【作者】 刘永峰；

【导师】 昝林森； 李奎；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 本研究以秦川牛、鲁西牛、南阳牛、郏县红牛、夏南牛、婆罗门牛、BMY牛、雪龙黑牛、秦杂牛组合、西鲁杂牛组合等群体为研究对象,运用电子克隆技术和DNA序列分析方法对部分基因进行了克隆;结合生物信息学方法,运用启动子分析技术对牛部分基因启动子的结构和功能进行了分析;结合生物软件,运用生物信息学方法分析了13个体尺相关基因的DNA和蛋白序列的特征;运用荧光定量PCR技术和生物信息学方法,对13个基因进行了组织表达谱分析;运用PCR-RFLP、等位基因PCR技术(AS-PCR)、创造酶切位点酶切技术(ACRS-PCR-RFLP)、DNA直接测序技术、飞行质谱MALDI-TOF技术分析了13个基因的多态性;通过生物统计学软件的应用,对检测的38个SNP位点进行了频率、平衡性、群体遗传性及与体尺性状的相关性进行了统计分析。筛选出对中国黄牛体尺性状有重要影响的功能基因和分子标记,以期发现对牛体尺性状的有效分子标记,为中国黄牛生长发育性状的分子标记辅助选择和优质高产新品种(系)牛的培育提供科学依据。本研究主要结果如下:1.采用电子克隆(e-PCR)、反转录PCR(RT-PCR)、基因组学等技术,从秦川牛组织中分离、克隆得到了牛UQCC基因编码区全长序列和内含子序列,得到牛GDF5基因和CDK6基因的启动子序列。2.对牛UQCC、GDF5、ADAMTSL3、ZBTB38、CDK6、HHIP、IHH、GHRL、PTHR1、INS、UCP2、col1A1和col1A2基因,通过生物信息学方法对其序列特征和功能进行了预测分析,得到了13个基因的序列及氨基酸信息、预测了13个基因蛋白质的二级和三级结构、信号肽序列、跨膜结构、功能域结构,并对这些蛋白进行了亚细胞定位,与其它物种该基因的蛋白进行了保守性分析,构建了系统发生树。3.采用荧光定量PCR技术,分析牛UQCC和ADAMTSL3基因的组织表达谱,发现牛UQCC基因在牛不同组织中由高到低表达的顺序为:脾脏、心脏、气管、大肠、脂肪、睾丸、肾脏、肌肉和膀胱;牛ADAMTSL3基因在牛不同组织中由高到低表达的顺序为:睾丸、心脏、脂肪、胃、小肠、肝脏、肺脏、气管、肾脏、脾脏、大肠和肌肉。结合生物信息学方法,分析、归纳了13个基因的组织表达谱信息。4.采用生物信息学方法,结合国外文献对人基因启动子序列结构和特征的报道,分析了牛GDF5基因和CDK6基因启动子的结构和功能,发现牛GDF5基因启动子没有TATA box和CAAT box结构,发现了不同于人该基因的转录因子AML-la、CdxA、GTATA-1、MZF1、CdxA和CdxA,推测断定牛GDF5基因启动子转录起始位点位于翻译起始密码子ATG上游-359bp位置,-457至-423序列中的GT重复序列可以增加基因的转录,其最小增强子处于-458和-377之间;发现牛CDK6基因启动子亦没有TATA box和CAAT box结构,发现了不同于人该基因的转录因子Ik-2、Ik-1、CREB和NF-E2,推测断定牛GDF5基因启动子转录起始位点位于翻译起始密码子ATG上游-90bp位置。5.采用PCR-RFLP酶切技术检测了牛UQCC和GDF5基因的3个位点的多态性,在牛群中进行基因分型,并与体尺性状进行关联分析,结果发现:牛UQCC基因的A2691T SNP基因型与总群体的尻长、胸深和坐骨端宽极显著相关(P<0.01),A3150G SNP基因型与总群体的体长和胸深显著相关(P<0.05),两位点联合基因型与总群体的体长、胸深和坐骨端宽显著相关(P<0.05);牛GDF5基因的T586C SNP基因型与普通牛总群体的体长和腰角宽极显著相关(P<0.01),与BMY牛群体的体长显著相关(P<0.05),与秦川牛的体长、体高和腰角宽极显著相关(P<0.01),与秦杂牛组合的体长、腰高和腰角宽显著相关(P<0.05)。6.采用AS-PCR技术分析了牛ADAMTSL3基因和ZBTB38基因的3个位点的多态性,在牛群中进行基因分型,并与体尺性状进行关联分析,结果发现:牛ADAMTSL3基因的T1532C SNP基因型与总群体的体尺指标无显著相关(P>0.05),C1899T SNP基因型与总群体的坐骨端宽显著相关(P<0.05),两位点联合基因型与总群体的体长、胸深、胸围和坐骨端宽显著相关(P<0.05);牛ZBTB38基因的A841G SNP基因型与总群体的体长显著相关(P<0.05),与秦杂牛组合群体腰高和胸围显著相关(P<0.05),与雪龙黑牛群体体高显著相关(P<0.05)。7.采用创造酶切位点PCR技术分析了牛CDK6基因的1个位点的多态性,在牛群中进行基因分型,并与体尺性状进行关联分析,结果发现:牛CDK6基因的T-1075C SNP基因型与总群体的体长和胸围显著相关(P<0.05)。8.采用DNA直接测序技术分析了牛ZBTB38基因的9个位点的多态性,通过单倍型分析、连锁不平衡分析,在牛群中进行基因分型,并与体尺性状进行关联分析,结果发现:牛ZBTB38基因9个SNP位点形成单倍型中频率大于0.03的有TTGCTGCGG(0.643)、TTATTGCGG(0.137)、CCGCTCTAA(0.130)和CCGCCGCGA(0.041)等4个;T2145C、T2286C、G2457C、C2583T、G2643A、G2676A等6个SNP位点可作为一个整体(r2>0.33),G2323A和C2325T可作为一个整体(r2>0.33);T2145C、T2286C、G2457C、C2583T、G2643A、G2676A等6个SNP与牛总群体体尺数据无显著相关(P>0.05),T2409C SNP与牛总群体体尺数据亦无显著相关(P>0.05),仅G2323A和C2325T SNP与总群体的体长、体高和尻长有显著相关关系(P<0.05)。9.