【作者】 潘和平；

【导师】 刘孟洲；

【作者基本信息】 甘肃农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 本研究利用PCR-SSCP和PCR-RFLP技术,对新疆巴州牦牛、天祝白牦牛、甘南牦牛、青海环湖牦牛四个不同类群及培育品种大通牦牛PRL、PRLR、GH、GHR、IGF-Ⅰ五个候选基因遗传多态性进行了测定,分析了等位基因频率、基因型频率、多态信息含量、杂合度和有效等位基因数等指标,并运用最小二乘法分析了所发现的基因多态位点与牦牛体尺、体重等生长发育性状的关系,初步探讨了PRL、PRLR、GH、GHR、IGF-Ⅰ基因作为影响生长发育性状候选基因的可能性,取得了如下研究结果:1.利用PCR-SSCP技术对类胰岛素生长因子-Ⅰ基因(IGF-Ⅰ)多态性研究表明:(1)首次在牦牛IGF-Ⅰ基因的第1内含子发现PCR-SSCP多态,IGF-Ⅰ基因第1内含子的PCR-SSCP多态性检测结果表明,该多态位点由一对等位基因A、B控制,序列比对发现在67bp处单碱基C的缺失,造成了该多态位点的出现。等位基因A(B)在大通牦牛、天祝白牦牛、甘南牦牛、青海环湖牦牛、新疆巴州牦牛群体中的基因频率分别为:0.9028(0.0972)、0.9234(0.0766)、0.8485(0.1515)、0.7929(0.2071)、0.9200(0.0800),等位基因A为五个群体中的优势等位基因。(2)IGF-Ⅰ基因第5外显子的PCR-SSCP分析结果表明,品种间基因型分布存在显著差异。在大通牦牛中检测到了从、BB和AB三种基因型,而在其它群体中只发现AA型,呈单态型,达到了纯合状态。经克隆测序表明:该位点的多态是由于在202bp处一个T→C的突变,导致等位基因A突变成等位基因B。(3)对IGF-Ⅰ基因上2个多态位点不同基因型与大通牦牛6月龄生长发育性状指标进行显著性检验,结果表明,IGF-Ⅰ基因第1内含子AA基因型的体重、体斜长显著高于AB、BB基因型(P＜0.05)。(4)IGF-Ⅰ基因的二个多态位点基因型与大通牦牛早期发育的分析表明,18月龄IGF-Ⅰ第5外显子位点AA基因型的个体体高最小二乘均值(91.43cm),与BB型(80.00cm)差异显著(P＜0.05)。在牦牛的早期选育中,AA型个体可以作为选留的对象。(5)六月龄大通牦牛群体体尺指标与甘南牦牛和天祝白牦牛群体比较可以看出不论公牛群还是母牛群,大通牦牛的平均体重显著大于甘南牦牛和天祝牦牛群体平均体重(P＜0.05):大通牦牛的平均体斜长显著比甘南牦牛和天祝白牦牛长(P＜0.05),而胸围相比大通牦牛略高于其它两个群体但差异不显著(P＞0.05)。在这些体尺指标上,大通牦牛表现出明显的优势。2.利用PCR-SSCP和PCR-RFLP技术对GH基因、GHR基因多态性进行了研究,结果表明:(1)GH基因第3外显子在183bp处发生T-C突变,该多态位点由一对等位基因A、B控制,其中A为优势等位基因,AA为优势基因型。五个群体的哈代—温伯格平衡检验结果显示,青海大通牦牛、青海环湖牦牛、天祝白牦牛、甘南牦牛处于非平衡状态。运用最小二乘法对三个牦牛群体进行分析发现GH基因第3外显子对体重有显著影响(P＜0.05),其中,最小二乘法得出的均值,AA型最大,AB型次之,最小的为BB型。另外,GH基因第3外显子对体长也有显著影响(P＜0.05),其中AA型体长最长,AB型次之,BB型体长最短。GH基因第3外显子对体高、胸围、管围无显著性影响(P＞0.05)。(2)GH基因第5外显子在64bp处发生A-C的突变。GH基因第5外显子位点上,A1为优势等位基因,A1A1为优势等位基因型。五个品种进行的哈代—温伯格平衡检验结果显示,甘南牦牛处于非平衡状态;运用最小二乘法对五个牦牛品种进行分析发现第5外显子对生长发育指标没有显著影响(P＞0.05)。(3)经过PCR-SSCP分析,GH基因保守区位点呈单态性,没有发现多态性信息。(4)经过PCR-RFLP分析,特异性GH-ⅠR引物对牦牛基因进行PCR扩增,得到一条长约1244bp的产物,用限制性内切酶AvaⅠ、HaeⅢ、HinfⅠ、PstⅠ、SmaⅠ进行酶切,AvaⅠ、HinfⅠ、HaeⅢ、PstⅠ均具有预期的酶切位点,没有发现多态性,而SmaⅠ不具有预期的酶切位点,经测序比较,发现在878位点处的SmaⅠ酶切位点“CCC|GGG”丢失。特异性GH-2R引物对牦牛基因组进行PCR扩增,得到长度约为1037bp的DNA片段。采用限制性内切酶HinfⅠ、SmaⅠ、PstⅠ对该PCR产物进行单酶酶切,结果发现HinfⅠ、SmaⅠ、PstⅠ均具有酶切位点,且电泳检测结果与预期相一致,不具有限制性片段长度多态性。而GH-2R在限制性内切酶HaeⅢ的切点中,电泳检测结果与预期不一致,经测序比较,发现在75位点处的酶切位点“CC|GG”丢失。3.利用PCR-SSCP技术对PRL、PRLR基因多态性进行了研究,结果表明:催乳素受体基因的第2外显子、第3外显子在大通牦牛、甘南牦牛、天祝白牦牛、青海环湖牦牛、新疆巴州牦牛5个品种中检测到了AA、AB和BB基因型,而且在PRLR-exon2中A等位基因为5个牦牛群体的优势等位基因,具有较高的基因频率;在PRLR-exon3中,B等位基因较A基因占优势。PRLR-exon2在大通牦牛、新疆牦牛、天祝白牦牛、青海环湖牦牛、甘南牦牛A基因频率都超过了0.500,分别为0.520、0.560、0.600、0.580和0.640。基因多态信息表现为中度多态。测序表明催乳素受体基因第二外显子在174bp处的突变,为A→G,表现多态性。PRLR-exon3,在大通牦牛、天祝白牦牛、甘南牦牛、青海环湖牦牛、新疆牦牛五个群体中都检测出AA、BB、AB三个基因型,而且B等位基因为优势等位基因。PRLR-exon3的PCR-SSCP结果及测序结果分析显示该位点的98bp处多态为C→G取代造成。等位基因A(B)在大通牦牛,天祝白牦牛、甘南牦牛、青环湖原牦牛、新疆巴州牦牛的基因频率分别为:0.460(0.540)、0.420(0.580)、0.500(0.500)、0.460(0.540)、0.450(0.550)。在PRLR-exon3中,除了在天祝白牦牛中表现为高度多态,其余牦牛品种多为中度多态,遗传力较高,作为遗传杂交可能有较强优势。更多还原

