【作者】 吴井生；

【导师】 王金玉；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 猪的繁殖性状,包括初情期、产仔数、初生重等,是现代猪生产中重要的经济性状,繁殖性能的高低直接影响到猪场的生产效率和经济效益；同时,也是一个低遗传力的数量性状,容易受到环境条件的影响。研究猪繁殖性状相关的基因对于揭示影响猪繁殖性能遗传机制和高繁殖力猪的选育具有重要意义。候选基因法是一种定位数量性状基因座(QTL)的主要方法,该方法是依据生物体内已知的或可能的生理生化过程来选择相关基因并对其与表型的关系进行探讨。利用候选基因法定位主效基因在畜禽中已有许多成功的范例,如鸡性连锁矮小基因、猪应激综合征基因、猪雌激素受体基因等。本研究利用候选基因法分析了繁殖性状相关基因FSHβ、FSHR、LHR、PGR、KISS-1、 GPR54与小梅山猪产仔性能的关系。采用混合基因组DNA池与测序技术对上述6个基因的单核苷酸多态性(Single nucleotide polymorphism, SNP)进行筛选；采用PCR-SSCP、 PCR-RFLP、错配PCR-RFLP、直接PCR等技术对SNPs进行大规模检测,并与小梅山猪的繁殖性状进行关联分析；采用RQ-PCR技术研究上述6个基因在小梅山猪下丘脑-垂体-性腺轴系统中的表达规律；采用ECLIA技术研究血清中FSH、LH、E2、P4等生殖激素在不同生长发育阶段中的变化规律。主要研究结果如下：1、以猪FSHβ基因为候选基因,对该基因的外显子区域进行扫描,共发现7个SNP。外显子1区域中未检测出突变位点,但在内含子1中检测出1个SNP,即C5807A；外显子2中检测出1个SNP,即C6699T;外显子3中检测出5个SNP,分别为A8753G、C8766T、 T8788C、A8880G、8943del;上述突变位点中,只有C6699T发生在CDS区域内,但未能引起氨基酸的改变,属于同义突变。小梅山猪、枫泾猪和大白猪FSHβ基因中分别有4、4和3个基因位点表现出多态性,并均为中度多态。连锁不平衡分析结果表明,上述7个突变位点间存在强连锁不平衡现象。相关分析结果表明,FSHβ基因FSHβ-1位点的BB型、FSHβ-2位点的DD型、FSHβ-3.1位点的FF型、FSHβ-3.2位点的HH型与单倍型组合H5H5(BDFH/BDFH)对2胎以上或所有胎次小梅山母猪的总产仔数(Total number born, TNB)和产活仔数(Number born alive, NBA)有显著或极显著影响；方差组分分析结果表明,2胎以上或所有胎次小梅山母猪的TNB和NBA的遗传在各位点上主要受到基因的加性效应的影响,单倍型组合H5H5为一个优势组合,可作为猪繁殖性状选育的一个分子标记。2、以猪FSHR基因为候选基因,对该基因的外显子区域进行扫描,共发现6个SNP。外显子1区域内检测到3个SNP,分别为C70T、C74G、C81T;外显子10中检测到3个SNP,分别为C1166T、T1491C、T1977C;其中,C74G的突变导致苏氨酸变为丝氨酸,即Thr13Ser, C1166T导致苏氨酸变为异亮氨酸,即Thr377Ile,其余突变均未能引起氨基酸的改变,属于同义突变。小梅山猪、枫泾猪和大白猪FSHR基因中分别有3、3和4个基因位点表现出多态性,小梅山猪FSHR-1位点为高度多态,其余为中度多态；枫泾猪的3个基因位点均为中度多态；大白猪FSHR-1位点为高度多态,其余为低度多态。相关分析结果表明,FSHR基因FSHR-1位点的AC型、FSHR-10.4位点的BB型、FSHR-10.5位点的DD型与单倍型组合H3H5对2胎以上或所有胎次小梅山母猪的TNB和NBA有显著或极显著影响；方差组分分析结果表明,2胎以上或所有胎次小梅山母猪的TNB和NBA的遗传在FSHR-1位点上主要受到基因显性效应的影响,在FSHR-10.4与FSHR-10.5位点上主要受到基因的加性效应的影响。3、以猪LHR基因为候选基因,对该基因的外显子区域进行扫描,共发现1个SNP。外显子11中1351bp处发生C→T的突变,即C1351T,未引起氨基酸的变化,属于同义突变。小梅山猪中检测出多态性,而枫泾猪和大白猪中未能检测出多态性。相关分析结果表明,LHR基因LHR-11.4位点的MN型对2胎以上或所有胎次小梅山母猪的TNB和NBA有显著或极显著影响；方差组分分析结果表明,2胎以上或所有胎次小梅山母猪的TNB和NBA的遗传在LHR-11.4位点上主要受到基因的显性效应的影响。4、以猪PGR基因为候选基因,对该基因的外显子区域进行扫描,共发现1个SNP。外显子1中1319bp处发生C→T的突变,即C1319T,未引起氨基酸的变化,属于同义突变。小梅山猪、枫泾猪和大白猪PGR基因中均检测出多态性。相关分析结果表明,对2胎以上或所有胎次小梅山母猪中,PGR基因PGR-1.6位点的AB型个体的TNB和NBA比AA型个体均高。5、以猪KISS-1基因为候选基因,对该基因的外显子区域进行扫描,共发现6个SNP。5’区域内检测到1个SNP,即A7G；外显子1中1个SNP,即G41T；内含子1中3个SNP,分别为633inde1、A859G、C1026A;外显子2中1个SNP,即G2343C。小梅山猪、枫泾猪和大白猪KISS-1基因中分别有4、5和5个基因位点表现出多态性,小梅山猪KISS-1基因中4个位点均为中度多态；枫泾猪中,KISS1-4位点为中度多态,其余位点均为低度多态；大白猪中KISS1-5位点为中度多态,其余位点均为低度多态。连锁不平衡分析结果表明,A7G与G41T、A7G与C1026A、G41T与C1026A间存在完全连锁不平衡现象。相关分析结果表明,KISS-1基因KISS1-3位点的AB型、KISS1-4位点的KL型、KISS1-6位点的MN型、KISS1-7位点的PQ型与单倍型组合H1H5(ALNQ/BKMP)对2胎以上或所有胎次小梅山母猪的TNB和NBA有显著或极显著影响；方差组分分析结果表明,2胎以上或所有胎次小梅山母猪的TNB和NBA的遗传在各位点上主要受到基因的显性效应的影响。6、以猪GPR54基因为候选基因,对该基因的外显子区域进行扫描,共发现3个SNP。外显子1中检测出1个SNP,即T245C;内含子2中1个SNP,即1984-1985bp间增加1个碱基C；外显子5中1个SNP,即T3295C,其中,T245C引起氨基酸的改变(亮氨酸→脯氨酸,即Leu35Pro)。小梅山猪、枫泾猪和大白猪GPR54基因中分别有3、2和3个基因位点表现出多态性,小梅山猪与枫泾猪各位点均为中度多态；大白猪GPR54基因中,GPR54-1.1位点为低度多态,其余2个位点为中度多态。连锁不平衡分析结果表明,1984add与T3295C间存在完全连锁不平衡现象。相关分析结果表明,GPR54基因GPR54-1.1位点的BB型、GPR54-3位点的CD型、GPR54-5.1位点的EF型与单倍型组合H1H1(BDE/BDE)对2胎以上或所有胎次小梅山母猪的TNB和NBA有显著或极显著影响；方差组分分析结果表明,2胎以上或所有胎次小梅山母猪的TNB和NBA的遗传在GPR54-1.1位点上主要受到基因的加性效应的影响,而在GPR54-3和GPR54-5.1位点上主要受到基因的显性效应的影响。7、对小梅山猪FSHβ、FSHR、LHR、PGR、KISS-1和GPR54基因在下丘脑-垂体-性腺轴系统中的表达规律进行研究。