【作者】 黄贺；

【导师】 李和平；

【作者基本信息】 东北林业大学 ， 野生动植物保护与利用， 2013， 博士

【摘要】 产仔数是养狐业的一个重要的经济性状,提高产仔数可以减少种狐饲养数量,降低生产成本,增加皮张产量,给养狐业带来巨大的经济效益；同时,提高产仔数也是北极狐育种工作的主要目标。北极狐的产仔性状是遗传力低的数量性状,采用常规的育种方法难以在短期内取得较大的遗传进展,而分子标记辅助选择(MAS)为显著改良产仔数性状提供了有效途径。到目前为止,ESR基因和FSHR基因与产仔性状的关系已经基本明确,并且在多种动物中进行了大量的研究,但是关于ESR基因和FSHR基因与北极狐产仔数关系的研究未见报道。因此,本研究以FSHR基因和ESR基因作为北极狐高产仔性状的候选基因,采用PCR技术对FSHR基因和ESR基因进行克隆测序,获得FSHR基因和ESR基因序列并进行序列分析和同源性比较；采用PCR-RFLP技术和PCR-SSCP技术对FSHR基因和ESR基因进行多态性检测,对多态位点进行测序和序列分析,确定变异类型,并将多态位点与北极狐的产仔数性状进行关联分析；采用realtime PCR技术研究不同FSHR基因和ESR基因型卵巢组织中FSHR和ESR的表达量及其对产仔数的影响;采用realtime PCR技术和免疫组化技术研究FSHR基因在北极狐发情周期卵巢中的表达规律,并对FSHR基因表达进行定位。研究结果如下：(1)北极狐ESR基因全长1824bp,包含ATG起始密码子,开放读码框编码607个氨基酸,序列中的A、T、G、C比例分别为25.23%、22.69%、25.92%、26.16%,G+C含量(52.08%)高于A+T的含量(47.92%)。DNA结合区存在八个保守的半胱氨酸残基(C),构成两个锌指结构(Zn-C4), D-box (EGCKA)、P-box (PATNQ),还存在一核定位信号(aa244-250) DRNRRKS。(2)北极狐FSHR基因序列长1947bp,包含ATG起始密码子,可编码648个氨基酸,序列中的A, T、G、C比例分别为25.17%、26.55%、21.42%、26.86%,G+C含量(48.28%)低于A+T的含量(51.72%)。(3)北极狐ESR基因的氨基酸序列与鸡、小鼠、羊、牛、人、猪的同源性分别为74%、77%、87%、88%、85%、89%,在进化关系上北极狐与猪的亲缘关系较近,与鸡最远,氨基酸组成分析表明丝氨酸含量最高,色氨酸含量最低。(4)北极狐FSHR基因的氨基酸序列与鸡、小鼠、羊、牛、人、猪的同源性分别为59%、73%、83%、83%、82%、84%,在进化关系上北极狐与人的亲缘关系较近,与鸡最远,氨基酸组成分析表明丝氨酸含量最高,酪氨酸含量最低。(5)北极狐ESR蛋白在56～60、88～89、108～121、154-172、198-205、215-220、238～239、263～266、315～337、353～359、366～374、394～420、454～503、518～526、548-556氨基酸存在15个显著的疏水区域并且不存在跨膜结构,氨基酸序列从196至267存在ZnF_C4模序结构域,氨基酸序列360至530存在HOLI结构域。(6)北极狐FSHR蛋白在5-16、25～33、288-296、304-309、332-338、340-349、357～366、374～379、402～417、443～456、485～553、565～572、587～600、618～647氨基酸存在14个显著的疏水区域,氨基酸序列367至389、402至424、444至466、490至512、532至554、575至597、607至629的存在的7个跨膜结构。(7)在北极狐的FSHR基因5’端侧翼没有发现多态性；在FSHR基因外显子10发现多态性,检测到AA、AB和BB三种基因型, AA基因型为优势基因型,A等位基因频率为0.82,B等位基因频率为0.18；BB基因型个体初产的TNB和NBA分别比AA基因型的高1.08只和1.58只(P<0.05),BB基因型个体经产的TNB和NBA分别比AA基因型的高1.72只和1.03只(P<0.05)。FSHR基因外显子10第120bp发现C→T单碱基变异。(8)北极狐ESR基因外显子1存在多态性,优势等位基因为A等位基因,A等位基因频率为0.68,B等位基因频率为0.32；BB基因型个体初产的TNB和NBA分别比AA基因型的高2.61只和1.25只(P<0.05),BB基因型个体经产的TNB和NBA分别比AA基因型的高2.08只和1.29只(P<0.05)。ESR基因外显子1第539bp发现G→C单碱基变异。(9) FSHR基因和ESR基因合并基因型表现出AA、AB、BB逐步递增的趋势,基因型AAAA的初产TNB和NBA分别为7.42和6.22,基因型ABAB的TNB和NBA分别为7.46和7.07,基因型BBBB的TNB和NBA分别为8.92和8.56,经产的TNB和NBA也基本存在同样的规律。(10)在发情期,北极狐BB基因型个体右侧卵巢FSHR的mRNA水平显著高于AA基因型个体(P<0.01),左侧卵巢BB基因型个体与AA基因型个体差异显著(P<0.05)。(11)在发情期,北极狐两侧卵巢ESR的mRNA水平在基因型间无显著差异(P>0.05)。(12)北极狐发情周期不同阶段卵巢组织中均有FSHR mRNA的存在,从发情后期、间情期、发情前期到发情期,FSHR mRNA的表达量呈上升趋势,发情期表达量最高,发情后期表达量最低。(13)北极狐发情周期不同阶段卵巢各期卵泡中均有FSHR阳性细胞分布,FSHR阳性细胞主要存在于卵泡细胞、颗粒细胞、卵泡膜细胞和初级卵母细胞中。从发情后期、间情期、发情前期到发情期,FSHR阳性细胞数量呈上升趋势,发情期最多,发情后期最少。阳性细胞数量随着卵泡的发育和卵泡体积的增大不断增多。更多还原

