RESUMO
The polypeptide N-acetylgalactosaminyltransferase-like protein 5 (GALNTL5) is a newly identified protein that is specifically expressed in testis tissue and participates in spermatogenesis. In this study, we characterized a novel bovine GALNTL5 splice variant, designated as GALNTL5-AS, by using real-time polymerase chain reaction (RT-PCR) and clone sequencing methods. The novel GALNTL5 isoform was derived from the complete transcript, GALNTL5-complete, via alternative splicing (AS). The pattern of the splice variant was exon skipping. Bovine GALNTL5 transcripts were expressed in the testis, as demonstrated by RT-PCR. The expression levels of both transcripts were higher in adult testes than in calf testes (P < 0.05). In addition, prediction analysis showed that the GALNTL5-AS transcript only encoded 122 amino acids and lost its glycosyltransferase 1 and Gal/GalNAc-T motifs, which may result in a dysfunctional protein compared with the predominant transcript GALNTL5-complete. This study improves our understanding of the bovine GALNTL5 gene function during bull sperm formation.
Assuntos
Processamento Alternativo , N-Acetilgalactosaminiltransferases/genética , Testículo/metabolismo , Motivos de Aminoácidos , Animais , Bovinos , Éxons , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Polymorphonuclear neutrophil (PMN) leukocytes are primary phagocytic cells of the bovine mammary gland and a first line of defense against invading pathogens during bovine mastitis infection. Cluster of differentiation 14 (CD14) is mainly expressed in macrophages and neutrophils and acts as a co-receptor that binds bacterial lipopolysaccharide (LPS) and recruits PMNs to CD14-LPS complexes in mammary epithelial cells. In this study, we identified a novel splice variant in PMNs, named CD14-SV, characterized by a deleted region from c.143-579 nt compared to the CD14 reference mRNA sequence. Moreover, a single nucleotide polymorphism (c.523 A>G) in exon 2 of CD14 was identified and found to modify the secondary structure and hydrophilicity of the CD14 protein. Association analysis also showed that the milk somatic cell score, an indicator of mastitis, of cows with the GG genotype was lower than that of cows with the AA and AG genotypes. Our findings suggest that the expression of CD14 in bovine blood PMNs is regulated by alternative splicing, and that CD14-SV is a candidate functional marker that may influence mastitis-resistance in dairy cows.
Assuntos
Bovinos/genética , Receptores de Lipopolissacarídeos/genética , Mastite Bovina/genética , Neutrófilos/metabolismo , RNA Mensageiro/genética , Processamento Alternativo , Animais , Feminino , Variação Genética , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Mastite Bovina/sangue , Polimorfismo de Nucleotídeo Único , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismoRESUMO
The sperm flagella 2 (SPEF2) gene is essential for development of normal sperm tail and male fertility. In this study, we characterized first the splice variants, promoter and its methylation, and functional single-nucleotide polymorphisms (SNPs) of the SPEF2 gene in newborn and adult Holstein bulls. Four splice variants were identified in the testes, epididymis, sperm, heart, spleen, lungs, kidneys, and liver tissues through RT-PCR, clone sequencing, and western blot analysis. Immunohistochemistry revealed that the SPEF2 was specifically expressed in the primary spermatocytes, elongated spermatids, and round spermatids in the testes and epididymis. SPEF2-SV1 was differentially expressed in the sperms of high-performance and low-performance adult bulls; SPEF2-SV2 presents the highest expression in testis and epididymis; SPEF2-SV3 was only detected in testis and epididymis. An SNP (c.2851G>T) in exon 20 of SPEF2, located within a putative exonic splice enhancer, potentially produced SPEF2-SV3 and was involved in semen deformity rate and post-thaw cryopreserved sperm motility. The luciferase reporter and bisulfite sequencing analysis suggested that the methylation pattern of the core promoter did not significantly differ between the full-sib bulls that presented hypomethylation in the ejaculated semen and testis. This finding indicates that sperm quality is unrelated to SPEF2 methylation pattern. Our data suggest that alternative splicing, rather than methylation, is involved in the regulation of SPEF2 expression in the testes and sperm and is one of the determinants of sperm motility during bull spermatogenesis. The exonic SNP (c.2851G>T) produces aberrant splice variants, which can be used as a candidate marker for semen traits selection breeding of Holstein bulls.