结果显示：小梅山猪从初生到5月龄间下丘脑-垂体-性腺轴下各组织中均检测到FSHp、FSHR、LHR、PGR、KISS-1和GPR54基因的表达；不同组织中,FSHβ基因在垂体中均高度表达,输卵管和卵巢则低度表达,4月龄时,FSHβ基因在垂体中的相对表达量是输卵管的54.557倍,其表达水平在不同生长阶段中呈现先上升后下降趋势,4月龄时达到高峰；4月龄前,FSHR基因在下丘脑中高度表达,4月龄之后,则卵巢和下丘脑均为高度表达,而子宫均为低度表达；LHR基因在不同组织中,均以下丘脑为高度表达,4月龄之后,卵巢亦为高度表达；PGR基因在不同组织中,均以子宫为高度表达,4月龄之后,下丘脑和卵巢亦高度表达,垂体均为低度表达；不同生长阶段中,各组织PGR基因表达水平均呈现先上升后下降的趋势,并且均在4月龄达到高峰,尤其是卵巢最为明显；4月龄之后,KISS-1与GPR54基因在下丘脑中高度表达,但4月龄之前,两者表达差异较大,KISS-1基因在子宫中高度表达,GPR54基因则波动很大；KISS-1与GPR54基因表达水平在下丘脑、垂体、卵巢和子宫中的趋势,与FSHβ基因之于垂体、FSHR基因之于卵巢与子宫、LHR基因之于卵巢与下丘脑、PGR之于各组织,是相同的。8、对小梅山猪性发育过程中血清中FSH、LH、E2和P4等生殖激素水平的变化规律加以研究,结果显示：初生时,血清中FSH、LH、E2和P4等生殖激素水平处于高位状态,1月龄～4月龄间可能略有波动,但均呈现上升趋势,至4月龄时达到最高水平,并且差异均达到显著或极限著水平,5月龄时,这4种生殖激素水平又迅速下降；根据上述4种生殖激素的变化规律,并结合生产实践,可以推测小梅山猪的初情期在3.5月龄-4.5月龄间。更多还原

【Abstract】 Reproductive traits of swine, including puberty, litter size, weight at birth, and so on, are important economic traits in modern pig production, which highly influence productive efficiency and economic profits. And also they are quantitative traits of low heredity, which are easily influenced by environmental factors. Identification of genes associated with reproductive traits could give an insight into the genetic mechanism of pig reproductive performance and be helpful for pig breeding with high reproductive performance. Candidate gene approach is one of the strategies in mapping quantitative traits. Some major genes have been mapped successfully by this method, e.g. sex-linkage dwarf gene of chicken, stress syndrome gene of swine and estrogen receptor gene of swine, and so on.To investigate the genes associated with swine reproductive traits, we selected FSHβ, FSHR, LHR, PGR, KISS-1and GPR54gene as candidate genes to analyze the relationship of those genes with reproductive performance in Xiaomeishan pig population. Mixed genome DNA pool and direct sequencing methods were used to screen the SNPs of those above six genes, PCR-SSCP, PCR-RFLP, mismatched PCR-RFLP, and direct PCR methods to detect at each SNP locus in a large scale, and then their relationship with reproductive traits in Xiaomeishan pig population were analyzed. The developmental patterns of FSHβ, FSHR, LHR, PGR, KISS-1and GPR54mRNA expression in the hypothalamic-pituitary-gonadal (HPG) axis in Xiaomeishan pig population and serum FSH, LH, E2and P4were determined by relative quantitative RT-PCR and ECLIA methods, respectively. The main results were as follows:1. All exons in FSHβ gene were screened, and seven SNPs in total were detected. No mutations were detected in exon1, but one SNP in intron1, that is, C5807A; one SNP in exon2, that is, C6699T; five SNPs in exon3, that is, A8753G, C8766T, T8788C, A8880G and8943delllbp. Only transition C6699T in those above SNPs was located in CDS region, but not causes the change of amino acid, and was a synonymous mutation. In Xiaomeishan pig, Fengjing pig and Large White pig populations, there were4,4and3loci displayed polymorphisms in FSHP gene, respectively, and all were highly informative loci. Those above seven SNPs were in intense state of linkage disequilibrium. It could be found that genotype BB at FSHβ-1locus, genotype DD at FSHβ-2locus, genotype FF at FSHβ-3.1, genotype HH at FSHβ-3.2and haplotype combination H5H5(BDFH/BDFH) had significant effect or highly significant effect on total number born (TNB) or number born alive (NBA) traits in Xiaomeishan sows after the