结合多重PCR技术,从牛65个多态位点中筛选了31目的位点,采用中通量多态分析方法——飞行质谱MALDI-TOF技术对分析了牛31个多态位点的多态性,通过单倍型分析、连锁不平衡分析,在牛群中进行基因分型,并与体尺性状进行关联分析,结果发现:8个SNP位点为假SNP ,23个为有效SNPs,并分布于位于2号、19号、21号、29号、4号(4个)、13号(4个)、15号(6个)、17号(2个)、22号(3个)染色体上;其中15号染色体上SNP58、SNP60、SNP63可作为一个整体(r2>0.33),SNP61与SNP63可作为一个整体(r2>0.33),22号染色体上的SNP22与SNP25可作为一个整体(r2>0.33);GDF5基因的SNP9、col1A2基因的SNP36和SNP37、ZBTB38基因的SNP46、UQCC基因的SNP49和SNP50、UCP2基因的SNP55、SNP58、SNP60、SNP61和SNP63,这11个SNPs与牛总群体体尺性状无显著相关(P>0.05),CDK6基因的SNP4基因型与总群体的腰高和胸围显著相关(P<0.05),CDK6基因的SNP5基因型与总群体的腰角宽显著相关(P<0.05),HHIP基因的SNP12基因型与总群体的体高显著相关(P<0.05),HHIP基因的SNP13基因型与总群体的体长和体高显著相关(P<0.05),IHH基因的SNP14基因型与总群体的胸深极显著相关(P<0.01),GHPL基因的SNP16基因型与总群体的体高显著相关(P<0.05),PTHR1基因的SNP22基因型与总群体的体长、体高、腰角宽、胸围和坐骨端宽显著相关(P<0.05),PTHR1基因的SNP25基因型与总群体的体长、腰角宽、胸围和坐骨端宽显著相关(P<0.05),col1A1基因的SNP30基因型与总群体的坐骨端宽显著相关(P<0.05),INS基因的SNP41基因型与总群体的尻长显著相关(P<0.05),GDF5基因的SNP52基因型与总群体的体长和体高显著相关(P<0.05),UCP2基因的SNP64基因型与总群体的腰角宽和胸围显著相关(P<0.05)。更多还原

【Abstract】 The e-PCR and DNA sequence analysis techniques were applied not only to clone and predict several bovine genes but also analyze partial stuctures and functions of these genes promoters in ten bovin breeds, including Qinchuan (QC), Luxi (LX), Nanyang (NY), Jiaxian Red (JR), Xianan (XN), Brahma (BH), Brahma crossed with descendant of male murry grey and female Yunnan yellow cattle (BMY), Angus crossed with descendant of male Japanese Black cattle and female Fuzhou cattle (XL), Qinchuan improvement steers (QI) and Simmental and Luxi crossbred steers (SL). Combining biological software and bioinformatics, the characteristics of 13 body measurement traits related to these genes sequences of DNA and protein were analyzed. The tissue expression of these 13 genes were analyzed by Real-time-PCR and bioinformatics, while the polymorphism of these genes were analyzed by RT-PCR, bioinformatics, PCR-RFLP, AS-PCR, CARS-PCR-RFLP and MALDI-TOF. The frequency, equitability, total heritability and relevance of body measurement traits of 13 SNPs were analyzed by Bio-statistics.In order to produce scientific proof for marker-assisted selection of growth and devolopment traits of chinese yellow cattle and built scientific basis for high-yield breeding skills of new breed, some of the effecitve function genes and molecular marks were selected in our study reaserch. The main resules of our study are as follwed:1. The whole cDNA sequence , intron of UQCC gene and partial promoter sequences of GDF5 and CDK6 gene were separated, cloned and identified from Qinchuan cattle by e-PCR, RT-PCR and genomics.2. The characteristics and functions of gene sequences and amino acid information in 13 genes, including UQCC, GDF5, ADAMTSL3, ZBTB38, CDK6, HHIP, IHH, GHRL, PTHR1, UCP2, collA1 and collA2, were predicted and analyzed by bioinformatics. The secondary and tertiary structure, signal peptide sequence, transmembrane region and functional domain of these 13 proteins were predicted and analyzed, too. The phylogenetic trees were also built by comparison with other species and conservative analysis of corresponding proteins in other species. 