Assuntos
Processamento Alternativo/genética , Bovinos/genética , Metilação de DNA/genética , Proteínas dos Microfilamentos/genética , Polimorfismo de Nucleotídeo Único/genética , Testículo/metabolismo , Animais , Epididimo/química , Masculino , Regiões Promotoras Genéticas/genética , Motilidade dos Espermatozoides , Cauda do Espermatozoide/química , Espermatogênese , Espermatozoides/química , Espermatozoides/metabolismo , Testículo/químicaRESUMO
Mutations, such as single nucleotide polymorphisms (SNPs), in the 5'-flanking and microRNA (miRNA) regulatory regions may result in altered gene expression levels and cause diseases. Alpha-2-macroglobulin (A2M) has the function of binding host or foreign peptides and particles, and thereby serves as a defense barrier against pathogens in the plasma and tissues of animals. To investigate the functional markers of the A2M gene associated with mastitis, the promoter was characterized and SNPs that affect promoter activity or binding affinity with the target miRNA were identified using the luciferase reporter assay and real-time quantitative PCR method. Results showed that the core promoter of A2M was found between the bases g.-2641 and g.-2479. Four novel SNPs (g.-724A>G, g.-665G>A, g.-535C>G and g.-520_-519insA) in the promoter region were completely linked. The activity of the mutant haplotype (GAGA) increased by 177% compared with that of the wild haplotype (AGC-). Bta-miR-2898 was upregulated by 6.25-fold in the mammary gland tissues of mastitis-infected cows compared with that of the healthy cows. One SNP (c.4659_4661delC) located in the 3'-untranslated region of the A2M gene may affect the binding affinity with the target bta-miR-2898. Five SNPs exhibited tight linkage. Association analysis showed that the milk somatic cell score for cows with the mutant haplotype (GAGA-) was lower than that for cows with the wild haplotype. Thus, the mutant type can be used as a potential functional marker for a mastitis resistance breeding program in dairy cows. Our findings provided the molecular basis for A2M transcriptional and post-transcriptional regulations. A close relationship between regulatory mutations and mastitis susceptibility of cows also was established.
Assuntos
Predisposição Genética para Doença , Mastite Bovina/genética , MicroRNAs/genética , alfa-Macroglobulinas/genética , Animais , Bovinos , Feminino , Células HEK293 , Haplótipos , Humanos , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Transfecção , Regiões não TraduzidasRESUMO
Phospholipase C zeta 1 (PLCζ1), which transcribes a key protein, has an important function in oocyte activation and embryo development because PLCζ1 can trigger a series of intracellular Ca2+ oscillations in mammals. In this study, a novel splice variant in the testis tissues of adult and fetal Chinese Holstein bulls was characterized by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing analysis. The novel splice variant PLCζ1-sv1 was derived from the PLCζ1 complete transcript (PLCζ1-complete) by alternative splicing; the alternative splicing pattern exhibited alternative 5'-splice sites. The full-length transcript, PLCζ1-complete, is the main transcript found in fetal and adult cow testis tissue. Quantitative real-time PCR (qPCR) analysis demonstrated that the expression levels of the PLCζ1-complete transcript were significantly higher than those of the PLCζ1-sv1 splice variant in bovine testis tissues. PLCζ1 protein sequencing analysis showed that the amino acids at positions 453 to 457 were deleted in PLCζ1-sv1, thereby terminating transcription prematurely. In summary, this study provided information to elucidate the structure and function of the bovine PLCζ1 gene.
Assuntos
Processamento Alternativo/genética , Fosfolipase C gama/genética , Testículo/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , China , Elementos Facilitadores Genéticos/genética , Éxons/genética , Masculino , Dados de Sequência Molecular , Motivos de Nucleotídeos/genética , Fosfolipase C gama/química , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
Phosphoenolpyruvate carboxykinase 1 (PCK1), also named PEPCK-C, is a multiple-function gene that is involved in gluconeogenesis, glyceroneogenesis, reproduction, female fertility, and development of obesity and diabetes. How its many functions are regulated was largely unknown. Therefore, we investigated mRNA expression and possible splice variants of PCK1 by screening cDNA in nine tissues from Holstein bulls and cows. PCK1 mRNA was highly expressed in the liver, kidney, ovary and testis; expression levels were low in the heart, spleen, and lung tissues. Expression of this gene was not detected in skeletal muscle. This led to the discovery of five novel bovine splice variants, named PCK1-AS1-PCK1-AS5. In PCK1-AS1, 51 nucleotides in the interior of exon 2 were spliced out. In PCK1-AS2, exons 2 and 3 were altered by the alternative 3' and 5' splice sites, respectively. PCK1-AS3 was truncated from the 3' end of exon 2 to the 5' end of exon 4. In PCK1-AS4, exon 5 was completely spliced out. In PCK1-AS5, exons 5 and 6 and the 5' end of exon 7 were spliced out. These splice variants (PCK1-AS1-PCK1-AS5) potentially encoded shorter proteins (605, 546, 373, 246 and 274 amino acids, respectively), when compared to the complete protein (622 amino acids). Considering the functional domains of the PCK1 protein, it is likely that these splice variants considerably affect the function of this protein; alternative splicing could be one of the mechanisms by which the diverse functions of PCK1 are regulated.
