【作者】 庞永红；

【导师】 陈宏；

【作者基本信息】 西北农林科技大学 ， 遗传学， 2011， 博士

【摘要】 秦川牛、南阳牛和郏县红牛品种是中国优秀的地方黄牛品种,原为役用,现向肉役兼用乃至肉牛方向培育。传统的肉牛育种方法周期长,效率低。况且肉牛的许多经济性状属多基因控制的数量性状,常规的育种手段很难取得突破性的进展。近年来发展起来的分子标记辅助选择（marker-assisted selection,MAS）技术,由于直接在DNA水平上对肉牛性状的基因型进行选择,不仅弥补了传统育种技术中选种准确率低的缺点,而且提高了育种效率。转基因技术还能根据人们的需求创造出一些非常规性的畜牧产品。因此,在DNA水平上寻找与肉牛经济性状紧密连锁的分子标记,研究优良基因的功能,采用现代分子育种新技术结合常规育种,是加快中国肉牛品种培育的大趋势。研究表明血管内皮生长因子A（Vascular endothelial growth facto-Ar,VEGF-A）、血管内皮生长因子B（vascular endothelial growth factor-B,VEGF-B）和它们的受体FMS样酪氨酸激酶（Fms-like tyrosine kinase,Flt-1）基因的单核苷酸多态性（single nucleotide polymorphisms,SNPs）标记与人类疾病易感性有关,也与动物生长性状关系密切;但在畜禽遗传标记方面缺乏系统研究。本研究以秦川牛、南阳牛、郏县红牛3个品种共675头黄牛为试验材料,采用PCR-SSCP及DNA测序技术首次检测了VEGF、VEGF-B和其受体Flt-1基因的编码区及其周边的非编码区共42个位点的遗传变异,并将发现的19个SNPs不同的基因型与南阳牛的生长性状进行了关联分析;首次以实时荧光定量PCR技术研究了VEGF和VEGF-B基因在黄牛不同组织、不同发育时期的mRNA表达规律;首次克隆了黄牛VEGF基因完整CDS区序列,并成功实现了该基因在原核细胞表达。这些结果为黄牛分子辅助标记和功能验证及转基因育种奠定了基础。本研究得到以下重要结果:（1）黄牛VEGF基因遗传变异及其与南阳牛生长性状的关联检测了3个黄牛品种VEGF基因外显子及其周边内含子共6个位点的遗传变异,发现了3个新的SNPs: rs10960392（56765T﹥C）,rs11075091（76860A﹥G）,rs109288296（6893T﹥C）。其中1个SNP （6765T> C）位于内含子2。另外2个SNPs（6860A>G,6893T> C）位于外显子3,表现出强烈的连锁不平衡,仅引起同义突变。以此定义了3种单倍型:A（C-A-T）, B（T-A-T）,C（C-G-C）,组成6种基因型: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）,BC（T-A-T/C-G-C）。使用单标记混合模型的关联分析法对SNP不同基因型与南阳牛0、6、12、18和24月龄的生长性状（体重、体高、体长、胸围和坐骨端宽）进行关联分析,结果发现南阳牛TC基因型（rs109603925,n=89）的初生重、6月龄体重、胸围明显高于TT（n = 6）与CC（n = 176）基因型个体（P﹤0.05）,但第12,第18和24月龄之间性状差异不显著（P﹥0.05）。这些结果表明VEGF基因内含子2上的一个SNP （rs109603925）影响黄牛的早期生长发育。该SNP位点有望成为DNA分子标记,应用于黄牛的分子育种。（2）黄牛VEGF-B基因遗传变异及其与南阳牛生长性状的关联检测了3个黄牛品种VEGF-B基因编码区及其侧翼区共6个位点的遗传变异,发现了4种新的SNPs:ss251344001（782 A>G）,ss251344002（1002 -- >CT）,ss251344003（1079 C>T）,ss251344004（2129 G>A）。将南阳牛不同时期（0、6、12、18和24月龄）的生长性状（体重、体高、体长、胸围、坐骨端宽）与4个SNPs组成的基因型进行关联分析,结果发现ss251344002（1002 -- >CT）SNP标记组成的3种基因型中,6月龄和12月龄的南阳牛,CT/CT（n = 89）和--/CT（n=107）基因型个体比--/--（n=79）基因型个体的体重更大（P﹤0.05）。而在18和24月龄的南阳牛中,这种差异并不显著（P﹥0.05）。这些结果表明该基因是分析牛早期生长发育潜在的候选基因。该SNP位点（ss251344002）可以作为辅助选择黄牛生长发育性状的分子标记。（3）黄牛Flt-1基因遗传变异及其与南阳牛生长性状的关联检测了3个黄牛品种Flt-1基因编码区及其侧翼区共30个位点。发现了12个SNPs:ss184956516（2260A﹥G）;ss184956517（32255C>A）; ss184956518（43654G>C）; ss184956519（44409C>G）,ss251343993（68859G>A）,ss251343994（68872G>A）,ss251343995（78152C>G）,ss251343996（86197A>T）,ss251343997（91417G>C）,ss251343998（128783C>G）,ss251343999（133972A>G）,ss251344000（145849C>T）。其中4个SNPs位于内含子,8个位于外显子。Flt- 1基因（XP₀01249769.2）外显子区域的8个SNPs均引起同义突变。使用单标记混合模型的关联分析法对SNPs不同基因型与南阳牛不同生长时期各性状进行了关联分析,发现12个SNPs位点的基因型并没有显著影响南阳牛不同生长时期的生长性状。故初步认为Flt - 1基因的SNPs不能作为辅助选择黄牛生长发育性状的分子标记。（4）黄牛VEGF和VEGF-B基因的组织表达以实时荧光定量PCR技术研究了VEGF和VEGF-B基因mRNA在黄牛不同年龄,不同组织的表达规律,发现VEGF、VEGF-B基因在黄牛的心、肝、脾、肺、肾、肌肉组织均有广泛的表达谱,但表达丰度最高的都是肌肉组织。黄牛胚胎时期大部分组织的表达量均高于成年组织。这些结果为黄牛分子育种奠定了分子生物学基础。（5）黄牛VEGF基因CDS区的克隆及原核表达克隆了黄牛VEGF基因CDS区的全序列,并构建了重组表达载体pET-28a-VEGF。经过IPTG诱导,在原核细胞BL21中表达出了目的蛋白。探索了目的蛋白在大肠杆菌中的最佳表达条件:37℃,IPTG浓度0.1 mmol/L,诱导3 h是最佳表达条件。本研究实现了VEGF在原核细胞BL21的大量表达,此结果为后续在真核细胞上功能验证及黄牛转基因育种奠定了基础。更多还原

【Abstract】 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（6765T﹥C）, rs110750917（6860A﹥G）, rs109288296（6893T﹥C）. One SNP （6765T﹥C） was detected in intron2 and the other two SNPs （6860A﹥G, 6893T﹥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 （p﹥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 （p﹥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.更多还原