3. Real-time-PCR showed the content order of UQCC in different cattle tissues and the order from high to low is spleen, heart, trachea, large intestine, fat, testis, kinney, muscle and bladder, while the content order of ADAMTSL3 is testis, heart, fat, stomach, small intestine, liver, lung, trachea, kidney, spleen, large intestine and muscle.4. Combinding abroad articles about human promter genes, bioinformatics were used to analyze the strcture and function of bovine GDF5 and CDK6 gene. Several different cattle transcription factors, named AML-LA, CdxA, GTATA-1 and MZF1, were found, which implied the transcriptional start site in GDF5 was at -359 bp position before ATG and the GT repetitive sequence between -457 bp and -423 bp could enhance gene transcription, while the samllest enhancer was between -458 bp and -377 bp and no TATA box or CAAT box was found. On the other hand, Lk-2, Ik-1, CREB and NF-E2 were found in CDK6 gene, with no TATA box or CAAT box in CDK6 either,which implied that the transcriptional start site was at -90 bp before ATG.5. PCR-RFLP technology were adopted to detect the polymorphism of three locus of UQCC gene and GDF5 gene and analysis of genotype identification in bovine population and their association with body measurement traits were conducted, the results show that the genotypes of A2691T SNP of bovine UQCC gene have a highly remarkable correlation with Rump Length (RL), Chest Depth (CD) and Pin Bone Width (PBW) in the total population (P<0.01), the genotypes of A3150G SNP have an significant association with Body Length (BL) and CD in the total population(P<0.05), joint genotypes are conspicuously relavant with BL, CD, PBW in the total population (P<0.05); the genotypes of T586C SNP of GDF5 gene have a highly remarkable correlation with BL, Hip Width (HW) in Bos Taurus population (P<0.01), they are also significantly correlated with BL in BMY population (P<0.05) and BL, Hip Height (HH) and HW in QI population (P<0.05).6. Three SNPs polymorphism of bovine ADAMTSL3 gene and ZBTB38 gene were studied using AS-PCR technology, at the same time, genotypes identification in bovine population and their association with body measurement traits were analyzed. The result show that the genotypes of T1532C SNP of bovine ADAMTSL3 gene have no significant correlation with body measurement in total population (P>0.05), and the genotypes of C1899T SNP there is a remarkable correlation with PBW(P<0.05), joint genotypes is significantly relavant with BL, CD, Heart Girth (HG) and PBW in the total population(P<0.05); the genotypes of A841G SNP of bovine ZBTB38 gene have a conspicuous association with BL in total population (P<0.05), HH and HG in the QI population (P<0.05) and Withers Height (WH) in XL population.7. ACRS-PCR-RFLP technology were adopted to study a SNP polymorphism of bovine CDK6 gene, genotype identification in bovine population and their association with body measurement were analyzed, the results show that the genotypes of T-1075C SNP of CDK 6 gene have a remarkable association with BL and HG in total population (P<0.05).8. The polymorphism of 9 locus of bovine ZBTB38 gene were studied through DNA sequencing. The analysis results of haplotype, linkage disequilibrium, genotype identification