Assuntos
Bovinos/genética , Fosfoenolpiruvato Carboxilase/genética , Sequência de Aminoácidos , Animais , China , Clonagem Molecular , DNA Complementar , Éxons , Feminino , Masculino , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Conformação Proteica , RNA/genética , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNARESUMO
Mastitis is an economically devastating disease affecting the dairy industry. Dairy cows with mastitis give reduced milk yield and produce milk that is unfit for consumption. The chemokine receptor CXCR1 is an excellent prospective genetic marker for mastitis resistance in cattle because it regulates neutrophil migration, killing, and survival during infection. We detected 4 single nucleotide polymorphisms (SNPs) of the CXCR1 gene in Chinese native cattle and analyzed their associations with milk traits. Screening for genetic variations in CXCR1 among 648 Chinese Holstein, Luxi Yellow, and Bohai Black cattle by created restriction site polymerase chain reaction (PCR), nested PCR, and DNA sequencing revealed 4 new SNPs with allelic frequencies ranging from 0.676 to 0.821, 0.706 to 0.803, 0.647 to 0.824, and 0.558 to 0.581. All four CXCR1 gene SNPs were located in exon II. Two SNPs, c.337A>G and c.365C>T, were nonsynonymous mutations [ATC (Ile) > GTC (Val) and GCC (Ala) > GTC (Val)], whereas two, c.291C>T and c.333C>T, were synonymous mutations [TTC (Gly) > TTT (Gly) and GGC (Phe) > GGT (Phe)]. Statistical analyses revealed the significant association of c.337A>G and c.365C>T with the somatic cell score, which suggests the possible role of these SNPs in the host response against mastitis. Our data suggest that combined genotypes CCAC/CCGC, CCAC/CTAT, and CCAT/CTAT (lowest somatic cell scores); CTAC/CTAT (highest protein rate); CCAC/CTGC (highest fat rate); and CCAT/CTAT (highest 305-day milk yield) can be used as possible candidates for marker-assisted selection in dairy cattle breeding programs.
Assuntos
Bovinos/genética , Lactação/genética , Polimorfismo de Nucleotídeo Único , Receptores de Interleucina-8A/genética , Animais , Animais Endogâmicos , Éxons , Feminino , Estudos de Associação Genética , Mastite Bovina/genética , Leite/metabolismo , Mutação , Característica Quantitativa HerdávelRESUMO
The mannan-binding lectin gene (MBL) participates as an opsonin in the innate immune system of mammals, and single nucleotide polymorphisms (SNPs) in MBL cause various immune dysfunctions. In this study, we detected SNPs in MBL2 at exon 1 using polymerase chain reaction single-strand conformation polymorphism analysis and DNA sequencing techniques in 825 Chinese Holstein cows. Four new SNPs with various allele frequencies were also found. The g.1164 G>A SNP was predicted to substitute arginine with glutamine at the N-terminus of the cysteine-rich domain. In the collagen-like domain, SNPs g.1197 C>A and g.1198 G>A changed proline to glutamine, whereas SNP g.1207 T>C was identified as a synonymous mutation. Correlation analysis showed that the g.1197 C>A marker was significantly correlated to somatic cell score (SCS), and the g.1164 G>A locus had significant effects on SCS, fat content, and protein content (P < 0.05), suggesting possible roles of these SNPs in the host response against mastitis. Nine haplotypes and nine haplotype pairs corresponding to the loci of the 4 novel SNPs were found in Chinese Holsteins. Haplotype pairs MM, MN, and BQ were correlated with the lowest SCS; MN with the highest protein yield; MM with the highest protein rate, and MN with the highest 305- day milk yield. Thus, MM, MN, and BQ are possible candidates for marker-assisted selection in dairy cattle breeding programs.
