Sihuan Zhang

Identification of novel alternative splicing transcript and expression analysis of bovine TMEM95 gene.

Transmembrane protein 95 (TMEM95) is closely related to male reproductive performance in cattle, but does not affect semen quality. Alternative splicing plays an important role in regulating biological function as well as in generating proteomic and functional diversity in metazoan organisms. Thus, the aim of this study was to clone and identify transcripts of the TMEM95 gene in cattle using RT-PCR, characterize them via bioinformatics analysis, and detect their expression patterns using qRT-PCR. Two transcripts of TMEM95 were identified in cattle, including TMEM95-SV1 and TMEM95-SV2. Bioinformatics predicted that TMEM95-SV1 has a leucine-rich repeat C-terminal domain and a Pfam: IZUMO. These regions are closely related to protein interactions and the acrosome reaction, respectively. Interestingly, the two transcripts were exclusively expressed in the testes and brain in male fetus cattle, and TMEM95-SV1 was expressed in the brain at significantly higher levels than in the testis (P<0.05, 4.06-fold) and TMEM95-SV2 in the brain (P<0.05, 4.95-fold). These findings enrich the understanding of the TMEM95 gene function and benefit for enhancing male reproduction in cattle industry.

Bovine pituitary homeobox 2 (PITX2): mRNA expression profiles of different alternatively spliced variants and association analyses with growth traits

Pituitary homeobox 2 (PITX2) plays crucial roles in embryogenesis, ontogenesis, growth, and development via the Wnt/beta-catenin and POU1F1 pathways. To better understand the characteristics and genetic effects of the cattle PITX2 gene, we identified alternative PITX2 splicings, examined the effects of the spliced variants on mRNA expression levels in tissues, and then used association analyses to explore the relationships between a PITX2 deletion genetic variant and growth traits in 750 native Chinese cattle. An unreported spliced variant of PITX2, designated here as PITX2-V1, was identified in cattle using in silico cloning and RT-PCR. The entire coding sequence of PITX2 is 978 bp, encoding 325 amino acids, whereas that of PITX2-V1 is 357 bp encoding 118 amino acids. Cattle PITX2 exhibited both a perfect homeodomain and an OAR domain, but PITX2-V1 lacked the homeodomain. Analyses with qRT-PCR showed that the expression level of PITX2 in cattle testis was very low, and PITX2-V1 was only very slightly expressed in the brain and testis. Furthermore, a 24 bp deletion was detected within PITX2 intron, and the different genotypes were significantly associated with growth traits (e.g., body height, body length, heart girth) in four cattle breeds (P < 0.05). These results are of direct benefit to future cattle breeding, and provide new insights into the characteristics and functions of cattle PITX2 gene.

Goat Boule: Isoforms identification, mRNA expression in testis and functional study and promoter methylation profiles

A conserved gene in meiosis, the Boule gene is involved in meiosis and spermatogenesis. The deletion of this gene in males blocks meiosis and results in infertility. Alternative splicing variants of the Boule gene have been identified in humans, bovines, and bats, but in dairy goats remains unknown. This study was therefore to detect splicing variants of the goat Boule gene and explore their potential roles in meiosis. Three isoforms, denoted as Boule-a, Boule-b, and Boule-c, were identified in the testes of goats using real-time PCR (RT-PCR) and cloning sequencing. Compared to the normal Boule gene, Boule-a was found to lack exons 7 and 8, which corresponds to a predicted variant, X4, on the NCBI database. Boule-b lacked exon 8, and Boule-c only retained exons 1 and 2. Of these three variants, two were novel isoforms of the Boule gene. Quantitative RT-PCR (qRT-PCR) showed that the Boule-a and Boule-b expression patterns were significantly different between the adult goat testes and the postnatal testes of 42 and 56 days. Overexpression of Boule-a and Boule-c in GC-1 spg cells of model mice significantly repressed CDC2 expression. Bisulfite sequencing PCR (BSP) results showed that the promoter region of the Boule gene was hypermethylated in goat testes. A negative correlation between the methylation levels of the Boule gene promoter and total mRNA expression of its transcripts was found. Our data showed alternative splicing and promoter methylation in the goat Boule gene, suggesting that this gene may play an important role in the regulation of Boule expression and in meiosis processing.