【Abstract】 In this study,the objects were Xinjiang,Tianzhu white,Gannan,around Qinghai Lake and Datong yak that was the bred breed.The genetic polymorphism of candidate gene in yak,such as PRL,PRLR,GH,GHR and IGF-Ⅰ,was researched with PCR-SSCP and PCR-RFLP.The body size and body weight of yak were used as the index for growth and development. Relative allele frequency,polymorphicsm information content（PIC）,heterozygosity and effective allele numbers of above yaks were analyzed.Relation between polymorphic loci observed and traints for growth and development was analyzed by least square method.Then possibility of effect of PRL,PRLR,GH,GHR and IGF-Ⅰon candidate gene for productive performance was researched.The results were as follows.1.On polymorphism IGF-Ⅰgene researched by PCR-SSCP（1） The polymorphism of PCR-SSCP on the 1^st intron in IGF-Ⅰgene of yak was discovered for the first time.By measuring the polymorphism of PCR-SSCP on the 1^st intron in IGF-Ⅰgene,the results indicated that this polymorphic locus was controlled by a pair of alleles,namely allele A and B.according to comparing sequence with BioEdit software,a single base,C,was delected in the 67 bp.So,the polymorphic locus appeared.The gene frequency of allele A or B in Xinjiang yak,Tianzhu white yak,Gannan yak,yak around Qinghai Lake and Datong yak was 0.9028（0.0972）,0.9234（0.0766）,0.8485（0.1515）, 0.7929（0.2071） and 0.9200（0.0800） respectively.Allele A was the predominant allele in five yak populations.（2） By analyzing the polymorphism of PCR-SSCP on the fifth extron in IGF-Ⅰgene,the results indicated,the distribution of genotype was significant difference among breeds.In Datong yak,there were three genotypes,that was AA,BB and AB.While in other yaks,only AA,without AB and BB,its distribution was monomorphism without polymorphic loci.So, they were heterozygosity.By cloning and sequencing,the polymorphism on the locus was resulted from mutation of allele A to B,which T mutated C in the 202 bp.（3） Different genotype of 2 polymorphic loci on IGF-Ⅰgene and index for productive traits were tested significantly.The results showed,the body weight and body length with AA on the 1^st intron in IGF-Ⅰgene were significantly higher than that with AB and BB（P＜0.05）.（4） By analyzing the genotype of 2 polymorphic loci on IGF-Ⅰgene and early growth of Datong yak,the results indicated that there was significant different（P＜0.05） in square mean of body height between AA individual（91.43） and BB individual（80.00） with IGF-Ⅰ（P5） at 18 months old.So,AA individual may be selected.（5） By comparing body size Datong yak,Gananan yak and Tianzhu white yak at the age of 6-month,the results showed,in regardless male or female yaks,the body weight of Datong yak was heaver than that of Gananan yak and Tianzhu white yak.The body length of Datong yak was obviously higher than that of Gananan yak and Tianzhu white yak.The heart girth of Datong yak was a little larger than that of Gananan yak and Tianzhu white yak.So,in respect of growth index,there was obvious advantage in Datong yak.2.On polymorphism of GH and GHR gene tresearched by PCR-SSCP and PCR-RFLP.（1） There was a mutation from T to C on the locus of GH3 in the 183 bp.In them,A was the predominant allele.By Hardy-Weinberg equilibrium testing in 5 yak breeds,there was not equilibrium in Tianzhu white yak,Gannan yak,yak around Qinghai Lake and Datong yak.3 yak breeds were analyzed through least square method,GH3 obviously affected the body weight.In them,as for the mean value calculated by least square method,AA maximum,AB next and BB minimum.In addition,GH3 obviously affected the body length,AA maximum, AB next and BB minimum.GH3 didn’t affect the body height,heart girth and cannon circumference significantly.