The Molecular Mechanism Of Genetic Variants In KDR Transcriptional Regularoty Region Affcting Intramuscular Fat Deposition In Pigs

Intramuscular fat (IMF) content is generally perceived as one of the key indicators influencing quality and sensory characteristics of pork. Kinase insert domain receptor (KDR), a vascular endothelial growth factor (VEGF) receptor, is widely regarded as having a principal role in mediating VEGF-induced responses in angiogenesis which provides oxygen and nutrient for adipose cells growing and deposition. In this research, our objective was to analyze the relationship between candidate gene KDR’s expression level and IMF content. After digging out some single nucleotide polymorphisms (SNPs) in the transcriptional regulatory region of KDR gene, we want to investigate whether the promoter polymorphisms in the KDR gene have effects on IMF deposition in the longissimus dorsi (LD) muscle. The results are as follows:Immunohistochemical sections showed that KDR protein specifically expressed in the capillaries which were chiefly distributed between fascicle and muscle fibers. Besides, it has not been examined in adipose tissue and muscle cells. KDR expression level was significantly higher in the high IMF content group, which revealed a significant correlation between KDR mRNA level and IMF content in LD muscle (r= 0.47, P<0.01). Three novel SNPs, c.-1316A>G, c.-1303C>T and c.-1108A>C, were revealed to have differential allele distribution between high and low IMF content groups by comparative sequencing of DNA pools. The three SNPs were completely linked, forming only ACA or GTC haplotypes, when genotyped in 105 Erhualian purebred pigs and 98 Duroc × Landrace × Yorkshire (D×L×Y) crossbred pigs. It is interesting to see ACA haplotype is present exclusively in Erhualian pigs while not in D×L×Y pigs。 These three completely linked SNPs were associated with IMF content. A-C-A haplotype associated with the higher IMF content, while G-T-C haplotype contributed to the lower IMF content. In addition, c.-998G>C was identified in both the Erhualian and crossbred pigs and was not associated to the IMF content. The c.-873G>A and c.-816A>G were also entirely interlocked and existed exclusively in Erhualian pigs, forming GA and AG haplotypes.The ACA promoter was found to have higher activity than GTC type for KDR transcription using either gene expression analysis or luciferase assay (P<0.05). Site-direct mutation analysis demonstrated that the substitution from G to A at c.-1316 site is the causation of promoter activity alteration. Furthermore, we detected CD31 and CD34, two blood vessel endothelial markers, expressed higher in ACA/ACA individuals (P<0.05). We concluded that the c.-1316A of ACA promoter might be a desirable form for improving IMF content, by promoting higher KDR gene expression and more blood vessel network.