Ailing Zhang

Differential gene expression in the endometrium on gestation day 12 provides insight into sow prolificacy

BackgroundErhualian pigs, one of Chinese Taihu pig breeds, are known to have the largest recorded litter size in the world. A lower prenatal death rate is the major contributing factor to the prolificacy of Taihu pigs. Cross-breeding experiments have demonstrated that Taihu sows exhibit a strong maternal effect and that their large litter sizes are mainly caused by maternal genes. The growth and development of porcine embryos on gestation day (GD) 12 are dependent on histotroph secreted by endometrium. Embryonic loss of Taihu pigs on GD12 is lower than that of Western pigs. Here, endometrial samples were collected from pregnant Erhualian sows (parity 3) and Landrace × Large White (LL) sows (parity 3) on GD12. Digital gene expression profiling (DGE) was used to measure the gene expression in the endometrium of the two breeds.ResultsA total of 13,612 genes were differentially expressed between the two breeds (P < 0.001, FDR < 0.001). Gene Ontology (GO) analysis showed that the differential genes involved in reproduction and growth. Pathway analysis revealed that the differentially expressed genes significantly enriched in 24 KEGG pathways. Quantitative real-time RT-PCR confirmed the differential expression of eight genes. Analyses of the differentially expressed genes suggested possible reasons for the difference in embryonic survival ratio between the two breeds. Specifically, these findings point to a higher ratio of PGE2:PGF2α in the endometrium of Erhualian pigs, which facilitates the establishment and maintenance of pregnancy. We also suggest that the differences in the uterine environment lead to higher uterine capacity in Erhualian pigs.ConclusionsThe DGE expression profiles of Erhualian and LL endometrium demonstrated differential expression of genes. Our results will increase understanding of the molecular mechanisms of the low rate of embryonic loss in Chinese Taihu pigs, facilitate the identification of major genes that affect litter size, and be valuable for porcine transcriptomic studies.

Selection of reference genes for studies of porcine endometrial gene expression on gestational day 12.

Comparing gene expression patterns in the endometrium on gestational day 12 (GD12) between Erhualian (ER) and Landrace×Large White (LL) pigs is helpful to understand the biological mechanisms of fecundity. Selecting genes that have stable expression levels as the internal standards in a comparative study is essential for identifying real gene-specific variation by quantitative RT-PCR (qRT-PCR). Five genes expressed in sow endometria on GD12 were evaluated for their suitability as internal control for relative quantification by qRT-PCR. These genes were beta-actin (ACTB), beta-2-microglobulin (B2M), phosphoglycerate kinase 1 (PGK1), RNA polymerase II polypeptide G (RPG), and ribosomal protein S20 (RPS20), which represent different functional classes. Our results indicated that ACTB, B2M, and PGK1 were not suitable as internal standards for normalization because of their huge variability between the two breeds. RPS20 and RPG were most stable, and the former is recommended to serve as the internal standard when the use of multiple housekeeping genes is unpractical.

Association analysis between variants in bovine NPY gene and growth traits in Nanyang cattle (Bos tarus)

Neuropeptide Y (NPY) is one of the most potent orexigenic factors that is implicated in the control of feeding behavior and energy homeostasis. Four Chinese indigenous cattle breeds (n=338) were used to detect single nucleotide polymorphisms (SNPs) in the coding regions with their intron-exon boundaries and the proximal flanking regions of NPY gene by PCR-SSCP and DNA sequencing. Five novel SNPs (38017 C>G, 34240 C>A, 34168 G>A, 32463 A>C and 32302 C>G) were identified. Polymorphisms of P3 lous in NPY gene, containing SNP4 and SNP5, were associated with body length and chest girth in Nanyang cattle aged 6, 12 and 18 months (P<0.05), but they had no significant effect on the two growth traits in Nanyang cattle aged 24 months (P>0.05). The results open a new field of study and extend the spectrum of genetic variation of the bovine NPY gene, which might contribute to cattle genetic resources and breeding.

