Chunyan Lu

A comparative profile of the microRNA transcriptome in immature and mature porcine testes using Solexa deep sequencing.

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that play key roles in many diverse biological processes such as spermatogenesis. However, no study has been performed on the miRNA transcriptome of developing porcine testes. Here, we employed Solexa deep sequencing technology to extend the repertoire of porcine testis miRNAs and extensively compare the expression patterns of sexually immature and mature porcine testes. Solexa sequencing of two small RNA libraries derived from immature (30 days) and mature (180 days) pig testis samples yielded over 25 million high‐quality reads. Overall, the two developmental stages had significantly different small RNA compositions. A custom data analysis pipeline identified 398 known and/or homologous conserved porcine miRNAs, 15 novel pig‐specific miRNAs and 56 novel candidate miRNAs. We further observed multiple mature miRNA variants and identified a new bidirectional transcribed miRNA locus, ssc‐mir‐181a. A total of 122 miRNAs were differentially expressed in the immature and mature testes, and 10 were validated using quantitative RT‐PCR. Furthermore, GO and KEGG pathway analyses of the predicted miRNA targets further illustrate the likely roles for these differentially expressed miRNAs in spermatogenesis. This study is the first comparative profile of the miRNA transcriptome in immature and mature porcine testes using a deep sequencing approach, and it provides a useful resource for future studies on the role of miRNAs in spermatogenesis and male infertility treatment.

Deep Sequencing and Screening of Differentially Expressed MicroRNAs Related to Milk Fat Metabolism in Bovine Primary Mammary Epithelial Cells

Milk fat is a key factor affecting milk quality and is also a major trait targeted in dairy cow breeding. To determine how the synthesis and the metabolism of lipids in bovine milk is regulated at the miRNA level, primary mammary epithelial cells (pMEC) derived from two Chinese Holstein dairy cows that produced extreme differences in milk fat percentage were cultured by the method of tissue nubbles culture. Small RNA libraries were constructed from each of the two pMEC groups, and Solexa sequencing and bioinformatics analysis were then used to determine the abundance of miRNAs and their differential expression pattern between pMECs. Target genes and functional prediction of differentially expressed miRNAs by Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes analysis illustrated their roles in milk fat metabolism. Results show that a total of 292 known miRNAs and 116 novel miRNAs were detected in both pMECs. Identification of known and novel miRNA candidates demonstrated the feasibility and sensitivity of sequencing at the cellular level. Additionally, 97 miRNAs were significantly differentially expressed between the pMECs. Finally, three miRNAs including bta-miR-33a, bta-miR-152 and bta-miR-224 whose predicted target genes were annotated to the pathway of lipid metabolism were screened and verified by real-time qPCR and Western-blotting experiments. This study is the first comparative profiling of the miRNA transcriptome in pMECs that produce different milk fat content.

MicroRNAs in the immune organs of chickens and ducks indicate divergence of immunity against H5N1 avian influenza

Chickens are susceptible to the highly pathogenic H5N1 strain of avian influenza virus (HPAIV), whereas ducks are not. Here, we used high‐throughput sequencing to analyse the microRNA expression in the spleen, thymus and bursa of Fabricius of H5N1‐HPAIV‐infected and non‐infected chickens and ducks. We annotated the genomic positions of duck microRNAs and we compared the microRNA repertoires of chickens and ducks. Our results showed that the microRNA expression patterns in the homologous immune organs of specific‐pathogen‐free (SPF) chickens and ducks diverge substantially. Moreover, there was larger divergence between the microRNA expression patterns in immune organs of HPAIV‐infected chickens than HPAIV‐infected ducks. Together, our results might help to elucidate the roles of microRNAs in the divergent immunity of chickens and ducks against H5N1 HPAIV.

