Yunliang Jiang

Identification of miRNAs associated with sexual maturity in chicken ovary by Illumina small RNA deep sequencing

BackgroundMicroRNAs have been suggested to play important roles in the regulation of gene expression in various biological processes. To investigate the function of miRNAs in chicken ovarian development and folliculogenesis, two small RNA libraries constructed from sexually mature (162-day old) and immature (42-day old) ovary tissues of Single Comb White Leghorn chicken were sequenced using Illumina small RNA deep sequencing.ResultsIn the present study, 14,545,100 and 14,774,864 clean reads were obtained from sexually mature (162-d) and sexually immature (42-d) ovaries, respectively. In total, 202 known miRNAs were identified, and 93 of them were found to be significantly differentially expressed: 42 miRNAs were up-regulated and 51 miRNAs were down-regulated in the mature ovary compared to the immature ovary. Among the up-regulated miRNAs, gga-miR-1a has the largest fold-change (6.405-fold), while gga-miR-375 has the largest fold-change (11.345-fold) among the down-regulated miRNAs. The three most abundant miRNAs in the chicken ovary are gga-miR-10a, gga-let-7 and gga-miR-21. Five differentially expressed miRNAs (gga-miR-1a, 21, 26a, 137 and 375) were validated by real-time quantitative RT-PCR (qRT-PCR). Furthermore, the expression patterns of the five miRNAs were analyzed in different developmental stages of chicken ovary and follicles of various sizes.ConclusionThe present study provides the first miRNA profile in sexually immature and mature chicken ovaries. Some miRNAs such as gga-miR-1a and gga-miR-21are expressed differentially in immature and mature chicken ovaries as well as among different sized follicles, suggesting an important role in the follicular growth or ovulation mechanism in the chicken.

Effect of dietary betaine supplementation on lipogenesis gene expression and CpG methylation of lipoprotein lipase gene in broilers

Experiments were conducted to investigate the effect of betaine supplementation on mRNA expression levels of lipogenesis genes and CpG methylation of lipoprotein lipase gene (LPL) in broilers. From 22xa0days of age, 78 broilers were feed basal diet without betaine and basal diet supplemented with 0.1% betaine, respectively, and at 56 and 66xa0days of age, the traits of 15 chickens (7 males and 8 females) of each group were recorded and abdominal fat pads were collected. The mRNA expression levels of several lipogenesis gene were analyzed by semi-quantitative RT-PCR and real-time quantitative RT-PCR (qPCR), respectively. The CpG methylation profile at the promoter region of LPL gene in 66-day-old broilers was determined by bisulfite sequencing. The average daily gain and percent abdominal fat traits were slightly improved in 56-day-old and 66-day-old broilers after dietary supplementation of betaine to diet. After adding 0.1% betaine to diet, the mRNA levels of fatty acid synthase (FAS) and adipocyte-type fatty acid-binding protein genes in abdominal adipose were significantly decreased in 56-day-old broilers, and those of LPL and FAS genes in abdominal adipose were significantly decreased in 66-day-old broilers comparing with the control group (Pxa0<xa00.05 and Pxa0<xa00.001). Moreover, in 66-day-old broilers fed 0.1% betaine diet, a different CpG methylation pattern was observed: the CpG dinucleotides of 1st, 6th, 7th, 8th and from 10th to 50th were less methylated; however, those of 2nd, 5th and 9th were more heavily methylated. The results suggest that transcription of some lipogenesis genes was decreased by betaine supplementation and betaine may decrease LPL mRNA expression by altering CpG methylation pattern on LPL promoter region.