second parity or sows in all parities, the heredity of which is mainly influenced by gene additive effect at each locus. So haplotype combination H5H5is a predominant genotype, and can be regarded as a molecular marker in pig breeding of reproductive traits.2. Six SNPs were discovered in swine FSHR gene. Amongst them, three SNPs were in exonl, that is, C70T, C74G and C81T; three SNPs in exon10, that is, C1166T, T1491C and T1977C, in which transversion C74G and transition C1166T caused the change of amino acid, that is, Thrl3Ser and Thr377Ile, respectively. In Xiaomeishan pig, Fengjing pig and Large White pig populations, there were3,3and4loci displayed polymorphisms in FSHR gene, respectively; FSHR-1locus in Xiaomeishan pig was highly informative locus, the other were reasonably informative loci; three loci in Fengjing pig were all reasonably informative loci; FSHR-1locus in Large white pig was highly informative locus, the other were slightly informative loci. It could be found that genotype AC at FSHR-1locus, genotype BB at FSHR-10.4locus, genotype DD at FSHR-10.5and haplotype combination H3H5had significant effect or highly significant effect on TNB or NBA traits in Xiaomeishan sows after the second parity or sows in all parities, the heredity of which is mainly influenced by gene dominant effect at FSHR-1locus and by gene additive effect at FSHR-10.4and FSHR-10.5loci.3. Only one SNP was found in exonll of swine LHR gene, that is, C1351T, which not caused the change of amino acid, and was a synonymous mutation. And it only existed in Xiaomeishan pig population, not in Fengjing pig and Large White pig populations. The results suggested that genotype MN at LHR-11.4locus had significant effect or highly significant effect on TNB or NBA traits in Xiaomeishan sows after the second parity or sows in all parities, the heredity of which is mainly influenced by gene dominant effect at LHR-11.4locus.4. Only one SNP was detected in exonl of swine PGR gene, that is, C1319T, which not caused the change of amino acid, and was a synonymous mutation. Polymorphism was detected in all three pig populations. The result showed that AB genotypic sows of Xiaomeishan pigs produced more TNB and NBA than AA genotypic sows after the second parity or in all parities.5. Six SNPs were found in swine KISS-1gene. Amongst them, one SNP was in5’-UTR, that is, A7G; one SNP in exon1, that is, G41T; three SNP2in intron1, that is, A859G, C1026A and633addl9bp; one SNP in exon2, that is, G2343C. In Xiaomeishan pig, Fengjing pig and Large White pig populations, there were4,5and5loci displayed polymorphisms in KISS-1gene, respectively. All four loci in Xiaomeishan pig were highly informative loci; KISS1-4locus in Fengjing pig and KISS1-5locus in Large White pig were reasonably informative loci, the other slightly informative loci. SNPs A7G and G41T, A7G and C1026A, G41T and C1026A were all in complete state of linkage disequilibrium. The results indicated that genotype AB at KISS1-3locus, genotype KL at KISS1-4locus, genotype MN at KISS1-6, genotype PQ at KISS1-7and haplotype combination H1H5(ALNQ/BKMP) had significant effect or highly significant effect on TNB or NBA traits in Xiaomeishan sows after the second parity or sows in all parities, the heredity