无毛大白猪生物学特征与无毛性状遗传机制研究
发布时间：2018-10-14 14:50
【摘要】：为充分了解新发现“无毛”表型大白猪群体,本研究以无毛表型猪为研究对象,以正常表型大白猪为对照,对“无毛”表型大白猪的生物学特性,包括生长性能、体尺指标、繁殖性能、屠宰性能和肉品质进行了测定,探讨了无毛性状的遗传规律,分析了皮肤组织学特性,同时采取候选基因关联研究策略,运用生物信息学方法,结合生物学软件分析候选基因的序列特征,采用PCR产物直接测序的方法,筛查与大白猪毛囊发育和毛囊密度密切相关的候选基因,为阐明大白猪无毛性状形成的分子机制奠定基础。无毛表型大白猪生物学特性研究结果显示,在相同饲养条件下,无毛表型仔猪初生重、达100 kg体重日龄、体尺指标、100 kg活体背膘厚及繁殖性能均与正常表型大白猪无显著差异(P0.05)。在屠宰性能上,“无毛”表型大白猪宰前活重、胴体重、屠宰率、背膘厚、瘦肉率、脂肪率、骨率、皮率均与正常表型大白猪无显著差异(P0.05);在肉质性状上,“无毛”表型大白猪肉色、p H值、大理石纹、肌内脂肪含量均与正常表型猪无显著差异(P0.05)。结果表明,“无毛”大白猪在主要生产性能方面均表现正常,无毛性状对猪的繁殖性能、生长发育和产肉性能均无显著影响。通过设置相同表型间交配及不同表型间正反交杂交组合试验,统计各杂交组合后代的表型,根据遗传学定理,分别对无毛性状是由一对基因或两对基因控制的情况进行逻辑推理,进而确定无毛性状的遗传规律。结果显示,大白猪的无毛性状不是由简单的一对或两对基因位点控制的,该结果表明无毛性状可能是由更多基因之间相互作用的结果。通过显微观察无毛表型大白猪被毛纤维结构和皮肤组织结构,结果显示:无毛表型大白猪毛干鳞片多呈鱼鳞状;毛干髓质所占比例较正常被毛小,而皮质所占比例相对较大。在不同阶段,无毛表型猪被毛纤维平均细度较被毛正常表型猪小,且均有极显著差异(P0.01)。不同部位真皮层厚度也较正常被毛表型猪薄,且单位面积毛囊密度较小。本研究发现大白猪的DKK1基因启动子区存在4个变异位点-1833C/T、-1808C/G、-1628AATA插入/缺失、-1268TT插入/缺失,共生成7种单体型。独立卡方检验结果显示:3个变异位点-1833C/T、-1808C/G、-1268ins/del的基因型在无毛表型和正常表型中均存在极显著差异(P0.01),由此可知,DKK1基因启动子区变异位点基因型分布与无毛表型相关联;利用启动子在线预测软件对DKK1基因启动子区变异位点预测发现,-1833CT和-1268(ins/del)变异均未破坏转录因子结合位点,位点-1808CG变异,由HNF-1C转录因子结合位点变为C/EBPalp和GR;而位点-1628(ins/del)的变异使得核心区将不包含TATA-box序列结构。此外,还发现大白猪β-catenin基因启动子区存在3个变异位点-1288GT、-1182TATT插入/缺失、-401TATT插入/缺失,共生成5种单体型,研究发现这些变异位点的基因型分布与无毛表型相关联;利用启动子在线预测软件对β-catenin基因启动子区变异位点预测发现,位点β-catenin-1288可结合转录因子为C/EBPalp,碱基G突变为T后会增加一个YY1转录因子结合位点;位点β-catenin-1182(ins/del)和β-catenin-401(ins/del)变异后使得该位点附近序列能够结合的转录因子随即丢失,此外β-catenin-1182位点缺失TATT四个碱基后,核心区也不存在TATA-box序列结构,因而可推测以上变异位点均可能影响基因的转录活性。根据DKK1基因启动子的序列特征,构建5个不同缺失片段的p GL3-DKK1双荧光素酶表达载体,分别转染293T细胞和Hela细胞,并进行双报告基因活性检测。结果显示,DKK1基因启动子对239T细胞具有偏好性,且5个重组p GL3-Basic载体荧光素酶活性均显著高于p GL3-Basic空载体,其中p-1679/+292 bp启动子片段活性最高,且显著高于其他缺失片段(P0.01);-215—+219 bp维持了基础转录活性,为核心启动子区域;-586—-953 bp区域可能存在负调控元件,在-953—-1679 bp区域可能存在正调控元件。进而构建变异位点碱基缺失质粒表达载体,转染293T细胞,荧光素酶活性活性检测结果显示:PGL3-4(-1628 4N del和-1268 2N del)的启动子活性最弱,其次为PGL3-2(-1628 4N del)、PGL3-3(-1268 2N del),且均极显著低于PGL3-1(P0.01),结果表明变异位点碱基的缺失对DKK1基因的转录活性有显著影响(P0.01)。依据β-catenin基因启动子的序列特征,构建3个不同缺失片段的p GL3-β-catenin双荧光素酶表达载体,分别转染293T细胞和Hela细胞,并进行双报告基因活性检测。结果均显示,试验组各组相对荧光素酶活性数值极低。进而采用RT-PCR和荧光定量PCR方法,对β-catenin基因在不同细胞中的表达进行相对定量,结果显示:表达量由高到低依次为293T、LNCap、MCF7、PC3、Hep G2细胞。因而可推测该基因在细胞中的表达具有特异性。
[Abstract]:In ord to fully understand that new findings, "No hair" Phenotypic big white pig population was studied with no wool-type pig as the research object, and the normal phenotype was large white pig as control. "No hair" The biological characteristics of large white pig, including growth performance, body scale index, reproductive performance, slaughtering performance and meat quality, were determined. The genetic law of non-wool traits was discussed, the histological characteristics of skin were analyzed, and the strategy of candidate gene association study was taken. Using bioinformatics method, combined with biological software to analyze the sequence characteristics of candidate genes, the candidate genes closely related to hair follicle development and hair follicle density of large white pigs were screened by PCR product direct sequencing. The results of the study on the biological characteristics of the non-hairy phenotype showed that in the same feeding condition, the birth weight of piglets with no wool-like phenotype reached 100 kg body weight day age, body scale index, and the thickness and reproductive performance of 100 kg living body were not significantly different from those of normal phenotypic big white pigs (P0.05). in slaughter performance, "No hair" There was no significant difference in carcass weight, carcass ratio, back thickness, lean meat rate, fat rate, bone rate and skin rate of large white pigs in large white pigs (P0.05). "No hair" There was no significant difference in the color of white pork, p H value, marble pattern and intramuscular fat content in normal phenotype pigs (P0.05). the results show that, "No hair" There was no significant effect