Table 1.
List of primer sets used in this work.
Fig 1.
Diagram representation of the alternative splicing pattern of NCF4 transcripts and results of SNP g.18174 A>G sequencing in the retained 48 bp sequence in intron 9 of the bovine NCF4 gene.
(A) Genomic structure of the bovine NCF4 gene. (B) Splicing pattern of the NCF4-TV splice variant and the sequencing result of SNP g.18174 A>G. The NCF4-TV transcript retains a 48 bp sequence from intron 9. The position of the A nucleotide in the start codon (ATG) is defined as +1.
Fig 2.
The amino acids sequence, protein structures and domains of the bovine NCF4-isoforms.
(A) The amino acids sequence of bovine NCF4-reference and TV transcripts. The NCF4-reference isoform encoded a 339 aa protein and NCF4-TV isoform encoded a 285 aa protein. The amino acids sequence of red and blue marks were differed between the two isoforms. (B) The secondary structures and domains of putative isoforms of NCF4-reference and NCF4-TV.
Fig 3.
Relative expression of NCF4-reference transcripts in normal and mastitic bovine mammary tissues.
Gene-specific transcript levels are normalized by the expression of the housekeeping gene β-actin in each sample.
Fig 4.
ESE motif threshold scores associated with NCF4 genotypes.
Bar graphs represent scores above the threshold for the ESE motifs in the A or G allele of locus 18174. The red square indicates the introduction of the G allele, relative to the A allele in locus g.18174 A>G, thereby increasing binding sites to the auxiliary splicing proteins: SRSF1, SRSF1 (IgM-BRCA1), and SRSF5. g.18174 A>G-A sequence: 5′ -tgctggacctttcccgaactctgatctctcccacccaggcgggagttcca-3′; g.18174 A>G-G sequence: 5′ -tgctggacctttcccgaactctggtctctcccacccaggcgggagttcca-3′.
Fig 5.
SNP g.18174 A>G induces aberrant NCF4-TV splicing.
(A) Schematic representation of the NCF4 mini-genes used in the functional splicing assay. The wild-type and mutant fragments contained 125 bp of intron 9 and 53 bp of exon 10; fragments harboring the A or G allele were separately cloned into the EcoRI and XhoI cloning sites of the pSPL3 vector. Two mini-gene expression vectors were transiently transfected into 293T cells. (B) RT-PCR analysis of the NCF4 spliced transcripts on a 2% agarose gel. RT-PCR products were amplified from the total RNA of 293T cells transfected with the wild-type and mutant (g.18174 A>G) NCF4 mini-gene constructs. The size of the RT-PCR product (441 bp) corresponded to the amplified portion of intron 9 (77 bp), the retained portion of intron 9 (48 bp), the amplification of exon 10 (53 bp), and the pSPL3 control plasmid (263 bp). The size of the RT-PCR product (393 bp) corresponded to the amplified portion of intron 9 (77 bp), the amplification of exon 10 (53 bp), and the pSPL3 control plasmid (263 bp). (C) Electrophoresis of RT-PCR products showing the presence and abundance of NCF4-TV transcript in bovine mammary samples with three NCF4 SNP g.18174 A>G genotypes. Expression of the NCF4-TV transcript is highest in mammary samples from GG animals, followed by those from AG and AA individuals.
Table 2.
Least squares mean and standard error of SCS in different genotypes of the NCF4 SNP g.18174 A>G in Chinese Holstein cows.