Differential Expression of PPARγ, FASN, and ACADM Genes in Various Adipose Tissues and Longissimus dorsi Muscle from Yanbian Yellow Cattle and Yan Yellow Cattle

The objective of this study was to investigate the correlation between cattle breeds and deposit of adipose tissues in different positions and the gene expressions of peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FASN), and Acyl-CoA dehydrogenase (ACADM), which are associated with lipid metabolism and are valuable for understanding the physiology in fat depot and meat quality. Yanbian yellow cattle and Yan yellow cattle reared under the same conditions display different fat proportions in the carcass. To understand this difference, the expression of PPARγ, FASN, and ACADM in different adipose tissues and longissimus dorsi muscle (LD) in these two breeds were analyzed using the Real-time quantitative polymerase chain reaction method (qRT-PCR). The result showed that PPARγ gene expression was significantly higher in adipose tissue than in LD in both breeds. PPARγ expression was also higher in abdominal fat, in perirenal fat than in the subcutaneous fat (p<0.05) in Yanbian yellow cattle, and was significantly higher in subcutaneous fat in Yan yellow cattle than that in Yanbian yellow cattle. On the other hand, FASN mRNA expression levels in subcutaneous fat and abdominal fat in Yan yellow cattle were significantly higher than that in Yanbian yellow cattle. Interestingly, ACADM gene shows greater fold changes in LD than in adipose tissues in Yan yellow cattle. Furthermore, the expressions of these three genes in lung, colon, kidney, liver and heart of Yanbian yellow cattle and Yan yellow cattle were also investigated. The results showed that the highest expression levels of PPARγ and FASN genes were detected in the lung in both breeds. The expression of ACADM gene in kidney and liver were higher than that in other organs in Yanbian yellow cattle, the comparison was not statistically significant in Yan yellow cattle.

The effect of leader peptide mutations on the biological function of bovine myostatin gene.

The growth of muscle fibers can be negatively regulated by bovine myostatin. The first two exons of myostatin gene code for the N-propeptide and its third exon codes for the C-polypeptide. Myostatin is secreted as a latent configuration formed by dimerization of two matured C peptides non-covalently linked with the N terminal pro-peptide. Pro-peptide has two distinct functions in guiding protein folding and regulating biological activity of myostatin. When the structure of the leader peptide is altered via mutations resulting in more tight binding with the mature peptide, myostatin function is inhibited, resulting in the changes of P21 and CDK2 expression levels which are related to the regulation of cell cycle. In the present study, the coding region of bMSTN (bovine myostatin) gene was amplified and mutated (A224C and G938A) through fusion PCR, and the mutated bMSTN gene (bMSTN-mut) was inserted in frame into the pEF1a-IRES-DsRed-Express2 vector and transfected into bovine fibroblast cells. The expression levels of bMSTN-mut, P21 and CDK2 (cyclin dependent kinase 2) were examined with qPCR and Western-blotting. Changes in cell cycle after transfection were also analyzed with flow cytometry. The results indicated that leader peptide mutation resulted in down-regulation of P21 expression levels and up-regulation of CDK2 expression levels. The flow cytometry results showed that the proportion of cells in the G0/G1-phase was lower and that of cells in the S-phase was higher in bMSTN-mut transfected group than that in the control group. The proliferation rate of bMSTN-mut transfected cells was also significantly higher than that of the control cells. In conclusion, the studies have shown that the pEF1a-IRES-DsRed-Express2-bMSTN-mut recombinant plasmid could effectively promote the proliferation of bovine fibroblast cells. The site-directed mutagenesis of bMSTN gene leader peptide and in vitro expression in bovine fibroblast cells could be helpful to further the studies of bMSTN in regulating bovine muscle cell growth and development.

RNA interference-mediated knockdown of DGAT1 decreases triglyceride content of bovine mammary epithelial cell line.

Diacylglyceroltransferase-1 (DGAT1) expresses in nearly all tissues, including the mammary gland. Mice lacking DGAT1 exhibit decreased triglyceride content in mammary tissue, and are resistant to diet-induced obesity and diabetes mellitus. Thus, DGAT1 has received considerable attention. In the present study, the function of DGAT1 was examined by liposome mediated RNA interference (RNAi) to knockdown the expression of endogenous DGAT1 expression in bovine mammary epithelial cells (BMEC) and the changes of the biological functions of cells were analyzed. The mRNA and protein levels, intracellular triglyceride (TG) content, and total protein of BMECs were analyzed by real-time PCR, Western blot, TG kit, and ultraviolet spectrophotometer, respectively, before and after RNAi treatment. The results indicated that knockdown of DGAT1 expression significantly reduced TG content in BMECs. This study further confirmed the importance of DGAT1 in triglyceride synthesis in bovine mammary tissue.

