【作者】 孙瑞萍；

【导师】 曹斌云；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 本研究以西农萨能奶山羊和波尔山羊2个品种共计260个个体为材料,利用生物信息学、DNA测序、DNA序列分析、PCR-SSCP技术研究了山羊PRLR基因(intron1、2,exon10)、PRL基因(5’端)、GHR基因(exon9、10)以及LHβ基因(5’端、exon2、exon3)共4个候选基因16个位点遗传变异,同时探讨山羊群体遗传多态性及其与经济性状(产奶量、产羔数)的关系,旨在获取相应的分子遗传学信息,找到与经济性状相关的DNA标记,为山羊遗传资源保护、开发与利用提供科学依据。本研究获得以下重要结果:1山羊PRLR基因遗传变异位点与经济性状的关系1.1 PRLR基因内含子Ⅰ和Ⅱ位点与经济性状的关系针对PRLR基因内含子Ⅰ和Ⅱ设计的2对引物中,引物P1不存在多态,3处突变导致引物P2位点多态,共发现GG、GH、HH 3种基因型,2个山羊群体都以GG型为主;其基因型和等位基因分布与西农萨能奶山羊的产奶量和产羔数之间不存在显著相关(P>0.05);该位点与波尔山羊产羔数之间存在显著相关(P<0.01),如:引物P2位点多态的GH基因型在第5胎产羔数极显著高于GG型个体(P<0.01),第3胎也显著高于GG型(P<0.05)。1.2 PRLR基因外显子10位点与经济性状的关系在针对PRLR基因外显子10设计的4对引物的PCR-SSCP分析中,发现仅引物P3不存在多态性,引物P4、P5与P6扩增片段具有多态性。应用P4扩增片段,在2个品种中仅检测到AA型和AB型,纯化测序表明AA型与AB型相比有2处突变,27C→T的突变没有导致氨基酸变化,第189处的碱基缺失导致氨基酸由组氨酸变为酪氨酸和苏氨酸。AA和AB基因型之间产奶量的最小二乘均值差异显著(P<0.05),AA基因型在各胎次产奶量和平均产奶量等指标上均高于AB基因型,其中第三胎的产奶量达到显著水平(P<0.05);应用P5扩增片段,只检测到三种基因型(CC、CD、DD),纯化测序表明CC型与DD型相比有2处突变(103C→A和106T→C),分别导致氨基酸由赖氨酸变为苏氨酸、苏氨酸变为异亮氨酸;三种基因型之间产奶量的最小二乘均值差异不显著(P>0.05)。在两个品种中,引物P5的多态都与产羔数相关。如:波尔山羊P5位点的各基因型与产羔数间存在显著相关,在第三胎产羔数指标上,CC基因型个体产羔数显著多于DD型(P<0.05)。应用P6扩增片段,在2个品种中也只检测到EE型和EF型,纯化测序表明EE型与EF型相比有1处突变(114A→G),该突变导致氨基酸有蛋氨酸变为缬氨酸;EE和EF基因型之间产奶量差异显著(P<0.05)。EE基因型的个体各胎次产奶量均比EF型高,其中在第3、4胎产奶量和前四胎平均产奶量3项指标上都达到显著水平(P<0.05)。2山羊GHR基因遗传变异位点与经济性状的关系在山羊GHR基因Exon9和Exon10位点中,仅发现Exon9存在1处突变位点:241G→T,在两个群体中只发现两个基因型(NN,NT),该位点仍在两个山羊中均为低度多态(PIC<0.25)。Exon9扩增位点对产奶量有显著影响:NN基因型在各胎次产奶量和平均产奶量等指标上均好于NT基因型,其中在第三胎的产奶量和第四胎产奶量2项指标上都达到显著水平(P<0.05)。在西农萨能山羊品种上,GHR基因座位的不同基因型与各胎次产羔数及平均产羔数等指标均无相关性(P>0.05)。在波尔山羊中,GHR基因NN基因型在各胎次产羔数上均好于NT基因型,且在第四胎产羔数上NN型个体优于NT型个体(P<0.05)。3山羊LHβ基因遗传变异位点与产羔数的关系应用PCR-SSCP技术对两个山羊品种进行LHβ基因多态性分析,在设计的6对引物中,山羊LHβ基因P1与P5(exon2)存在153C→A和64T→C突变位点,它们与山羊的产羔数之间存在显著相关,P1引物位点多态与波尔山羊第3胎产羔数和第4胎产羔数显著相关:PP型个体第3胎和第4胎产羔数均极显著高于PQ型个体(P<0.01);此位点多态在西农萨能奶山羊第4胎和第5胎产羔的差异显著(P<0.05);在引物P5位点,不同基因型与产羔数之间存在显著关联:在波尔山羊中,LL型个体第4胎产羔数极显著高于LM型(P<0.01),第5胎产羔数显著高于LM型(P<0.05)。更多还原

【Abstract】 The bioinformatics, DNA sequencing, DNA sequence analysis, PCR-SSCP techniques were applied to analyze the genetic variations of key function gene and their association with economic traits in 260 goat, which comprised of two breed, namely XinongSannen dairy goat and Boer goat. The four key function gene included PRLR gene (intron1,2,exon 10), PRL gene (5’UTR), GHR(exon9,10), LHβgene(5’UTR,exon2,exon3) .The economic traits comprised of milk yield and little size . The association between genetic variations economic traits were analyzed by using general linear model (GLM) or adjusted linear model, as well as population genetic structure and distribution of genotype and allele were calculated by chi-square and quantitative genetic method. These will benefit for the protection, development and application of goat genetic resource ,application of DNA marker related to economic traits on marker-assist-selection(MSA), and improvement and promotion of goat. The important results were in the following:1 Association of genetic variation of PRLR gene with economic traits in goat1.1 Association of genetic variation of intron1and 2 with economic traits in goatTwo pairs of primer were designed to amplify intron1 and 2, of PRLR gene , The results showed that the products amplified by primer P1 didn’t display polymorphisms; the polymorphism of P2 locus was resulted by 3 mutation. In this locus ,the genotyp GG,GH,HH were detected in analyzed populations and the predominant GG genotype in two breed. It was not significantly found that the distribution of genotypes and alleles associated with milk yield and litter size of Xinong Sannen dairy goat. But the correlations were observed between this and litter size of Boer goat: the fifth litter size of individuals with genotype GH were significant higher than that of genotype GG(P<0.01), the third litter size of individuals with genotype GH were significant higher than that of genotype GG(P<0.05). 