無毛大白豬生物學(xué)特征與無毛性狀遺傳機(jī)制研究
發(fā)布時間：2018-10-14 14:50
【摘要】：為充分了解新發(fā)現(xiàn)“無毛”表型大白豬群體,本研究以無毛表型豬為研究對象,以正常表型大白豬為對照,對“無毛”表型大白豬的生物學(xué)特性,包括生長性能、體尺指標(biāo)、繁殖性能、屠宰性能和肉品質(zhì)進(jìn)行了測定,探討了無毛性狀的遺傳規(guī)律,分析了皮膚組織學(xué)特性,同時采取候選基因關(guān)聯(lián)研究策略,運(yùn)用生物信息學(xué)方法,結(jié)合生物學(xué)軟件分析候選基因的序列特征,采用PCR產(chǎn)物直接測序的方法,篩查與大白豬毛囊發(fā)育和毛囊密度密切相關(guān)的候選基因,為闡明大白豬無毛性狀形成的分子機(jī)制奠定基礎(chǔ)。無毛表型大白豬生物學(xué)特性研究結(jié)果顯示,在相同飼養(yǎng)條件下,無毛表型仔豬初生重、達(dá)100 kg體重日齡、體尺指標(biāo)、100 kg活體背膘厚及繁殖性能均與正常表型大白豬無顯著差異(P0.05)。在屠宰性能上,“無毛”表型大白豬宰前活重、胴體重、屠宰率、背膘厚、瘦肉率、脂肪率、骨率、皮率均與正常表型大白豬無顯著差異(P0.05);在肉質(zhì)性狀上,“無毛”表型大白豬肉色、p H值、大理石紋、肌內(nèi)脂肪含量均與正常表型豬無顯著差異(P0.05)。結(jié)果表明,“無毛”大白豬在主要生產(chǎn)性能方面均表現(xiàn)正常,無毛性狀對豬的繁殖性能、生長發(fā)育和產(chǎn)肉性能均無顯著影響。通過設(shè)置相同表型間交配及不同表型間正反交雜交組合試驗(yàn),統(tǒng)計(jì)各雜交組合后代的表型,根據(jù)遺傳學(xué)定理,分別對無毛性狀是由一對基因或兩對基因控制的情況進(jìn)行邏輯推理,進(jìn)而確定無毛性狀的遺傳規(guī)律。結(jié)果顯示,大白豬的無毛性狀不是由簡單的一對或兩對基因位點(diǎn)控制的,該結(jié)果表明無毛性狀可能是由更多基因之間相互作用的結(jié)果。通過顯微觀察無毛表型大白豬被毛纖維結(jié)構(gòu)和皮膚組織結(jié)構(gòu),結(jié)果顯示:無毛表型大白豬毛干鱗片多呈魚鱗狀;毛干髓質(zhì)所占比例較正常被毛小,而皮質(zhì)所占比例相對較大。在不同階段,無毛表型豬被毛纖維平均細(xì)度較被毛正常表型豬小,且均有極顯著差異(P0.01)。不同部位真皮層厚度也較正常被毛表型豬薄,且單位面積毛囊密度較小。本研究發(fā)現(xiàn)大白豬的DKK1基因啟動子區(qū)存在4個變異位點(diǎn)-1833C/T、-1808C/G、-1628AATA插入/缺失、-1268TT插入/缺失,共生成7種單體型。獨(dú)立卡方檢驗(yàn)結(jié)果顯示:3個變異位點(diǎn)-1833C/T、-1808C/G、-1268ins/del的基因型在無毛表型和正常表型中均存在極顯著差異(P0.01),由此可知,DKK1基因啟動子區(qū)變異位點(diǎn)基因型分布與無毛表型相關(guān)聯(lián);利用啟動子在線預(yù)測軟件對DKK1基因啟動子區(qū)變異位點(diǎn)預(yù)測發(fā)現(xiàn),-1833CT和-1268(ins/del)變異均未破壞轉(zhuǎn)錄因子結(jié)合位點(diǎn),位點(diǎn)-1808CG變異,由HNF-1C轉(zhuǎn)錄因子結(jié)合位點(diǎn)變?yōu)镃/EBPalp和GR;而位點(diǎn)-1628(ins/del)的變異使得核心區(qū)將不包含TATA-box序列結(jié)構(gòu)。