Identification of a Novel Polymorphism in Bovine lncRNA ADNCR Gene and Its Association with Growth Traits

ABSTRACT Adipocyte differentiation-associated long noncoding RNA (ADNCR) is a newly discovered lncRNA. It plays function by targeting miR-204 to significantly regulates the expression of the target SIRT1 gene in preadipocytes both at the level of mRNA and protein, thereby inhibiting adipogenesis. The tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) strategy is fast and accuracy at a negligible cost for SNP genotyping in large samples. In the study, a novel SNP g.1263T>A in intron 1 of bovine ADNCR gene was found. Herein, the T-ARMS-PCR assay was applied to detect the genotypes of the novel SNP of bovine ADNCR gene in 1017 individuals from seven cattle breeds and validated the accuracy by DNA sequencing assay of ninety animals representing three different genotypes. The concordance between two different methods was 100%. The association analysis indicated that this locus was significantly associated with the body weight (P = 0.010), chest girth (P = 0.014) and rump length (P = 0.038) in Jinnan cattle, hucklebone width (P = 0.032) in Qinchuan cattle, the cannon circumference (P = 0.019) in Jinjiang cattle, respectively. These novel findings may be used for marker-assisted selection (MAS) and contribute to the performance of beef cattle in the future.

Reduced representation bisulfite sequencing (RRBS) of dairy goat mammary glands reveals DNA methylation profiles of integrated genome-wide and critical milk-related genes

DNA methylation (DNAm), a major element of epigenetics, plays critical roles in individual development. Reduced representation bisulfite sequencing (RRBS) is an effective and economical method for analyzing the DNA methylation of a single base. The aims of this study were to determine the DNAm profiles of the methylation contexts (CGs and non-CGs) of lactation and dry periods of goat mammary glands using the RRBS, and to identify potential milk-related genes. The proportion of CG was the highest among all the sequence contexts. The highest CG levels (72.44% to 75.24%) occurred in the 3′ UTR region, followed by the gene body region (61.14% to 65.45%). The non-CG levels were low compared to the CG levels. Bioinformatic analysis demonstrated that the CGs were mainly enriched at high methylation levels (>90%), while non-CGs were enriched at low methylation levels. Methylation levels of 95 and 54 genes in the lactation period were up- or downregulated, respectively, relative to the dry period, such as PPARα, RXRα and NPY genes. The bisulfite sequencing PCR results showed that the methylation level of goat PPARα gene during the lactation period was significant lower than in the dry period, while the methylation level of the RXRα gene was lower in the dry period than in the lactation period. Meanwhile, the methylation levels of human PPARα and NPY genes were significantly higher in MCF-7 than in MCF-10A cells. These findings provide essential information for DNA methylation profiles of goat mammary gland and detect some potential milk-related genes in dairy goats.

Identification of novel isoforms of dairy goat EEF1D and their mRNA expression characterization.

Eukaryotic translation elongation factor 1 delta (EEF1D) gene encodes guanine nucleotide exchange protein eEF1Bδ, which participates in the eukaryotic protein synthesis, and plays important roles in regulating cell cycling and milk production. This study firstly focused on detecting the isoforms of dairy goat EEF1D gene and their mRNA expression characterization. Herein, two novel isoforms, EEF1Da and EEF1Dc, were identified in dairy goat. The entire coding sequences of EEF1Da and EEF1Dc isoforms were 843bp and 267bp in length, respectively. Goat EEF1Da had complete conserved domains of elongation factor 1 (EF1) family, and the evolution of goat EEF1Da isoform was agreed with the evolution of species. Expression pattern analysis of different isoforms revealed relatively ubiquitous expression of EEF1D and EEF1Da. While EEF1Dc only expressed in heart, lung, kidney, adipose and muscle. Combining with the analysis results of cloning, qRT-PCR and bioinformatics, EEF1Da is the major alternative splicing form of EEF1D gene. Interestingly, qRT-PCR result showed that the highest expression of EEF1D was in adipose, which is the major component of mammary. This result was consistent with the early research that EEF1D expressed highly in the mammary, which indicated that EEF1D played a potential key role in regulating adipose development and milk production. All these findings would provide a foundation for the further research of EEF1D gene and development of dairy goat industry.