Genome-Wide Gene Expression Profiles in Lung Tissues of Pig Breeds Differing in Resistance to Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is an infectious disease characterized by severe reproductive deficiency in pregnant sows, typical respiratory symptoms in piglets, and high mortality rate of piglets. In this study, we employed an Affymetrix microarray chip to compare the gene expression profiles of lung tissue samples from Dapulian (DPL) pigs (a Chinese indigenous pig breed) and Duroc×Landrace×Yorkshire (DLY) pigs after infection with PRRSV. During infection with PRRSV, the DLY pigs exhibited a range of clinical features that typify the disease, whereas the DPL pigs showed only mild signs of the disease. Overall, the DPL group had a lower percentage of CD4+ cells and lower CD4+/CD8+ratios than the DLY group (p<0.05). For both IL-10 and TNF-α, the DLY pigs had significantly higher levels than the DPL pigs (p<0.01). The DLY pigs have lower serum IFN-γ levels than the DPL pigs (p<0.01). The serum IgG levels increased slightly from 0 dpi to 7 dpi, and peaked at 14 dpi (p<0.0001). Microarray data analysis revealed 16 differentially expressed (DE) genes in the lung tissue samples from the DLY and DPL pigs (q≤5%), of which LOC100516029 and LOC100523005 were up-regulated in the PRRSV-infected DPL pigs, while the other 14 genes were down-regulated in the PRRSV-infected DPL pigs compared with the PRRSV-infected DLY pigs. The mRNA expression levels of 10 out of the 16 DE genes were validated by real-time quantitative RT-PCR and their fold change was consistent with the result of microarray data analysis. We further analyzed the mRNA expression level of 8 differentially expressed genes between the DPL and DLY pigs for both uninfected and infected groups, and found that TF and USP18 genes were important in underlying porcine resistance or susceptibility to PRRSV.

Different expression patterns of PRRSV mediator genes in the lung tissues of PRRSV resistant and susceptible pigs.

Porcine reproductive and respiratory syndrome (PRRS) has caused severe economic loss in most swine-producing countries. The resistance to PRRS virus (PRRSV) infection varies among pig breeds and lines. In this study, we found that the Chinese Dapulian pigs (DPL) were more resistant to PRRSV than commercial Duroc×Landrace×Yorkshire (DLY) crossbred pigs in that lower rectal temperature and lower PRRSV copy number in the serum were detected in the former. Analysis of the mRNA expression of five PRRSV mediator genes (SIGLEC1, NMMHC-IIA, CD163, VIM and HSPG2) in the lung tissues indicated differences in expression between DLY and DPL pigs. In uninfected porcine lung tissues, the levels of SIGLEC1, NMMHC-IIA, CD163 and VIM genes were significantly higher in DLY than in DPL pigs (P<0.05); in PRRSV-infected pigs, the expression levels of NMMHC-IIA and CD163 mRNA were significantly higher in DPL pigs compared to uninfected ones (P<0.05), whereas these levels were not different in DLY pigs or between infected DPL and DLY pigs. Thus, the different expression of PRRSV mediator genes is likely related to pig resistance to PRRSV.

Identification of differentially expressed genes in ovaries of chicken attaining sexual maturity at different ages

In poultry as well as in other birds, sexual maturity is one of the important factors influencing female reproduction and egg production. In this study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) differential display approach was used to identify genes related to sexual maturity. Using 54 EcoR I/Mse I selective primer combinations, totally 403 differentially expressed transcript-derived fragments (TDFs) were isolated, 27 of which belong to 25 unigenes. By real-time quantitative PCR (qPCR), the expression pattern of 13 genes was confirmed; among them, four genes including ZNF183 (Pxa0<xa00.01), KIAA0700, CCT6A, and 23e 15 (Pxa0<xa00.05) are significantly up-regulated and one gene (Loc418883) is significantly down-regulated (Pxa0<xa00.01) in sexually mature ovaries compared to immature ones. The mRNA expression dynamics of ZNF183, CCT6A, 23e 15 and Loc418883 were further investigated in ovaries of 70-, 300- and 500-day-old commercial egg-laying hens: the expression level of CCT6A was the highest in 300-day-old hens (Pxa0<xa00.05), while that of Loc418883 in 500-day-old hens was significantly higher than the other two stages (Pxa0<xa00.01). The expression levels of ZNF183 and 23e 15 in ovary increase significantly from 70-day-old hens (Pxa0<xa00.01) and 300-day-old (Pxa0<xa00.05) to 500-day-old hens, respectively. The consistence of CCT6A expression and egg-laying performance suggests that CCT6A likely plays important role in sexual maturity in hens.