on the reproductive performance, growth and meat performance of pigs. By setting the reciprocal cross-hybridization combination test between the same phenotypes and different phenotypes, the phenotype of each hybrid combination progeny is counted, and according to the genetic theorem, the non-hair property is controlled by a pair of genes or two pairs of gene control respectively, Furthermore, the genetic law of hair-free traits was determined. The results showed that the hair-free character of the large white pig was not controlled by a simple one or two pairs of gene sites, and the results showed that the non-hair trait could be the result of interaction between more genes. The results showed that the dry scale of white pig hair was much smaller than that of the normal hair, while the proportion of the cortex was relatively large. In different stages, the average fineness of wool fiber was smaller than that of normal phenotypic pig, and there was very significant difference (P <0.01). The thickness of dermis layer of different sites is also thinner than that of normal hair-phenotype pigs, and the density of hair follicles in unit area is small. Four mutation sites-1833C/ T,-1808C/ G,-1628AATA insertion/ deletion,-1268TT insertion/ deletion were found in the promoter region of DKK1 gene in large white pigs. The results of independent card test showed that the genotypic distribution of the mutation sites in the promoter region of DKK1 gene was associated with the non-hair phenotype in three variant sites-1833C/ T,-1808C/ G,-1268ins/ del, respectively (P0.01). Using the promoter online prediction software to predict the mutation site of the promoter region of DKK1 gene, both 1833CT and -1268 (ins/ del) variants did not destroy transcription factor binding sites, and the site-1808CG mutation was changed from the binding site of HNF-1C transcription factor to C/ EBPalp and GR. The mutation of site-1628 (ins/ del) makes the core region not contain the TATA-box sequence structure. In addition, three mutation sites-1288GT,-1182TATT insertion/ deletion,-401TATT insertion/ deletion were found in the promoter region of the pig-catenin gene of the large white pig, and 5 types of haplotypes were generated. Using the promoter online prediction software to predict the mutation site of the promoter region of the promoter region, the transcription factor C/ EBPalp can be combined with the transcription factor, and a YY1 transcription factor binding site can be added after the mutation of the base G is T; The transcription factors which could bind to the site near the site were lost immediately after the mutation of the sites-catenin-1182 (ins/ del) and p27-catenin-401 (ins/ del), and no TATA-box sequence structure was present in the core region after the deletion of the four bases of the TATT-catenin-1182 site. Therefore, it was estimated that the above mutation sites could affect the transcription activity of the gene. According to the sequence characteristics of DKK1 gene promoter, we constructed a double luciferase expression vector pGL3-DKK1 with five different deleted fragments, respectively transfect 293T cells and Jurkat cells, and conduct double reporter gene activity detection. The results showed that the promoter of DKK1 gene had preference for 239T cells, and the luciferase activity of 5 recombinant pGL3-Basic carriers was significantly higher than that of the p-GL3-Basic empty vector, in which the promoter fragment activity of p-1679/ + 292 bp was the highest and was significantly higher than that of other deleted fragments (P0.01).