（2） There was a mutation from A to C on mutation locus of GH4 gene in the 64bp.A was the predominant allele.By Hardy-Weinberg equilibrium testing in 5 yak breeds,there was not equilibrium in Gannan yak.Through analyzing 5 yak breeds with least square method,the results indicated that GH3 didn’t affect the body weight significantly.（3） By PCR-SSCP analyzing,there was not polymorphism on GH5 locus.（4） By PCR-RFLP analyzing,the primer GH-1R was used to multiply the gene in yak with PCR.1244bp production was obtained.This fragment was cut by restriction enzyme,such as AvaⅠ,HaeⅢ,HinfⅠ,PstⅠand SmaⅠ,there were anticipative restriction enzyme cutting side, while without polymorphism.SmaⅠdidn’t have restriction enzyme cutting side.By sequencing and comparing,it was found that enzyme cutting side "CCC|GGG" at 878 locus on SmaⅠwas lost,so,there was not enzyme cutting side of SmaⅠ.in this experiment. The primer GH-2R was used to multiply the genome in yak with PCR.1037 bp DNA fragment was obtained.This fragment was cut by restriction enzyme,such as HaeⅢ,HinfⅠ, SmaⅠand PstⅠ.the results indicated that there were enzyme cutting sides on HinfⅠ,SmaⅠ and PstⅠ.by electrophoresis testing,it was found that the results were consistent with the anticipation.However,there was not restriction polymorphism.GH-2R was found that enzyme cutting side "CC|GG" at 75 locus was lost in restriction enzyme HaeⅢcutting side.3.Polymorphism of PRL、PRLR gene researched by PCR-SSCPAs for the second and third extrons in PRL receptor gene,there were AA,AB and BB genotype in Xinjiang yak,Tianzhu white yak,Gannan yak,yak around Qinghai Lake and Datong yak.A allele in PRLR-exon2 was the predominant allele in 5 yak populations. PRLR-exon2 gene frequency in Xinjiang yak,Tianzhu white yak,Gannan yak,yak around Qinghai Lake and Datong yak was above 0.500,the A gene frequency was 0.520,0.560,0.600, 0.580 and 0.640 respectively.The PIC was middle polymorphism,which had a certain genetic effect.Sequencing indicated that there ware a mutation in the second extron of PRLR gene,it was A to G in the 174bp,which resulted in locus mutation and polymorphism.As for PRLR-exon3 gene,there were AA,AB and BB genotype in Xinjiang yak,Tianzhu white yak, Gannan yak,yak around Qinghai Lake and Datong yak.B allele was the predominant allele. By analyzing PCR-SSCP production of PRLR-exon3 gene,the polymorphism resulted from the base C substituting G in the 98bp.A（B） allele frequency in Xinjiang yak,Tianzhu white yak,Gannan yak,yak around Qinghai Lake and Datong yak was 0.460（0.540）,0.420（0.480）, 0.500（0.500）,0.460（0.540） and 0.450（0.550） respectively.In PRLR-exon3,the polymorphism in Tianzhu white yak was higher,the polymorphism in other yak was middle. The heritability was higher.Crossbreeding vigor was strong.更多还原