本文選題：綿羊 + 基因表達(dá)　； 參考：《甘肅農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：繁殖力是綿羊的重要經(jīng)濟(jì)指標(biāo)之一,直接影響綿羊經(jīng)濟(jì)效益。母羊的產(chǎn)羔數(shù)是衡量綿羊繁殖力的重要指標(biāo),因此,提高綿羊的產(chǎn)羔數(shù)對(duì)發(fā)展養(yǎng)羊業(yè)十分重要。綿羊的產(chǎn)羔性狀是微效多基因控制的數(shù)量性狀,通過(guò)與數(shù)量性狀位點(diǎn)相連鎖的分子標(biāo)記實(shí)現(xiàn)對(duì)基因型的直接選擇,將傳統(tǒng)選育方法和現(xiàn)代分子育種方法相結(jié)合運(yùn)用于育種實(shí)踐,將會(huì)極大地提高選擇效率,進(jìn)一步提高綿羊繁殖性能。本研究以與綿羊繁殖性狀相關(guān)的NGF、TrkA、KIT、KITLG、LIF、LIFR、NPM1、NCOA1、ADAMTS1和NOGGIN等10個(gè)基因?yàn)檠芯繉?duì)象,利用Real-time PCR分析其在湖羊心、肝、脾、肺、腎、瘤胃、十二指腸、背最長(zhǎng)肌、脂肪、下丘腦、垂體和卵巢等12種組織中的表達(dá)特征,并采用DNA混合池測(cè)序、限制性長(zhǎng)度片段多態(tài)性(Restriction fragment length polymorphism,RFLP-RCR)和SNPscan分型技術(shù)研究以上10個(gè)基因全部外顯子及其側(cè)翼區(qū)SNPs與綿羊(湖羊,n=556;小尾寒羊,n=444)產(chǎn)羔性狀的關(guān)聯(lián)性,主要研究結(jié)果如下:(1)NGF、TrkA、KIT、KITLG、LIF、LIFR、NPM1、NCOA1、ADAMTS1和NOGGIN基因在湖羊心、肝、脾、肺、腎、瘤胃、十二指腸、背最長(zhǎng)肌、脂肪、下丘腦、垂體和卵巢等12種組織中廣泛表達(dá),其中NGF在卵巢、下丘腦和心臟中的表達(dá)量較高,在背最長(zhǎng)肌、瘤胃、肺、肝臟和脂肪表達(dá)量較低;TrkA基因在卵巢和肺中的表達(dá)量最高,而在背最長(zhǎng)肌、瘤胃、脂肪、下丘腦、垂體、脾、腎和十二指腸表達(dá)量較低;KITLG基因在心臟、卵巢和肺中表達(dá)量較高,在肝臟、瘤胃、脾臟和背最長(zhǎng)肌中表達(dá)量最低;KIT基因在卵巢、肝臟和肺中表達(dá)量最高,在脾臟、腎臟、瘤胃、十二指腸和脂肪組織中的表達(dá)量較低,在背最長(zhǎng)肌中最低;LIF基因在卵巢中表達(dá)量最高,其次為下丘腦、垂體和肝臟,在脾、肺、腎、瘤胃、十二指腸、背最長(zhǎng)肌和脂肪組織中表達(dá)較少;LIFR基因在卵巢、心臟、垂體、腎臟和肺中表達(dá)量較高,在其它組織中的表達(dá)量較低;NPM1在卵巢和肝臟中的表達(dá)量最高,表達(dá)量較低的組織為背最長(zhǎng)肌、脂肪、垂體和肺;NCOA1基因在卵巢、垂體、肝臟和肺中表達(dá)量最高,而在背最長(zhǎng)肌中表達(dá)量最低。ADAMTS1基因卵巢、肺、肝臟和瘤胃中表達(dá)量最高,在背最長(zhǎng)肌中呈現(xiàn)最低水平;NOGGIN基因的表達(dá)量在卵巢、脾臟和肺中最高,而在腎臟和背最長(zhǎng)肌中最低。以上結(jié)果顯示所研究候選基因均在卵巢中表達(dá)量較高,提示它們可能與卵巢的功能相關(guān)。(2)利用DNA池測(cè)序法對(duì)以上10個(gè)基因所有外顯子及側(cè)翼區(qū)SNPs進(jìn)行掃描,共檢測(cè)到78個(gè)突變位點(diǎn),其中在KIT基因上發(fā)現(xiàn)3個(gè)突變位點(diǎn),KITLG上發(fā)現(xiàn)6個(gè)突變位點(diǎn),ADAMTS1上7個(gè)突變位點(diǎn),NCOA1上6個(gè),NPM1上14個(gè),LIF上2個(gè),LIFR上23個(gè),NGF上4個(gè),TrkA上13個(gè),在NOGGIN基因上沒(méi)有檢測(cè)到SNP。分析突變類型,發(fā)現(xiàn)4個(gè)錯(cuò)義突變,分別位于KIT基因第10外顯子上g.70224398TA位點(diǎn)(Leu-His),ADAMTS1基因第9外顯子上的g.127756130GA(Met-Val),LIFR基因第7外顯子上g.35835474 GT(Ala-Ser),第12外顯子g.35835329 GA(Asn-Ser)。同時(shí)發(fā)現(xiàn)8個(gè)同義突變,分別為ADAMTS1第2外顯子上的g.127751615CT和g.127753565TC,在第5外顯子上g.127753643CT和g.127753727CT,NCOA1基因第8外顯子上的g.32072394CT,NGF基因在第1外顯子上的g.91651197GA,LIFR基因在外顯子6上的g.35835329GA,NPM1基因第5外顯子上的g.3247689AT。其余的突變位點(diǎn)均位于內(nèi)含子上。(3)利用PCR-RFLP方法對(duì)1000只湖羊和小尾寒羊母羊群體NGF基因的g.91651197GA,TrkA基因的g.105281586CT和g.105284246GC,KITLG基因的g.124502403CT和g.124511398TC等5個(gè)SNP位點(diǎn),進(jìn)行基因分型,并分析多態(tài)位點(diǎn)與綿羊產(chǎn)羔數(shù)關(guān)聯(lián)性,結(jié)果表明:在小尾寒羊和湖羊群體中,NGF基因g.91651197GA位點(diǎn)、TrkA基因的g.105281586CT和KITLG基因g.124511398TC位點(diǎn)與產(chǎn)羔數(shù)顯著相關(guān)(P0.05),而TrkA基因的g.105284246GC突變位點(diǎn)僅在湖羊群體中表現(xiàn)出不同基因型個(gè)體的產(chǎn)羔數(shù)差異顯著(P0.05)。分析了NGF和TrkA基因聚合效應(yīng)對(duì)綿羊產(chǎn)羔數(shù)的影響,結(jié)果發(fā)現(xiàn)兩個(gè)基因的GACTCC基因型組合為最優(yōu)組合基因型,表明NGF和TrkA基因組合共同影響綿羊產(chǎn)羔數(shù)。(4)對(duì)以上9個(gè)基因中檢測(cè)到的78個(gè)SNPs位點(diǎn)進(jìn)行評(píng)估,發(fā)現(xiàn)有62個(gè)SNPs位點(diǎn)可以利用SNPscan分型法進(jìn)行基因分型,對(duì)于分型結(jié)果與產(chǎn)羔數(shù)關(guān)聯(lián)性分析表明:以上9個(gè)基因均有SNP位點(diǎn)與綿羊產(chǎn)羔數(shù)相關(guān),KIT基因在g.70199073AG,LIFR基因的g.35845474 CT、g.3584563TC和g.35853637TG,NCOA1基因g.32140565 GA位點(diǎn)與小尾寒羊的產(chǎn)羔數(shù)存在著顯著相關(guān)(P0.05);NPM1基因的g.3246266 TG位點(diǎn)和NCOA1基因g.31928230 CT位點(diǎn)與湖羊產(chǎn)羔數(shù)顯著相關(guān)(P0.05)。單倍型分析中,在9個(gè)基因中共檢測(cè)到了50個(gè)不同的單倍型,其分布并不是均衡的,單倍型頻率最高的是KITLG基因的單倍型H8,其單倍型頻率為0.89,為優(yōu)勢(shì)單倍型,頻率最低的單倍型為L(zhǎng)IFR基因的H22,其頻率為0.0161。單倍型H3、H14、H17、H20、H23、H29、H47和H48與產(chǎn)羔性狀顯著相關(guān)(P0.05)。