Molecular characterization and identification of a novel polymorphism of 200 bp indel associated with age at first egg of the promoter region in chicken follicle-stimulating hormone receptor (FSHR) gene

Follicle-stimulating hormone receptor (FSHR) plays an important role in animal follicular development. Polymorphisms in FSHR promoter region likely impact transcription and follicle growth and maturation. In this study, a fragment of ~1.9xa0kb of cFSHR promoter for Zang, Xianju, Lohmann Brown, Jining Bairi and Wenchang breeds (line) was obtained. Totally 49 variations were revealed, of which 39 are single nucleotide substitutions, one is nucleotide substitution of (TTG) to (CAC) and nine are indels. Polymorphism at −874 site (a 200xa0bp indel mutation) was identified, and their effects on egg production traits as well as gene expression were analyzed. At this site, allele I+ was dominant in Lohmann Brown and Xinyang Brown (a synthetic egg-laying line) lines, but very rare in three Chinese indigenous chicken breeds, namely Jining Bairi, Wenchang, Zang and one synthetic boiler line (Luqin). In Xinyang Brown population, the polymorphism was associated with age at first egg (AFE) (Pxa0<xa00.05) and its effect on egg number at 37xa0weeks of age (E37) and egg number at 57xa0weeks of age (E57) was not significantly different (Pxa0>xa00.05). The cFSHR mRNA level was not significantly different between three genotypes in small white and small yellow follicles of Xinyang Brown hens, however, allele I+ tends to increase cFSHR transcription.

Folate Deficiency during Early-Mid Pregnancy Affects the Skeletal Muscle Transcriptome of Piglets from a Reciprocal Cross

Folate deficiency (FD) during pregnancy can cause fetal intrauterine growth restriction in pigs, of which the skeletal dysplasia is a major manifestation. Factors influencing muscle development are very important in the formation of porcine meat quality trait. However, the effect of folate deficiency on skeletal muscle development and its molecular mechanisms are unknown. The objective of this study is to determine the effect of maternal folate deficiency on the skeletal muscle transcriptome of piglets from a reciprocal cross, in which full-sibling Landrace (LR) and full-sibling Chinese local breed Laiwu (LW) pigs were used for reciprocal cross matings, and sows were fed either a folate deficient or a normal diet during early-mid gestation. In addition, the difference in the responsiveness of the piglets to folate deficiency during early-mid pregnancy between reciprocal cross groups was investigated. Longissimus dorsi (LD) muscle samples were collected from newborn piglets and a 4 × 44K Agilent porcine oligo microarray was used for transcriptome analysis of porcine LD muscle. The results showed that folate deficiency during early-mid pregnancy affected piglet body weight, LD muscle fiber number and content of intramuscular triglyceride. The microarray results indicated that 3154 genes were differentially expressed between folate deficient and normal piglets from the LR♂ × LW♀ cross, and 3885 differentially expressed genes (DEGs) in the ones from the LW♂ × LR♀ cross. From functional analyses, sow folate deficiency affected almost all biological processes in the progeny. Lipid metabolism-related genes and associated metabolic pathways were regulated extensively by folate deficiency, especially in LR♂ × LW♀ cross piglets. Most of the genes that are regulated by folate deficiency in the LD muscle of piglets were different between LR♂ × LW♀ and LW♂ × LR♀ crosses, suggesting some epigenetic effects of FD exist in genes underlying myogenesis and intramuscular fat deposition in piglets.