Assuntos
Bovinos/genética , Estudos de Associação Genética , Lectina de Ligação a Manose/genética , Leite/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Característica Quantitativa Herdável , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , China , Éxons/genética , Frequência do Gene/genética , Haplótipos/genética , Heterozigoto , Análise dos Mínimos Quadrados , Lectina de Ligação a Manose/química , Dados de Sequência Molecular , Polimorfismo Conformacional de Fita Simples/genética , Análise de Sequência de DNA , Coloração pela PrataRESUMO
The complement system helps in the direct lysis of invading pathogens and modulates phagocytic, humoral and cellular immune responses. Complement 4 is a critical component in complement activity and protection against many bacterial pathogens because it is essential to classical and lectin activation pathways. We used reverse transcription and PCR to investigate alternative splicing and expression of the complement component 4 (C4A) gene in Chinese Holstein cattle. The PCR products were cloned and sequenced. A novel splice variant involving intron 10 was identified, which we named C4A-AS. To examine how C4A gene activity is affected by bovine mastitis, six Chinese Holstein cattle were divided into healthy (non-mastitic) and Staphylococcus aureus-induced mastitic groups. Real-time quantitative PCR (qRT-PCR) revealed that the C4A-complete and C4A-AS transcripts are expressed at significantly different levels in healthy cows, while there were no significant differences in the mastitic group (P = 0.257). Expression of C4A-AS increased significantly when mastitis developed. We also examined the expression of C4A-complete and C4A-AS in several tissues (liver, heart, spleen, lung, kidney, tongue, and muscle). The two transcripts were expressed in all of these tissues but there were no significant differences in expression between healthy and mastitic cows. We therefore conclude that the C4A-complete transcript is the main transcript under normal physiological conditions, while C4A-AS is augmented when mastitis develops.
Assuntos
Processamento Alternativo/genética , Bovinos/genética , Bovinos/imunologia , Complemento C4a/genética , Indústria de Laticínios , Mastite Bovina/genética , Mastite Bovina/imunologia , Animais , China , Feminino , Mastite Bovina/microbiologia , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Staphylococcus aureus/fisiologiaRESUMO
Based on first-principles calculations, electronic properties of edge-functionalized zigzag graphene nanoribbons (ZGNRs) on SiO(2) substrate are presented. Metallic or semiconducting properties of ZGNRs are revealed due to various interactions between edge-hydrogenated ZGNRs and different SiO(2)(0001) surfaces. Bivalent functional groups decorating ZGNRs serve as the bridge between active edges of ZGNRs and SiO(2). These functional groups stabilize ZGNRs on the substrate, as well as modify the edge states of ZGNRs and further affect their electronic properties. Bandgaps are opened owing to edge state destruction and distorted lattice in ZGNRs.
RESUMO
Transferrin (Tf) is a ß-globulin protein that transports iron ions in mammalian cells. It contributes to innate immunity to microbial pathogens, primarily by limiting microbial access to iron. Thus, polymorphisms present in bovine Tf could potentially underlie inherited differences in mastitis resistance and milk production traits. We detected three novel single-nucleotide polymorphisms of the Tf gene in Chinese native cattle by screening for genetic variation of Tf in 751 individuals of three Chinese cattle breeds, namely China Holstein, Luxi Yellow and Bohai Black, using PCR-RFLP and DNA sequencing techniques. The three new SNPs, g.-1748G>A ss250608649, g.13942T>C ss250608650, and g.14037A>G ss250608651, had allele frequencies of 85.9, 86.3 and 92.5%, 64.5, 73.3 and 65.0%, and 67.6, 73.7 and 60.0%, respectively. SNP g.-1748G>A was located in the 5' flanking region of Tf. SNP g.14037A>G was located in intron 8 of Tf. SNP g.13942T>C, located in exon 8 of Tf, was a synonymous mutation (TTA > CTA), encoding a leucine (326 aa) in the Tf protein. Associations of the Tf SNPs with milk traits were also analyzed. Significant (P < 0.05) relationships among the Tf polymorphisms, somatic cell scores (SCS), and milk productive traits were observed. Cows with genotypes TT (g.13942T>C), GG (g.-1748G>A) and AG (g.14037A>G) had a lower SCS and higher protein levels and 305-day milk yield. Nineteen combinations of different haplotypes from the three SNPs were identified in Chinese Holstein cattle. The haplotype combination ATA/GCA, GCA/GCA and GCG/ GTA was dominant in cows with a lower SCS, a higher protein level and a higher 305-day milk yield, respectively. Moreover, the gene expression level of Tf was higher in mastitis-affected mammary tissues than in normal mammary tissues. These results suggest that the Tf gene affects milk production, as well as mastitis-resistance traits, in Chinese Holsteins.