An efficient and rapid method for gene cloning from eukaryotic genomic DNA using overlap-PCR: With an example of cattle Ghrelin gene

In this study, overlap-PCR, an efficient and rapid method, was used to clone cattle Ghrelin gene CDS (coding sequence) from genomic DNA. The procedure included seven primers and three-step PCRs. Cattle Ghrelin gene consists of four exons and the CDS contains 351 bps. In the first step three PCRs were performed to generate extended exon1, exon2, and exon3 that contained overlapped nucleotides and were used as the templates for second ligation PCR. Secondly, exon1 and exon2 were spliced together. And it was same with exon3 and exon4. Lastly, the four exons were linked together with outermost primers and the templates from the second step. Comparison analysis on the obtained CDS of Ghrelin gene and cDNA by RT-PCR showed that the two sequences were same. As an efficient and rapid method, overlap-PCR is feasible and acceptable for gene cloning from genomic DNA.

Association of polymorphisms in the Pit-1 intron 5 with body measurements in Chinese Cattle

The Pit-1 gene was studied as a candidate for genetic markers of growth traits in Chinese cattle in this study. The single-strand conformation polymorphism method was used to identify polymorphism in the Pit-1 intron 5. One single nucleotide polymorphism, Pit1I5, was found in intron 5. The frequencies of allele A of Nanyang cattle (NY), Qinchuan cattle (QC), Jiaxianhong cattle (JX), Luxi cattle (LX) and Holstein cow (H) populations were 0.444, 0.477, 0.538, 0.523 and 0.475, respectively. Associations of the polymorphisms with growth traits in Nanyang cattle were analyzed using a general linear model procedure. The following parameters were greater in individuals with AA genotype than with AB genotype: body weight, average day gain, body height, chest girth at six, 12, 18 and 24 months (P allele B in other age groups. Therefore, genotype AA maybe a dominant genotype and allele A may be a dominant allele. These results imply that allele A of Pit-1 gene may likely affect growth traits positively. Key words : Beef cattle, growth traits, Pit- 1 intron 5, polymorphism.

Single Nucleotide Polymorphism (SNP) Analysis of orexin Gene 5' Regulatory Region in Chinese Indigenous Cattle Populations

Abstract Orexin is an important neuropeptide that influences livestocks appetite and food intake and is closely related with livestocks growth and development. The variations in the orexin gene 5′ regulatory region might have an influence on the gene expression. Based on the hypothesis, five overlapped fragments including 1 794 bp of orexin gene 5′ regulatory region were investigated for single nucleotide polymorphisms (SNPs) by PCR-SSCP and DNA sequencing in three indigenous cattle populations. A total of five SSCP patterns observed revealed ten SNPs in the region. Two SSCP patterns genotypes (A and B) were exhibited in 0-2 fragment and three (A, B and C) were found in 0-5 fragment. 0-2 contained four SNPs, viz., −583 T>C, −479 OT, −474 A>T, and −451 A>G. In another lous 0-5, six SNPs were identified (-1 610 OG, −1 585 G>A, −1 550 T>C, −1 548 A>C, −1 438 OT, and −1 431 OA). Seven SNPs were found in transcription factor binding sites and four out of them existed in the core sequences. The SNPs at −479, −474 and −451 did not change the putative recognition core sequences of their factors. But the mutation at −583 changed the binding sequence of EVI1 into NFA and created one new binding site for ZFHX simultaneously. In three populations, the frequencies of A, B and C genotypes of 0-2 were 0.2367, 0.4842 and 0.2791, respectively. And the A pattern of 0-5 was preponderant (0.7549) and the other B pattern was not (0.2451). But the frequencies of different SSCP variants varied across three cattle populations. B genotype in 0-2 had significant associations to body weight (BW) and daily weight gain (DWG) in Nanyang cattle 6- and 12-mon aged and might serve as one potential candidate genetic marker for growth and development.