此外,還發(fā)現(xiàn)大白豬β-catenin基因啟動子區(qū)存在3個變異位點(diǎn)-1288GT、-1182TATT插入/缺失、-401TATT插入/缺失,共生成5種單體型,研究發(fā)現(xiàn)這些變異位點(diǎn)的基因型分布與無毛表型相關(guān)聯(lián);利用啟動子在線預(yù)測軟件對β-catenin基因啟動子區(qū)變異位點(diǎn)預(yù)測發(fā)現(xiàn),位點(diǎn)β-catenin-1288可結(jié)合轉(zhuǎn)錄因子為C/EBPalp,堿基G突變?yōu)門后會增加一個YY1轉(zhuǎn)錄因子結(jié)合位點(diǎn);位點(diǎn)β-catenin-1182(ins/del)和β-catenin-401(ins/del)變異后使得該位點(diǎn)附近序列能夠結(jié)合的轉(zhuǎn)錄因子隨即丟失,此外β-catenin-1182位點(diǎn)缺失TATT四個堿基后,核心區(qū)也不存在TATA-box序列結(jié)構(gòu),因而可推測以上變異位點(diǎn)均可能影響基因的轉(zhuǎn)錄活性。根據(jù)DKK1基因啟動子的序列特征,構(gòu)建5個不同缺失片段的p GL3-DKK1雙熒光素酶表達(dá)載體,分別轉(zhuǎn)染293T細(xì)胞和Hela細(xì)胞,并進(jìn)行雙報(bào)告基因活性檢測。結(jié)果顯示,DKK1基因啟動子對239T細(xì)胞具有偏好性,且5個重組p GL3-Basic載體熒光素酶活性均顯著高于p GL3-Basic空載體,其中p-1679/+292 bp啟動子片段活性最高,且顯著高于其他缺失片段(P0.01);-215—+219 bp維持了基礎(chǔ)轉(zhuǎn)錄活性,為核心啟動子區(qū)域;-586—-953 bp區(qū)域可能存在負(fù)調(diào)控元件,在-953—-1679 bp區(qū)域可能存在正調(diào)控元件。進(jìn)而構(gòu)建變異位點(diǎn)堿基缺失質(zhì)粒表達(dá)載體,轉(zhuǎn)染293T細(xì)胞,熒光素酶活性活性檢測結(jié)果顯示:PGL3-4(-1628 4N del和-1268 2N del)的啟動子活性最弱,其次為PGL3-2(-1628 4N del)、PGL3-3(-1268 2N del),且均極顯著低于PGL3-1(P0.01),結(jié)果表明變異位點(diǎn)堿基的缺失對DKK1基因的轉(zhuǎn)錄活性有顯著影響(P0.01)。依據(jù)β-catenin基因啟動子的序列特征,構(gòu)建3個不同缺失片段的p GL3-β-catenin雙熒光素酶表達(dá)載體,分別轉(zhuǎn)染293T細(xì)胞和Hela細(xì)胞,并進(jìn)行雙報(bào)告基因活性檢測。結(jié)果均顯示,試驗(yàn)組各組相對熒光素酶活性數(shù)值極低。進(jìn)而采用RT-PCR和熒光定量PCR方法,對β-catenin基因在不同細(xì)胞中的表達(dá)進(jìn)行相對定量,結(jié)果顯示:表達(dá)量由高到低依次為293T、LNCap、MCF7、PC3、Hep G2細(xì)胞。因而可推測該基因在細(xì)胞中的表達(dá)具有特異性。
[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.
【學(xué)位授予單位】：河北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S828
本文編號：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.
【學(xué)位授予單位】：河北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S828
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 蘇蕊;張文廣;常子麗;王瑞軍;李金泉;;內(nèi)蒙古絨山羊毛囊不同發(fā)育時期BMP2、Noggin及SHH基因在皮膚中的表達(dá)[J];揚(yáng)州大學(xué)學(xué)報(bào)(農(nóng)業(yè)與生命科學(xué)版);2009年01期
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