of which is mainly influenced by gene additive effect at each locus.6. Three SNPs were detected in swine GPR54gene. Amongst them, one SNP was in exon1, that is, T245C; one SNP in intron1, that is,1984addlbp; one SNP in exon5, that is, T3295C, in which transition T245C caused the change of amino acid, that is, Leu35Pro. In Xiaomeishan pig, Fengjing pig and Large White pig populations, there were3,2and3loci displayed polymorphisms in KISS-1gene, respectively. GPR54-1.1locus in Large White pig was slightly informative locus, the other in three pigs were all reasonably informative loci. SNPs1984addlbp and T3295C were in complete state of linkage disequilibrium. The results indicated that genotype BB at GPR54-1.1locus, genotype CD at GPR54-3locus, genotype EF at GPR54-5.1and haplotype combination H1H1(BDE/BDE) had significant effect or highly significant effect on TNB or NBA traits in Xiaomeishan sows after the second parity or sows in all parities, the heredity of which is mainly influenced by gene additive effect at GPR54-1.1and by gene dominant effect at GPR54-3and GPR54-5.1loci.7. The developmental patterns of FSHp, FSHR, LHR, PGR, KISS-1and GPR54mRNA expression in the hypothalamic-pituitary-gonadal (HPG) axis in Xiaomeishan pig population were studied. The results showed that FSHp, FSHR, LHR, PGR, KISS-1and GPR54mRNA were all tested in different tissues in HPG axis from birth to5-month-old in Xiaomeishan pig. FSHβ gene presented high expression in pituitary and low expression in oviduct and ovary. FSHβ gene expression in pituitary was54.557times higher than in oviduct, and it increased with the age, attained the peak at4-month old, and then declined. FSHR gene performed high expression in hypothalamus before4-month-old, and high in ovary and hypothalamus after4-month-old, and low in uterus during the sexual development phases. LHR gene showed high expression in hypothalamus in all phrases, and after4-month-old also high in ovary. PGR gene showed high expression in uterus in all phrases, after4-month-old high in hypothalamus and ovary, but low in pituitary in all phrases, and it increased with the age, attained the peak at4-month old, and then declined in all tissues, especially in ovary. KISS-1and GPR54genes presented high expression in hypothalamus after4-month-old. But there were big difference between them before4-month-old, KISS-1gene high in uterus and GPR54gene different. We also found that the tendency of KISS-1and GPR54gene expression in hypothalamus, pituitary, ovary and uterus were the same as FSHp gene in pituitary, FSHR gene in ovary and uterus, LHR gene in ovary and hypothalamus, and PGR gene in all tissue.8. The patterns of serum FSH, LH, E2and P4during sexual development phrases in Xiaomeishan pig population were also studied. The results showed that at birth serum FSH, LH, E2and P4were all in high levels, there was a slight fluctuation from1-4month old, but presented a increasing trend, and attained a peak at4-month-old, had significant or highly significant difference, at5-month-old they all rapidly decreased. Based on the changes of those four above hormones in association with the practice, it could be speculated that the puberty time of Xiaomeishan pig was between3.5-month-old and4.5-month-old.更多还原