[Abstract]:Fecundity is one of the important economic indicators of sheep, which directly affects the economic benefit of sheep. The number of lambs of the ewes is an important index to measure the fecundity of the sheep. Therefore, it is very important for the sheep to increase the number of lambs to develop the sheep industry. The lambing character of the sheep is a quantitative trait controlled by the micro effect gene, which is linked to the quantitative trait loci. The direct selection of the genotypes by molecular markers, the combination of traditional breeding methods and modern molecular breeding methods used in breeding practice will greatly improve the selection efficiency and further improve the reproductive performance of sheep. This study is based on 10 NGF, TrkA, KIT, KITLG, LIF, LIFR, NPM1, NCOA1, ADAMTS1 and NOGGIN, which are related to the reproductive traits of sheep. As an object of study, Real-time PCR was used to analyze its expression in 12 tissues of Hu sheep heart, liver, spleen, lung, kidney, kidney, rumen, duodenum, dorsal longest muscle, fat, hypothalamus, pituitary and ovary, and DNA mixed pool sequencing was used to limit polymorphisms of Restriction fragment length polymorphism, RFLP-RCR and SNPscan. Study the association of all 10 gene exons and their flank SNPs with sheep (Hu sheep, n=556, small tail Han sheep, n=444). The main results are as follows: (1) NGF, TrkA, KIT, KITLG, LIF, LIFR, NPM1, NCOA1, ADAMTS1, and base, the liver, spleen, lung, kidney, rumen, duodenum, dorsal longest muscle, fat, inferior colliculus The expression of NGF in the ovary, the hypothalamus, and the heart is highly expressed in the ovary, the hypothalamus, and the heart. The expression of the rumen, the rumen, the lungs, the liver and the fat is low. The expression of the TrkA gene in the ovary and lung is the highest, while the expression of the longest muscle in the back, the stomach, the fat, the hypothalamus, the hypophysis, the spleen, the kidney and the duodenum is more than that of the dorsi back. Low expression of KITLG gene in the heart, ovary and lung, the lowest expression in the liver, rumen, spleen and the longest muscle in the spleen, and the highest expression of the KIT gene in the ovaries, liver and lungs, low expression in the spleen, kidney, rumen, duodenum and adipose tissue, and the lowest in the longest muscle in the back, and the highest expression of the LIF gene in the ovary. Second, the hypothalamus, hypophysis, and liver were expressed in the spleen, the lung, the kidney, the rumen, the duodenum, the duodenum, the longest muscle and the adipose tissue. The expression of LIFR gene in the ovary, heart, the pituitary, kidney and lung was high, and the expression in other tissues was low; the expression of NPM1 in the ovary and liver was the highest, and the lower expression of the tissue was the longest muscle in the back. The highest expression of the NCOA1 gene in the ovary, the pituitary, the liver, and the lungs, and the lowest expression of the.ADAMTS1 gene in the longest muscle in the dorsum, the highest expression in the lung, liver, and rumen, and the lowest level in the longest muscle in the back; the NOGGIN gene is expressed in the ovary, the spleen, and the lungs, and in the kidney and the longest muscle in