Dynamic Changes in the Follicular Transcriptome and Promoter DNA Methylation Pattern of Steroidogenic Genes in Chicken Follicles throughout the Ovulation Cycle

The molecular mechanisms associated with follicle maturation and ovulation are not well defined in avian species. In this study, we used RNA-seq to study the gene expression profiles of the chicken follicles from different developmental stages (pre-hierarchical, pre-ovulatory and post-ovulatory). Transcriptomic analysis revealed a total of 1,277 and 2,310 genes were differentially expressed when follicles progressed through the pre-hierarchical to hierarchical and pre-ovulatory to post-ovulatory transitions, respectively. The differentially expressed genes (DEG) were involved in signaling pathways such as adherens junction, apoptosis and steroid biosynthesis. We further investigated the transcriptional regulation of follicular steroidogenesis by examining the follicle-specific methylation profiles of Star (steroidogenic acute regulatory protein), Cyp11a1 (cytochrome P450, family 11, subfamily a, polypeptide 1) and Hsd3b (hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1), genes encoding the key enzymes for progesterone synthesis. The varied patterns of DNA methylation in proximal promoters of Star and Cyp11a1but not Hsd3b in different follicles could play a major role in controlling gene expression as well as follicular steroidogenic activity. Finally, the promoter-reporter analysis suggests that TGF-β could be involved in the regulation of Hsd3b expression during ovulation. Together, current data not only provide novel insights into the molecular mechanisms of follicular physiology in chicken follicles, but also present the first evidence of epigenetic regulation of ovarian steroidogenesis in avian species.

Identification of a short interspersed repetitive element insertion polymorphism in the porcine MX1 promoter associated with resistance to porcine reproductive and respiratory syndrome virus infection

The myxovirus resistance (Mx) proteins belong to the dynamin superfamily and are important for innate host defence against RNA viruses. In this study, we demonstrate that positive elements are present in the two promoter regions of -2713 to -2565 and -688 to -431 in the porcine MX1 gene. Sequencing and alignment of the amplified porcine MX1 gene promoter region identified a short interspersed repetitive element (SINE) insertion of 275 bp at site -547. At this site, allele B (an insertion of 275 bp) is dominant in Chinese indigenous pig breeds but has a workable minor allele frequency in western lean-type pig breeds. Luciferase activity was compared between promoters with and without the insertion of the 275-bp fragment in transiently transfected MARC-145 cells. The insertion of the 275-bp fragment increased the luciferase activity significantly (P < 0.05) both prior to and post-porcine reproductive and respiratory syndrome (PRRS) virus inoculation. These results suggest that the SINE insertion polymorphism at site -547 of the MX1 gene promoter region is a potential DNA marker for PRRS resistance in pigs.

Identification of a single nucleotide promoter polymorphism regulating the transcription of ubiquitin specific protease 18 gene related to the resistance to porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome (PRRS), characterized by reproductive failure in sows and respiratory disease and mortality in piglets, is a major infectious disease that causes great economic loss throughout the world. Previous studies revealed that the overexpression of porcine ubiquitin specific protease 18 (USP18) gene inhibits PRRSV replication in vitro. The objective of this study is to compare the promoter activity of USP18 in Chinese indigenous Dapulian (DPL) pigs and Duroc×Landrace×Yorkshire (DLY) commercial pigs and screen single nucleotide polymorphism (SNP) affecting porcine USP18 transcription. We found that the promoter activity was significantly higher in DPL pigs than DLY commercial pigs (p<0.05), deletion of the promoter from -1790 to -1314bp decreased the transcriptional activity by roughly 60% (p<0.05) and a SNP G-1533A in this region increased the mRNA expression both prior to and post PRRSV infection in MARC-145 cells. Population genetics analysis showed that allele A was only detected in Chinese pig breeds which are generally resistant to PRRSV. These results suggest that the SNP G-1533A polymorphism in the promoter region of porcine USP18 gene is a potential DNA marker for the resistance to PRRSV.