1.2 Association of genetic variation of exon10 with economic traits in goatFour pairs of primers were designed to detect single nucleotide polymorphisms of exon 10 of PRLR gene by PCR-SSCP. The results showed that only the products amplified by primer P3 didn’t display polymorphisms. For primer P4, two genotypes (AA and AB) were detected in two breed. Sequencing revealed two mutations of PRLR gene in the genotype AA in comparison to the genotype AB, The mutation of27C→T did not cause any amino acid change, The deletion of 189th result in two amino acid changes of His→Tyr and Thr. The difference of the least squares means (LSM) for milk yield between AA and AB was significant (P<0.05) in Saanen dairy goat, the third lactation milk yield of individuals with genotype AA was higher than that of genotype AB(P<0.05).For primer P5, three genotypes(CC,CD,DD) was detected in Saanen dairy goat and Boer goat. Sequencing revealed two mutations(103C→A and 106T→C) of PRLR gene in the genotype CC in comparison to the genotype DD. The former mutation resulted in an amino acid change of Lys→Thr, and the later mutation resulted in an anino acid change Thr→Ile. The difference of LSM for milk yield among CC、CD and DD was non-significant(P>0.05). the correlations were observed between this and litter size of Boer goat and Xinong Sannen : the third litter size of individuals with genotype CC were significant higher than that of genotype DD(P<0.05).For primer P6, two genotypes(EE and EF) were detected in Saanen dairy goat. Sequencing revealed one mutation(114A→G) of PRLR gene in the EE in comparison to the genotype EF ,and this mutation resulted in an amino acid change of Met→Val. he difference of the LSM for milk yield between EE and EF was significant (P<0.05) in Saanen dairy goat, The milk yield of individuals with genotype EE was higher than that with genotype EF in third, fourth lactation milk yield and average milk yield (P<0.05).Based on these results ,it was indicated that PRLR gene had significant positive effects on milk performance of dairy goats.2 Association of genetic variation of GHR gene with economic traits in goatThe 241G→T produced the polymorphism of exon9 of goat GHR gene. In analyzed populations ,two genotypes was observed and PIC values was low(PIC<0.25). It was significantly found that the distribution of genotypes and alleles associated with milk yield and litter size of Xinong Sannen dairy goat: the milk yield of individuals with genotype NN was higher than that with genotype NT in third, fourth lactation milk yield and average milk yield (P<0.05).The correlations were observed between this and litter size of Boer goat : the third litter size of individuals with genotype NN were significant higher than that of genotype NT(P<0.05).3 Association of genetic variation of LHβgene with economic traits in goatApplying the PCR-SSCP technique and sequence to analyze the polymorphism of LHβgene, the results show that P1-153C→A and P564T→C were found in the populations ,respectively. The associations of the mutation with litter size were observed: for primer P1, genotype PP were significant higher than that of genotype PQ(P<0.01)in Boer goat and . genotype PP were significant higher than that of genotype PQ(P<0.01)in the third and the fourth litter size of individuals of Boer goat and in the fifth and the fourth litter size of individuals of Xinong Sannen dairy goat. For primer P5, the correlations were observed between the polymorphism and litter size of two breed: the fifth litter size of individuals with genotype LL were significant higher than that of genotype LM(P<0.01), the fourth litter size of individuals with genotype LL were significant higher than that of genotype GG(P<0.05).更多还原