the back. The above results showed that the candidate genes were highly expressed in the ovary, suggesting that they may be related to the function of the ovary. (2) the SNPs of all exons and flanking regions of the above 10 genes were scanned by DNA pool sequencing, and 78 mutation sites were detected, of which 3 mutation sites were found on the KIT gene and 6 on KITLG. A mutation site, 7 mutation sites on ADAMTS1, 6 on NCOA1, 14 on NPM1, 2 on LIF, 23 on LIFR, 4 on NGF, 13 on TrkA, and no detection of SNP. analysis mutation type on NOGGIN gene, and 4 missense mutations located in g.70224398TA loci (Leu-His) on the exon of KIT gene tenth, respectively, and ninth exons of the gene ninth. 756130GA (Met-Val), g.35835474 GT (Ala-Ser) on exon seventh of LIFR gene, and twelfth exon g.35835329 GA (Asn-Ser). 8 synonymous mutations were found, respectively, g.127751615CT and g.127753565TC on the exons of ADAMTS1 second, respectively, on the fifth exon and the eighth exons of the gene. The g.91651197GA in exon first, the LIFR gene in exon 6 on g.35835329GA, and the rest of the g.3247689AT. mutation sites on the exon fifth of the NPM1 gene are on the intron. (3) the g.91651197GA of the NGF gene of the 1000 sheep and the Small Tail Han sheep population by PCR-RFLP method, g.105281586CT and g.105284246GC of the TrkA gene, KITL, and KITL. 5 SNP loci, such as g.124502403CT and g.124511398TC of the G gene, were genotyping, and the association of polymorphic loci with lambing number of sheep was analyzed. The results showed that in the Small Tail Han sheep and the lake sheep population, the g.91651197GA locus of the NGF gene, the g.105281586CT and KITLG g.124511398TC loci of the TrkA gene were significantly correlated with the number of lambs (P0.05), and TrkA. The g.105284246GC mutation site of the gene showed a significant difference in the number of lambing of the different genotypes in the lake sheep population (P0.05). The effect of the NGF and TrkA gene aggregation effect on the lambing number of sheep was analyzed. The results showed that the combination of the two genes was the optimal combination type, which showed that the combination of NGF and TrkA gene combinations affected the cotton. The number of lambs of sheep. (4) evaluation of 78 SNPs loci detected in the above 9 genes. It was found that 62 SNPs loci could be genotyping by SNPscan typing. The correlation analysis between the typing results and the number of lambs showed that the above 9 genes were related to the number of lambs of the sheep and the KIT gene in g.70199073AG, the g.35 of the LIFR gene. 845474 CT, g.3584563TC and g.35853637TG, the g.32140565 GA locus of the NCOA1 gene was significantly correlated with the lambing number of Small Tail Han sheep (P0.05). The g.3246266 TG locus and NCOA1 gene of the NPM1 gene were significantly related to the number of lambs in lake sheep. In haplotype analysis, 50 different haplotypes were detected in the 9 genes. The distribution is not balanced. The haplotype frequency is the highest of the haplotype H8 of the KITLG gene. The haplotype frequency is 0.89, the haplotype is the dominant haplotype. The haplotype of the lowest frequency is the H22 of the LIFR gene, and the frequency is 0.0161. haplotype H3, H14, H17, H20, H23, H29.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S826

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