C, incubation at 39°C enhanced myotube length (P < 0.01) and diameter (P < 0.001) at 3 days after differentiation (D3). This enlargement of the myotubes incubated at 39°C can be explained by differences in the fusion index (56.7 vs. 46.2%, P < 0.05) and nuclei number per myotube (18.1 vs. 10.8, P < 0.001) compared to the control cells at D3. Additionally, a higher density of myotubes at D3 in cultures exposed to a higher temperature were related to higher levels of Pax-7 (P < 0.05) compared to the control cells incubated continuously at 37°C. These results indicated a higher proliferative capacity in cells exposed to mild heat stress compared to the control cells. On the other hand, mild heat stress enhanced protein levels of slow myosin heavy chain isoform (P < 0.01) and cytochrome c oxidase subunit IV (P < 0.01) compared to the control cells at D3. These discrepancies in protein expression indicated maintenance of slow muscle fiber type characteristics in myotubes incubated at 39°C. Our results suggest that mild heat stress plays a significant role in myogenic mechanisms related to muscle mass and development.

Differential expression of KRT83 regulated by the transcript factor CAP1 in Chinese Tan sheep

Keratin 83 (KRT83) is an important keratin protein in hair development. In this study, expression of KRT83 was compared among different tissues and between 1-month-old lambs and 48-month adult of Chinese Tan sheep, which showed different fleece phenotypes. The results showed that KRT83 was only expressed in skin, and KRT83 mRNA level in skin was significantly higher in Tan lambs than in adult sheep. To further understand the expression regulation of KRT83 by transcription factors in Tan sheep, amplified sequences coving different ranges of KRT83 promoter region were inserted into a pGL3-basic vector and then transfected into sheep primary fibroblast cells. Luciferase assay indicated that the sequence from -218bp to -10bp in the KRT83 promoter induced the highest transcription activity of the vector in the fibroblast cells. Transcription factor adenylate cyclase-associated protein 1 (CAP1) was predicted by online tools within this region. Electrophoretic mobility shift assay (EMSA) confirmed binding of the purified CAP1 protein to the target core region from -88bp to -10bp, because mutation in the target core sequence resulted in failure of CAP1 binding to the target region. Moreover, overexpression of CAP1 protein led to repression of the KRT83 promoter activity in sheep primary fibroblast cells, and expression of CAP1 was lower in lambs than in adult sheep. Therefore, we concluded that CAP1 is a key transcription factor involved in negative regulation of KRT83 expression in Tan sheep skin. Our study provides new insights into the transcriptional regulation of KRT83 and further hints of its critical role in curly hair phenotype in sheep.

Integrated miRNA-mRNA analysis reveals regulatory pathways underlying the curly fleece trait in Chinese tan sheep

BackgroundTan sheep is an indigenous Chinese breed well known for its beautiful curly fleece. One prominent breed characteristic of this sheep breed is that the degree of curliness differs markedly between lambs and adults, but the molecular mechanisms regulating the shift are still not well understood. In this study, we identified 49 differentially expressed (DE) microRNAs (miRNAs) between Tan sheep at the two stages through miRNA-seq, and combined the data with that in our earlier Suppression Subtractive Hybridization cDNA (SSH) library study to elucidate the mechanisms underlying curly fleece formation.ResultsThirty-six potential miRNA-mRNA target pairs were identified using computational methods, including 25 DE miRNAs and 10 DE genes involved in the MAPK signaling pathway, steroid biosynthesis and metabolic pathways. With the differential expressions between lambs and adults confirmed by qRT-PCR, some miRNAs were already annotated in the genome, but some were novel miRNAs. Inhibition of KRT83 expression by miR-432 was confirmed by both gene knockdown with siRNA and overexpression, which was consistent with the miRNAs and targets prediction results.ConclusionOur study represents the comprehensive analysis of mRNA and miRNA in Tan sheep and offers detailed insight into the development of curly fleece as well as the potential mechanisms controlling curly hair formation in humans.

Distinct cardiac responses to heat stress between two broiler lines identified by transcriptome analysis

We appreciate the work of Shurnevia Strickland in Dr. Schmidt’s lab at University of Delaware for constructing the transcriptome libraries. This work is supported by Agriculture and Food Research Initiative Foundational Award from the United States Department of Agriculture National Institute of Food and Agriculture (Award Number: 2011-6700330228) and Hatch project number #5358. We also thank the FAANG Awards to support travel fee for JZ to this meeting. Acknowledgements References Conclusions 0 20 40 60 80 100 120 140 160 180 200

RNA-seq analysis reveals distinct splenic response of two chickens lines to Newcastle disease virus

Leghorn 2 dpi Leghorn 6 dpi Jibin Zhang1, Michael G. Kaiser, Melissa S. Herrmann 1, Rodrigo A. Gallardo2, David A. Bunn3, Huaijun Zhou3, Susan J Lamont1 1 Department of Animal Science, Iowa State University, Ames, IA 50010 USA 2 Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616 3 Department of Animal Science, University of California, Davis, CA 95616