-215 bp + 219 bp maintained basal transcription activity as a core promoter region; a negative regulatory element may be present in the region of -586a-953bp, and a positive regulatory element may exist in the region of -953a-1679 bp. It was found that the promoter activity of PGL3-4 (-1628 4N del and-1268 2N del) was the most weak, followed by PGL3-2 (-1628 4N del), PGL3-3 (-1268 2N del) and was significantly lower than PGL3-1 (P0.01). The results showed that the deletion of variant sites had a significant effect on the transcription activity of DKK1 gene (P0.01). According to the sequence characteristics of the promoter of the p27-catenin gene, a pGL3-CoV-catenin double luciferase expression vector with three different deleted fragments was constructed, 293T cells and Jurkat cells were transfected respectively, and the double reporter gene activity detection was carried out. Results showed that the relative luciferase activity values were very low in the test groups. Furthermore, RT-PCR and fluorescence quantitative PCR were used to compare the expression of p27-catenin gene in different cells. The results showed that the expression level was 293T, LNCa7, MCF7, PC3, Hep G2 cells in order from high to low. It is therefore possible to speculate that the gene has specificity in the expression of cells.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828
本文编号：2270798
[Abstract]:In ord to fully understand that new findings, "No hair" Phenotypic big white pig population was studied with no wool-type pig as the research object, and the normal phenotype was large white pig as control. "No hair" The biological characteristics of large white pig, including growth performance, body scale index, reproductive performance, slaughtering performance and meat quality, were determined. The genetic law of non-wool traits was discussed, the histological characteristics of skin were analyzed, and the strategy of candidate gene association study was taken. Using bioinformatics method, combined with biological software to analyze the sequence characteristics of candidate genes, the candidate genes closely related to hair follicle development and hair follicle density of large white pigs were screened by PCR product direct sequencing. The results of the study on the biological characteristics of the non-hairy phenotype showed that in the same feeding condition, the birth weight of piglets with no wool-like phenotype reached 100 kg body weight day age, body scale index, and the thickness and reproductive performance of 100 kg living body were not significantly different from those of normal phenotypic big white pigs (P0.05). in slaughter performance, "No hair" There was no significant difference in carcass weight, carcass ratio, back thickness, lean meat rate, fat rate, bone rate and skin rate of large white pigs in large white pigs (P0.05). "No hair" There was no significant difference in the color of white pork, p H value, marble pattern and intramuscular fat content in normal phenotype pigs (P0.05). the results show that, "No hair" There was no significant effect on the reproductive performance, growth and meat performance of pigs. By setting the reciprocal cross-hybridization combination test between the same phenotypes and different phenotypes, the phenotype of each hybrid combination progeny is counted, and according to the genetic theorem, the non-hair property is controlled by a pair of genes or two pairs of gene control respectively, Furthermore, the genetic law of hair-free traits was determined. The results showed that the hair-free character of the large white pig was not controlled by a simple one or two pairs of gene sites, and the results showed that the non-hair trait could be the result of interaction between more genes. The results showed that the dry scale of white pig hair was much smaller than that of the normal hair, while the proportion of the cortex was relatively large. In different stages, the average fineness of wool fiber was smaller than that of normal phenotypic pig, and there was very significant difference (P <0.01). The thickness of dermis layer of different sites is also thinner than that of normal hair-phenotype pigs, and the density of hair follicles in unit area is small. Four mutation sites-1833C/ T,-1808C/ G,-1628AATA insertion/ deletion,-1268TT insertion/ deletion were found in the promoter region of DKK1 gene in large white pigs. The results of independent card test showed that the genotypic distribution of the mutation sites in the promoter region of DKK1 gene was associated with the non-hair phenotype in three variant sites-1833C/ T,-1808C/ G,-1268ins/ del, respectively (P0.01). Using the promoter online prediction software to predict the mutation site of the promoter region of DKK1 gene, both 1833CT and -1268 (ins/ del) variants did not destroy transcription factor binding sites, and the site-1808CG mutation was changed from the binding site of HNF-1C transcription factor to C/ EBPalp and GR. The mutation of site-1628 (ins/ del) makes the core region not contain the TATA-box sequence structure. In addition, three mutation sites-1288GT,-1182TATT insertion/ deletion,-401TATT insertion/ deletion were found in the promoter region of the pig-catenin gene of the large white pig, and 5 types of haplotypes were generated. Using the promoter online prediction software to predict the mutation site of the promoter region of the promoter region, the transcription factor C/ EBPalp can be combined with the transcription factor, and a YY1 transcription factor binding site can be added after the mutation of the base G is T; The transcription factors which could bind to the site near the site were lost immediately after the mutation of the sites-catenin-1182 (ins/ del) and p27-catenin-401 (ins/ del), and no TATA-box sequence structure was present in the core region after the deletion of the four bases of the TATT-catenin-1182 site. Therefore, it was estimated that the above mutation sites could affect the transcription activity of the gene. According to the sequence characteristics of DKK1 gene promoter, we constructed a double luciferase expression vector pGL3-DKK1 with five different deleted fragments, respectively transfect 293T cells and Jurkat cells, and conduct double reporter gene activity detection. The results showed that the promoter of DKK1 gene had preference for 239T cells, and the luciferase activity of 5 recombinant pGL3-Basic carriers was significantly higher than that of the p-GL3-Basic empty vector, in which the promoter fragment activity of p-1679/ + 292 bp was the highest and was significantly higher than that of other deleted fragments (P0.01).-215 bp + 219 bp maintained basal transcription activity as a core promoter region; a negative regulatory element may be present in the region of -586a-953bp, and a positive regulatory element may exist in the region of -953a-1679 bp. It was found that the promoter activity of PGL3-4 (-1628 4N del and-1268 2N del) was the most weak, followed by PGL3-2 (-1628 4N del), PGL3-3 (-1268 2N del) and was significantly lower than PGL3-1 (P0.01). The results showed that the deletion of variant sites had a significant effect on the transcription activity of DKK1 gene (P0.01). According to the sequence characteristics of the promoter of the p27-catenin gene, a pGL3-CoV-catenin double luciferase expression vector with three different deleted fragments was constructed, 293T cells and Jurkat cells were transfected respectively, and the double reporter gene activity detection was carried out. Results showed that the relative luciferase activity values were very low in the test groups. Furthermore, RT-PCR and fluorescence quantitative PCR were used to compare the expression of p27-catenin gene in different cells. The results showed that the expression level was 293T, LNCa7, MCF7, PC3, Hep G2 cells in order from high to low. It is therefore possible to speculate that the gene has specificity in the expression of cells.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828
【参考文献】
相关期刊论文 前1条
1 苏蕊;张文广;常子丽;王瑞军;李金泉;;内蒙古绒山羊毛囊不同发育时期BMP2、Noggin及SHH基因在皮肤中的表达[J];扬州大学学报(农业与生命科学版);2009年01期
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