Study On Genetic Variations, Cloning And Expression Of Bovine VEGF, VEGF-B And FLT-1 Genes

Qinchuan, Nanyang and Jiaxian Red cattle are excellent local cattle breeds in China. Originally, these three cattle breeds were evolved from draft animals, in recent years, these breeds were used for draft and meat dual-purpose, but now were used solely for beef cattle type. It would take several more years with low efficiency to develop beef cattle breed by conventional breeding strategy. It is very difficult to make great genetic progress in bovine quantitative traits using conventional breeding approaches, because many economic traits in beef cattle are quantitative traits, which are controlled by multiple genes. In recent years, the marker-assisted selection (MAS) can improve the breeding efficiency in beef cattle and compensate for the low rate of accuracy in bovine conventional breeding strategy because MAS is directly based on genotypes and DNA level. While, transgenic technology can also create some of unconventional livestock products according to the needs of people. It is very important for the marker-assisted selection technology to identify the suitable candidate markers correlated with economically relevant traits and investigate the function of genes at a molecular level in these breeds. This is a trend to speed up the cultivation and breeding of Chinese beef cattle breeds using the combination of modern molecular breeding technology and conventional breeding technology.Many reports suggested the Vascular endothelial growth factor-A (VEGF-A), Vascular endothelial growth factor-B (VEGF-B), and their receptor Fms-like tyrosine kinase (Flt-1) gene were associated with animal growth traits. Many polymorphisms of VEGF, VEGF–B and Flt-1 gene have been detected in their coding and surrounding DNA sequences in humans, and these polymorphisms could be regarded as a major genetic risk factor for certain diseases. To date, few polymorphisms within these genes have been reported in livestock.PCR-SSCP and DNA sequencing methods were for the first time employed to screen the genetic variation of 42 loci from VEGF, VEGF–B and Flt-1 gene in 675 individuals belonging to three Chinese indigenous cattle breeds including Qinchuan, Jiaxian Red and Nanyang breed. We have detected 19 single nucleotide polymorphisms (SNPs) from their coding and surrounding DNA sequences of VEGF, VEGF–B and Flt-1 gene and evaluated the association between genotypes of 16 SNPs and the growth traits in Nanyang cattle breed. The mRNA expression level of VEGF and VEGF-B gene were for the first time examined in bovine different tissues and different embryonic development periods using real-time quantitative PCR analysis. The full CDS sequence of bovine VEGF gene was cloned. The recombinant plasmid pET-28a-VEGF induced by IPTG have for the first time successfully expressed the target protein in Escherichia coli BL21 (DE3). These results provide a basis for the breeding strategies through MAS, functional verification and transgenic breeding in Chinese domestic cattle. The results are as follows.(1) Association of polymorphisms of the VEGF with growth traits in Nanyang breedThe genetic variation of 6 loci of VEGF gene were detected in 675 individuals belonging to three Chinese indigenous cattle breeds including Qinchuan, Jiaxian Red and Nanyang breed. Three new SNPs were found in three Chinese cattle breeds: rs109603925(6765Tï¹¥C), rs110750917(6860Aï¹¥G), rs109288296(6893Tï¹¥C). One SNP (6765Tï¹¥C) was detected in intron2 and the other two SNPs (6860Aï¹¥G, 6893Tï¹¥C) were in exon3 with strong linkage disequilibration. However, two synonymous mutations of exon3 were identified. Three haplotypes were described as: A(C-A-T), B(T-A-T)and C(C-G-C). The three haplotypes constituted six genotypes, including AA (C-A-T/C-A-T), AB (C-A-T/T-A-T), BB (T-A-T/T-A-T), AC (C-A-T/C-G-C), CC (C-G-C/C-G-C) and BC (T-A-T/C-G-C). The relationship between the different genotypes of each SNP and the various traits recorded (birth weight, body weight, body height, body length, heart girth and height at the hip cross) in Nanyang cattle breed (0, 6, 12, 18 and 24 month old) was evaluated using a single-marker mixed-model association analysis. Nanyang cattle with the TC genotype of rs109603925 (n = 89) showed higher birth weight, 6-month-old body weight and heart girth than those with the TT (n=6) and CC genotype (n=176) (p﹤0.05). At the traits of 12, 18, and 24 months, the difference was not significant between the above three genotypes in Nanyang samples (pï¹¥0.05). The analysis showed that the SNP (rs109603925) in intron 2 of the VEGF gene was significant contribution to early development of Chinese cattle. These findings indicated that this SNP can be a molecular marker and will be utilized to the molecular breeding strategies through marker assisted selection (MAS) in Chinese cattle.