绵羊繁殖相关候选基因的表达和SNP扫描及其与产羔数关联性分析

发布时间：2018-05-27 00:30

本文选题：绵羊 + 基因表达　；参考：《甘肃农业大学》2017年博士论文

【摘要】：繁殖力是绵羊的重要经济指标之一,直接影响绵羊经济效益。母羊的产羔数是衡量绵羊繁殖力的重要指标,因此,提高绵羊的产羔数对发展养羊业十分重要。绵羊的产羔性状是微效多基因控制的数量性状,通过与数量性状位点相连锁的分子标记实现对基因型的直接选择,将传统选育方法和现代分子育种方法相结合运用于育种实践,将会极大地提高选择效率,进一步提高绵羊繁殖性能。本研究以与绵羊繁殖性状相关的NGF、TrkA、KIT、KITLG、LIF、LIFR、NPM1、NCOA1、ADAMTS1和NOGGIN等10个基因为研究对象,利用Real-time PCR分析其在湖羊心、肝、脾、肺、肾、瘤胃、十二指肠、背最长肌、脂肪、下丘脑、垂体和卵巢等12种组织中的表达特征,并采用DNA混合池测序、限制性长度片段多态性(Restriction fragment length polymorphism,RFLP-RCR)和SNPscan分型技术研究以上10个基因全部外显子及其侧翼区SNPs与绵羊(湖羊,n=556;小尾寒羊,n=444)产羔性状的关联性,主要研究结果如下:(1)NGF、TrkA、KIT、KITLG、LIF、LIFR、NPM1、NCOA1、ADAMTS1和NOGGIN基因在湖羊心、肝、脾、肺、肾、瘤胃、十二指肠、背最长肌、脂肪、下丘脑、垂体和卵巢等12种组织中广泛表达,其中NGF在卵巢、下丘脑和心脏中的表达量较高,在背最长肌、瘤胃、肺、肝脏和脂肪表达量较低;TrkA基因在卵巢和肺中的表达量最高,而在背最长肌、瘤胃、脂肪、下丘脑、垂体、脾、肾和十二指肠表达量较低;KITLG基因在心脏、卵巢和肺中表达量较高,在肝脏、瘤胃、脾脏和背最长肌中表达量最低;KIT基因在卵巢、肝脏和肺中表达量最高,在脾脏、肾脏、瘤胃、十二指肠和脂肪组织中的表达量较低,在背最长肌中最低;LIF基因在卵巢中表达量最高,其次为下丘脑、垂体和肝脏,在脾、肺、肾、瘤胃、十二指肠、背最长肌和脂肪组织中表达较少;LIFR基因在卵巢、心脏、垂体、肾脏和肺中表达量较高,在其它组织中的表达量较低;NPM1在卵巢和肝脏中的表达量最高,表达量较低的组织为背最长肌、脂肪、垂体和肺;NCOA1基因在卵巢、垂体、肝脏和肺中表达量最高,而在背最长肌中表达量最低。ADAMTS1基因卵巢、肺、肝脏和瘤胃中表达量最高,在背最长肌中呈现最低水平;NOGGIN基因的表达量在卵巢、脾脏和肺中最高,而在肾脏和背最长肌中最低。以上结果显示所研究候选基因均在卵巢中表达量较高,提示它们可能与卵巢的功能相关。(2)利用DNA池测序法对以上10个基因所有外显子及侧翼区SNPs进行扫描,共检测到78个突变位点,其中在KIT基因上发现3个突变位点,KITLG上发现6个突变位点,ADAMTS1上7个突变位点,NCOA1上6个,NPM1上14个,LIF上2个,LIFR上23个,NGF上4个,TrkA上13个,在NOGGIN基因上没有检测到SNP。分析突变类型,发现4个错义突变,分别位于KIT基因第10外显子上g.70224398TA位点(Leu-His),ADAMTS1基因第9外显子上的g.127756130GA(Met-Val),LIFR基因第7外显子上g.35835474 GT(Ala-Ser),第12外显子g.35835329 GA(Asn-Ser)。同时发现8个同义突变,分别为ADAMTS1第2外显子上的g.127751615CT和g.127753565TC,在第5外显子上g.127753643CT和g.127753727CT,NCOA1基因第8外显子上的g.32072394CT,NGF基因在第1外显子上的g.91651197GA,LIFR基因在外显子6上的g.35835329GA,NPM1基因第5外显子上的g.3247689AT。其余的突变位点均位于内含子上。(3)利用PCR-RFLP方法对1000只湖羊和小尾寒羊母羊群体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个SNP位点,进行基因分型,并分析多态位点与绵羊产羔数关联性,结果表明:在小尾寒羊和湖羊群体中,NGF基因g.91651197GA位点、TrkA基因的g.105281586CT和KITLG基因g.124511398TC位点与产羔数显著相关(P0.05),而TrkA基因的g.105284246GC突变位点仅在湖羊群体中表现出不同基因型个体的产羔数差异显著(P0.05)。分析了NGF和TrkA基因聚合效应对绵羊产羔数的影响,结果发现两个基因的GACTCC基因型组合为最优组合基因型,表明NGF和TrkA基因组合共同影响绵羊产羔数。(4)对以上9个基因中检测到的78个SNPs位点进行评估,发现有62个SNPs位点可以利用SNPscan分型法进行基因分型,对于分型结果与产羔数关联性分析表明:以上9个基因均有SNP位点与绵羊产羔数相关,KIT基因在g.70199073AG,LIFR基因的g.35845474 CT、g.3584563TC和g.35853637TG,NCOA1基因g.32140565 GA位点与小尾寒羊的产羔数存在着显著相关(P0.05);NPM1基因的g.3246266 TG位点和NCOA1基因g.31928230 CT位点与湖羊产羔数显著相关(P0.05)。单倍型分析中,在9个基因中共检测到了50个不同的单倍型,其分布并不是均衡的,单倍型频率最高的是KITLG基因的单倍型H8,其单倍型频率为0.89,为优势单倍型,频率最低的单倍型为LIFR基因的H22,其频率为0.0161。单倍型H3、H14、H17、H20、H23、H29、H47和H48与产羔性状显著相关(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.
【学位授予单位】：甘肃农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S826

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