in bovine population and their association with body measurement traits show that, from the nine SNPs, four SNPs haplotype frequencies, including TTGCTGCGG (0.643), TTATTGCGG (0.137), CCGCTCTAA (0.130) and CCGCCGCGA (0.041), are higher than 0.03; SNPs of T2145C, T2286C, G2457C, C2583T, G2643A, G2676A could be seen as an integrity (r2>0.33), G2323A and C2325T could be seen as an integrity (r2>0.33), SNPs of T2145C, T2286C, G2457C, C2583T, G2643A, G2676A have no correlation with boty measurement traits in total bovine population (P>0.05), only SNPs of G2323A and C2325T have an significant association with body measurement traits of BL,WH and RL in the total population.9. Thirty one locus were selected from bovine 65 SNPs to anyalze their polymorphism using MALDI-TOF and multi-PCR technology. Haplotype, linkage disequilibrium, genotyope identification in bovine population and their association with body measurement traits were analyzed. The results show that 8 locus of SNP are faulse, 23 SNPs are effective and located on the chromosome of 2, 19, 21, 29, 4 (4 SNPs), 13 (4 SNPs), 15 (6 SNPs), 17 (2 SNPs) and 22 (3 SNPs) respectively. From all these effective SNPs, three SNPs on chromosome 15, including SNP 58, SNP 60 and SNP 63, could be seen as an integrity (r2>0.33); SNP22 and SNP25 of chromosome 22 could be seen as an integrity (r2>0.33); eleven SNPs, including SNP9 of GDF5 gene, SNP36 and SNP37 of col1A2 gene, SNP46 of ZBTB38 gene, SNP49 and SNP50 of UQCC gene, SNP55, SNP58, SNP 60, SNP61 and SNP 63 of UCP2 gene, have no significant correlation with body measurement traits in total bovine population (P>0.05); the genotypes of SNP4 on CDK6 gene are significantly associated with HH and HG in the total population (P<0.05), the genotypes of SNP5 on CDK6 gene have prominent association with PBW in the total pouplation (P<0.05), genotypes of SNP12 on HHIP gene are significantly relavant with WH in the total population (P<0.05), genotypes of SNP13 on HHIP gene are significantly relavant with BL and WH in the total population (P<0.05), genotypes of SNP14 on IHH gene have highly significant difference with CD in the total population (P<0.01), genotypes of SNP16 on GHPL gene are have a remarkable association with WH in the total population (P<0.05), genotypes of SNP22 on PTHR1 gene are significantly associated with BL, WH, PBW, HG and HW in the total population (P<0.05), genotypes of SNP25 on PTHR1 gene are significantly relavant with BL, HW, HG and PBW in the total population (P<0.05), genotypes of SNP30 on col1A1 gene are significantly associated with PBW in the total population (P<0.05), genotypes of SNP41 on INS gene have a distinct association with RL in the total population (P<0.05), the genotypes of SNP52 on GDF5 gene have a remarkable correlation with BL and WH in the total population (P<0.05), genotypes of SNP64 on UCP2 gene have significant correlation with HW and HG in the total population (P<0.05).更多还原