Construction of fat1 Gene Expression Vector and Its Catalysis Efficiency in Bovine Fetal Fibroblast Cells

The FAT-1 protein is an n-3 fatty acid desaturase, which can recognize a range of 18- and 20-carbon n-6 substrates and transform n-6 polyunsaturated fatty acids (PUFAs) into n-3 PUFAs while n-3 PUFAs have beneficial effect on human health. Fat1 gene is the coding sequence from Caenorhabditis elegans which might play an important role on lipometabolism. To reveal the function of fat1 gene in bovine fetal fibroblast cells and gain the best cell nuclear donor for transgenic bovines, the codon of fat1 sequence was optimized based on the codon usage frequency preference of bovine muscle protein, and directionally cloned into the eukaryotic expression vector pEF-GFP. After identifying by restrictive enzyme digests with AatII/XbaI and sequencing, the fusion plasmid pEF-GFP-fat1 was identified successfully. The pEF-GFP-fat1 vector was transfected into bovine fetal fibroblast cells mediated by Lipofectamine2000TM. The positive bovine fetal fibroblast cells were selected by G418 and detected by RT-PCR. The results showed that a 1,234 bp transcription was amplified by reverse transcription PCR and the positive transgenic fat1 cell line was successfully established. Then the expression level of fat1 gene in positive cells was detected using quantitative PCR, and the catalysis efficiency was detected by gas chromatography. The results demonstrated that the catalysis efficiency of fat1 was significantly high, which can improve the total PUFAs rich in EPA, DHA and DPA. Construction and expression of pEF-GFP-fat1 vector should be helpful for further understanding the mechanism of regulation of fat1 in vitro. It could also be the first step in the production of fat1 transgenic cattle.

Differential Expression of miR-34c and Its Predicted Target Genes in Testicular Tissue at Different Development Stages of Swine

To verified the target genes of miR-34c, bioinformatics software was used to predict the targets of miR-34c. Three possible target genes of miR-34c related to spermatogenesis and male reproductive development: zinc finger protein 148 (ZNF148), kruppel-like factor 4 (KLF4), and platelet-derived growth factor receptor alpha (PDGFRA) were predicted. Then, the expression of miR-34c and its target genes were detected in swine testicular tissue at different developmental stages by quantitative polymerase chain reaction. The results suggested that the expression of PDGFRA has the highest negative correlation with miR-34c. Then immunohistochemical staining was done to observe the morphology of swine testicular tissue at 2-days and 3, 4, 5-months of age, which indicated that PDGFRA was mainly expressed in the support cells near the basement membrane during the early development stages of testicular tissue, but that the expression of PDGFRA was gradually reduced in later stages. Therefore, western blot analyzed that the highest expression of PDGFRA was generated in 2-days old testicular tissues and the expression levels reduced at 3 and 4-months old, which correlated with the results of immunohistochemical staining. In conclusion, PDGFRA is a target gene of miR-34c.

Establishment and comparison of three different codon optimization of fat1 gene in transgenic mammary epithelial cell lines

Dactylellina cionopaga is a trapping fungus that produces adhesive columns and a two-dimensional network. The factors that influence protoplast preparation and regeneration of D. cionopaga were analyzed, and poly ethylene glycol (PEG)-CaCl 2 - or Agrobacterium tumefaciens -mediated transformation was conducted to develop a transformation system for the fungus and provide a tool for studying the function of nematode infection-related genes. The results indicate that between 4.175±1.025×10 6 and 3.08±1.4×10 7 , protoplasts/ml were obtained under optimized conditions and that the protoplasts could be regenerated on potato dextrose agar (PDA), RA and IM regeneration media. D. cionopaga transformation using PEG-CaCl2 or A. tumefaciens displayed 4.2 to 11 resistant colonies/μg DNA using 10 6 protoplasts and 180-270 resistant colonies using 10 6 conidia. Molecular analysis and microscopy of randomly selected transformants showed that the target genes were integrated into the genome of D. cionopaga and that green fluorescence could be detected in transformants containing pK2-BarGFP, which carried a glufosinate ammonium resistance gene and the enhanced green fluorescence protein gene. The methods used in this study for protoplast preparation and convenient Agrobacterium -mediated transformation of D. cionopaga represent useful tools for genetic research on this nematophagous fungus. This is the first report on protoplast generation and transformation of D. cionopaga. Key words: Nematophagous fungi, Dactylellina cionopaga, Agrobacterium tumefaciens- mediated transformation, PEG-CaCl2-mediated transformation, protoplast preparation and regeneration.