(2) Association of SNPs of the bovine VEGF-B gene with growth traits in Nanyang breedWe analyzed the genetic polymorphism of 6 loci of the coding and surrounding DNA sequences of bovine VEGF–B gene in three Chinese indigenous cattle breeds. Four novel SNPs were detected in three breeds: ss251344001(782A>G), ss251344002(1002-->CT), ss251344003 (1079C>T), ss251344004(2129G>A). In this study, Association analysis between 4 SNPs and growth traits (birth weight, body weight, body height, body length, heart girth, hucklebone width) were analyzed in Nanyang cattle at 6, 12, 18 and 24-month-old respectively. Nanyang cattle at 6, 12-month-old with the CT/CT (n = 89) and --/CT genotype of ss251344002 (n=107) showed heavier body weight than those with the --/-- genotype (n=79) (p﹤0.05). However, at 18 and 24-month-old, the difference between three genotypes was not significant (pï¹¥0.05). These results suggest that VEGF-B is a potential candidate gene for bovine growth traits and the SNP (ss251344002) is a molecular marker which contributes to early development of Chinese cattle.(3) Association of SNPs in the Flt-1 gene with growth traits in Nanyang breedWe analyzed, for the first time, the genetic variation of 30 loci of the coding region and the non-coding region of bovine Flt-1gene in three Chinese cattle breeds. Twelve novel SNPs were detected in bovine Flt-1gene in the three breeds: ss184956516(2260AG), ss184956517(32255C>A), ss184956518(43654G>C), ss184956519(44409C>G), ss2513 43993(68859G>A), ss251343994(68872G>A), ss251343995(78152C>G), ss251343996 (86197A>T), ss251343997(91417G>C), ss251343998(128783C>G), ss251343999(133972 A>G), ss251344000(145849C>T). Of which, four SNPs were located in introns and eight SNPs in exons of bovine Flt-1gene. All the eight SNPs studied here in exon regions of Flt-1 gene (XP₀01249769.2) cause eight synonymous mutations. The relationship between the different genotypes of each SNP and the various traits recorded was evaluated using a single-marker mixed-model association analysis. Statistical results showed no significant association between the Flt-1 polymorphism and growth traits in Nanyang breed. We concluded that the identified SNPs of the Flt-1 gene were not considered as a DNA marker for bovine growth traits in marker-assisted selection.(4) Tissue expression of VEGF and VEGF-B gene in Chinese cattleThe mRNA expression of VEGF and VEGF-B gene were examined in bovine different tissues during embryonic development periods in Chinese cattle by real-time quantitative PCR technology. The results showed that both VEGF and VEGF-B gene had a wide tissue distribution in heart, liver, spleen, lung, kidney and skeletal muscle tissues in Chinese cattle. We found that both VEGF and VEGF-B had the highest expression value in skeletal muscle. Both VEGF and VEGF-B mRNA expression abundance during different tissues of embryonic development stage were significantly higher than that in adults. The result of the tissue expression of VEGF and VEGF-B could provide a basis for molecular breeding of Chinese cattle.(5) Cloning and prokaryotic expression of the bovine CDS of VEGF geneWe have cloned the full CDS sequence of the bovine VEGF gene, and constructed prokaryotic expression recombinant plasmid vector of pET-28a-VEGF. The recombinant plasmid pET-28a-VEGF induced by IPTG have successfully expressed the target protein in Escherichia coli BL21(DE3) after optimizing the expression condition. The optimized induction condition was: at 37℃induced with 0.1 mmol/L IPTG for 3 h.The results showed that VEGF protein formed inclusion bodies in bacterial expression system, suggesting that this assay can be used to purify VEGF protein and hence provide a basis for studying the applications of VEGF protein in eukaryote expression, gene functional verification and transgenic breeding in Chinese cattle.