Assuntos
Leite , Polimorfismo de Nucleotídeo Único/genética , Transferrina/genética , Animais , Bovinos , Variação Genética/genética , Genótipo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição/genética , Análise de Sequência de DNARESUMO
Bovine lactoferrin (bLF) is a member of the transferrin family; it plays an important role in the innate immune response. We identified novel splice variants of the bLF gene in mastitis-infected and healthy cows. Reverse transcription-polymerase chain reaction (RT-PCR) and clone sequencing analysis were used to screen the splice variants of the bLF gene in the mammary gland, spleen and liver tissues. One main transcript corresponding to the bLF reference sequence was found in three tissues in both healthy and mastitis-infected cows. Quantitative real-time PCR analysis showed that the expression levels of the LF gene's main transcript were not significantly different in tissues from healthy versus mastitis-infected cows. However, the new splice variant, LF-AS2, which has the exon-skipping alternative splicing pattern, was only identified in mammary glands infected with Staphylococcus aureus. Sequencing analysis showed that the new splice variant was 251 bp in length, including exon 1, part of exon 2, part of exon 16, and exon 17. We conclude that bLF may play a role in resistance to mastitis through alternative splicing mechanisms.
Assuntos
Lactoferrina/genética , Glândulas Mamárias Animais/imunologia , Mastite Bovina/metabolismo , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/genética , Processamento Alternativo , Animais , Bovinos , Éxons , Feminino , Expressão Gênica , Lactoferrina/imunologia , Lactoferrina/metabolismo , Fígado/imunologia , Fígado/metabolismo , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/imunologia , Mastite Bovina/microbiologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/imunologia , Baço/metabolismo , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/imunologiaRESUMO
Heat stress (HS) seriously affects animal performance. In view of global warming, it is essential to understand the regulatory mechanisms by which animals adapt to heat stress. In this study, our aim was to explore the genes and pathways involved in heat stress in sheep. To this end, we used transcriptome analysis to understand the molecular responses to heat stress and thereby identify means to protect sheep from heat shock. To obtain an overview of the effects of heat stress on sheep, we used the hypothalamus for transcriptome sequencing and identified differentially expressed genes (DEGs; false discovery rate (FDR) < 0.01; fold change > 2) during heat stress. A total of 1423 DEGs (1122 upregulated and 301 downregulated) were identified and classified into Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Heat stress triggered dramatic and complex alterations in gene expression in the hypothalamus. We hypothesized that heat stress induced apoptosis and dysfunction in cells and vital organs and affected growth, development, reproduction, and circadian entrainment via the calcium signaling pathway, which influences ribosome assembly and function. Real-time PCR was used to evaluate the expression of the genes regulating important biological functions or whose expression profiles were significantly changed after acute heat stress (FDR < 0.01; fold change > 4), and the results showed that the expression patterns of these genes were consistent with the results of transcriptome sequencing, indicating that the credibility of the sequencing results. Our data indicated that heat stress induced calcium dyshomeostasis, blocked biogenesis, caused ROS accumulation, impaired the antioxidant system and innate defense, and induced apoptosis through the P53 signaling pathway activated by PEG3, decreased growth and development, and enhanced organ damage. These data is very important and helpful to elucidate the molecular mechanism of heat stress and finally to find ways to deal with heat stress damage in sheep.
Assuntos
Regulação da Expressão Gênica/fisiologia , Resposta ao Choque Térmico/fisiologia , Hipotálamo/metabolismo , Ovinos/metabolismo , Transcriptoma/fisiologia , Animais , Perfilação da Expressão Gênica/métodosRESUMO
OBJECT: The purpose of this study was to investigate functional alterations of the brain in the early stage of spinal cord injury (SCI) and further investigate how these functional alterations relate to SCI patients' sensorimotor functions. METHODS: Twenty-five patients with SCI and 25 matched healthy controls underwent imaging by using resting-state functional magnetic resonance imaging (fMRI). The amplitude of low-frequency fluctuations (ALFF) were used to characterize regional neural function, and the seed-based functional connectivity (FC) was used to evaluate the functional integration of the brain network. RESULTS: Compared to healthy controls, patients with SCI showed decreased ALFF in the bilateral primary sensorimotor cortex, and increased ALFF in the bilateral cerebellum and right orbitofrontal cortex (OFC). The ALFF value in the left cerebellum was negatively correlated with the clinical total motor score in patients with SCI. Furthermore, SCI patients mainly showed decreased inter-hemispheric FC between the bilateral primary sensorimotor cortex, as well as increased intra-hemispheric FC within the motor network, including the primary sensorimotor cortex, premotor cortex, supplementary motor area (SMA), thalamus and cerebellum. Subsequent correlation analyses revealed that increased FC within the primary sensorimotor cortex, SMA, and cerebellum negatively correlated with the total American Spinal Cord Injury Association (ASIA) motor score. CONCLUSIONS: Our findings provide evidence that SCI can induce significant regional and network-level functional alterations in the early stage of the disease. We hypothesized these alterations may be an adaptive phenomenon following SCI, reflecting a compensatory mechanism during the early stage of SCI.