【Abstract】 The litter size of arctic fox is one of the important economic characters, increasing litter size can reduce the breeding number of fox, increase the output of the skin, bring huge economic benefits to fully; At the same time, improve the litter size is also the main goal of the arctic fox breeding work. Arctic foxes breed character is very low heritability of quantitative traits, it is difficult to get large genetic progress in the short term by the conventional breeding methods, and marker-assisted selection (MAS) provides an effective way. This study serves FSHR gene and ESR gene as candidate genes, cloning FSHR gene and ESR gene by PCR, and sequence analysis and homology comparison; detecting FSHR gene and ESR gene polymorphism by PCR-RFLP and PCR-SSCP technology sequence analysis of polymorphic loci, determining the mutation type, and polymorphic loci associated with litter size traits of arctic fox analysis; researching expression of different FSHR and ESR genotype of FSHR and ESR gene in ovarian tissue by realtime PCR technology and its effects on litter size traits. The results are as follows:(1) ESR gene sequence is1824bp, contains the start codon ATG, open reading frame encoding607amino acids, the sequence of A, T, Q C ratio were25.23%,22.69%,25.92%,26.16%, G+C content (52.08%) higher than that of the A+T content (47.92%). DNA binding area, there are eight conservative cysteine residues (C), constitute two zinc (zinc-C4), D-box (EGCKA), P-box (PATNQ), there is a signal DRNRRKS (aa244-250).(2)Arctic foxes FSHR gene sequence is1947bp and contains a start codon ATG, encoding648amino acids, the sequence of A, T, G, C ratio were25.17%,26.55%,21.42%,26.86%, G+C content (48.28%) is lower than the A+T content (51.72%).(3) the arctic fox ESR gene sequence of amino acids with chickens, sheep, cows, mice, pigs, people of homology respectively is74%,77%,87%,88%,75%,91%, arctic fox on evolutionary relationships are the closest animal relatives to pigs, and farthest to chickens, amino acid composition analysis showed that the highest levels is serine,tryptophan is the lowest.(4) FSHR gene sequence of amino acids with chickens, sheep, cows, mice, pigs, people of homology respectively is59%,73%,83%,83%,82%,92%, arctic fox on evolutionary relationships are the closest animal relatives to pigs, and farthest to chickens, amino acid composition analysis showed that the highest levels is serine, the tyrosine is minimum.(5)Arctic fox ESR protein in56～60,88～88,89～121,154～154,172～205,215～220,215～220,263～263,266～337,353～359,353～337,394～394,420～503,518～526,518～503amino acids exist15significant hydrophobic area and there is no cross membrane structure, amino acid sequence from196to196are ZnF_C4model sequence structure domain, the amino acid sequence from360to530is HOLI structure domain.(6)FSHR protein in5～16,25～33,288～296,304-309,332～332,338～349,357～366,357 ～349,402～402,417～456,485～485,553～572,587～600,587～600amino acids exist14significantly hydrophobic regions, amino acid sequence from367to389,402to424,402to466,490to512,490to554,575to597,575to629exist seven transmembrane structure.(7) the FSHR gene5’end of the wing of the arctic fox found no polymorphism; found polymorphism in the arctic fox FSHR gene exon10, detected three genotypes AA, AB, and BB, AA genotype is advantage in the arctic fox group, the frequency of A allele is0.82, the frequency of B is0.18; TNB and NBA of primiparous arctic foxes with BB genotype is1.08and1.58respectively than AA genotype (P<0.05), TNB and NBA of parous arctic foxes with BB genotype is1.72and1.03respectively than AA genotype (P<0.05).There is C→T mutation at120bp in FSHR gene exon10.(8) ESR gene exon1of Arctic foxes exist polymorphism, A alleles is advantages, A allele frequency is0.68, the B allele frequency is0.32; TNB and NBA of primiparous arctic foxes with BB genotype is2.61and1.25respectively than AA genotype (P<0.05), TNB and NBA of parous arctic foxes with BB genotype is2.08and1.29respectively than AA genotype (P<0.05). There is G→C mutation at539bp in ESR gene exon1.(9)Combined genotype AA, AB, and BB show gradually increasing trend, primiparous TNB and NBA of genotype AAAA are7.42and6.22respectively, genetic type ABAB are7.46and7.07respectively, genotype BBBB are8.92and8.56respectively, parous TNB and NBA are also in the same rule.(10) FSHR mRNA level of BB genotype of the right ovary was significantly higher than that of AA genotype in the oestrum (P<0.01), BB genotype has significant difference with AA genotype in the left ovary (P<0.05)(11)ESR mRNA level of both ovary among the defferent genotypes have no significant difference (P>0.05).(12) FSHR mRNA of arctic foxes espresses during the different stage of estrous cycle in ovarian, the expression quantity of FSHR mRNA is on the rise from metaestrus to estrus, the highest expression quantity in the estrus,the lowest expression quantity in metaestrus.(13) FSHR positive cells distribute in the different stages of follicle of arctic foxes during the estrus cycle, FSHR positive cells mainly exist in the follicular cell and granulosa cell and theca cells and primary oocyte. From metaestrus to estrus, FSHR positive cells number is on the rise, the highest number in estrus, the lowest number in metaestrus. Number of positive cells increase with follicular development and increasing of follicular volume.更多还原