【作者】 张焱如；

【导师】 芒来；

【作者基本信息】 内蒙古农业大学 ， 动物遗传育种与繁殖， 2005， 博士

【摘要】 本研究以不同品种马为研究对象,利用 PCR 技术,首次成功地克隆了 6 个品种马的生长激素基因 1923bp 的片段(GenBank 登录号:AY837571),包括全部外显子和内含子。测序后,对其序列结构、核苷酸组成及所编码的氨基酸进行了初步研究,并利用 DNAMAN、DNAStar、Mega2.1、PAUP4.0 等生物学软件,构建了马 GH 基因进化树,同时对各品种马 GH 基因序列进行了 PCR-SSCP 和 PCR-RFLP 分析。研究结果表明: 1、马 GH 基因含有 5 个外显子和 4 个内含子,5 个外显子大小分别为:10 bp、161 bp、117 bp、162 bp 和 201 bp;4 个内含子大小分别为 253 bp、213 bp、199 bp 和 272 bp。外显子数目、大小、位置与哺乳类其他物种相比,表现出高度保守性。 2、马 GH 基因序列核苷酸组成富含 GC(60%以上),且外显子 GC 含量(61%)明显高于 5’端和 3’端的非编码区。编码氨基酸的密码子不同位点 GC 含量也有显著差异,表现为第一、第三位碱基富含 GC,且第一位碱基的 GC 含量接近于全序列,第三位碱基 GC 含量远高于其它位点,达 84.1%,第二位碱基富含 AT。密码子碱基组成的差异因物种不同而异,具有种属特异性。 3、马 GH 基因外显子在编码氨基酸时,对同义密码子的使用表现有强烈的偏好性。不同物种对同义密码子使用的偏倚程度与偏好类型各不相同,也具有种属特异性。 4、马 GH 基因所编码的蛋白质中,20 种氨基酸的含量差异很大。亮氨酸(L)含量最高,达 14.28%;色氨酸(W)最低,为 0.93%。极性氨基酸酸含量(54.99%)高于非极性氨基酸(45.01%)。此外,半胱氨酸(C)的含量与其他哺乳类完全一样,均为 5 个,而且半胱氨酸的位置在不同物种中高度保守,说明半胱氨酸对维持 GH 的空间结构起到极其关键的作用。 5、马 GH 氨基酸位点的进化演变表明,马各品种间氨基酸差异数很少,仅有 0～5个氨基酸不同,说明相互间的进化距离很小。马与人及其它动物相比较,进化距离由近至远依次为猪、狗、牛、羊和人。 6、采用不同方法构建的马 GH 基因进化树表明,马 GH 基因不同区段进化速率不同,外显子区域进化速率最慢;内含子序列中碱基变异频繁,进化较快。进化树分枝显示,乌珠穆沁马、三河马、锡尼河马三个品种与其它品种间已形成明显的进化分歧。 7、PCR-SSCP 分析结果表明,在所检测的 5’端侧翼区、第一内含子、第二外显子和第五外显子四个位点中,仅第五外显子检测到多态,该多态位点由 A 和 B 两种等位基因控制。GH 基因第 1719bp 处一个 T→C 的突变和 1743 处 G→A 的突变,导致等位基因 B 变为等位基因 A。除纯血马外,A 基因在各品种中均为优势等位基因,B 基因为纯血马的优势基因。在此多态位点,乌珠穆沁马、乌审马和巴尔虎马处于 Hardy-Weinberg 平衡状态,纯血马、三河马和锡尼河马则未达到 Hardy-Weinberg 平衡状态。 8、对马 GH 基因全序列进行 PCR-RFLP 分析,采用四种限制性内切酶 BstⅪ、HindⅢ、SmaⅠ和 StuⅠ进行酶切,仅后两种酶检测到多态。SmaⅠ位点出现 A、B 两种等位基因,在 GH 基因序列的 1256bp 处发生 T→C 的转换,导致产生 1 个 SmaⅠ的酶切位点 CCC↓GGG,从而使基因型 AA 变为基因型 BB。在该位点乌审马 B 基因频率最高,为 0.667,纯血马 A 等位基因频率最高,达 0.643。乌审马、乌珠穆沁马和巴尔虎马处于 Hardy-Weinberg 平衡状态,锡尼河马、三河马和纯血马处于非平衡状态。 StuⅠ位点存在 C 和 D 两种等位基因,在 GH 序列的 1353bp 处发生了 C→T的变化,导致产生一个 StuⅠ的酶切位点 AGG↓CCT,使基因型 CC 变为基因型DD;C 基因为锡尼河马的优势等位基因,基因频率达 0.63,D 基因是纯血马的优势等位基因,基因频率为 0.74。该位点上,只有乌审马处于 Hardy-Weinberg平衡状态,其余品种均未达到 Hardy-Weinberg 平衡。 9、本研究首次对几个品种马 GH 基因序列、分子进化及其多态性进行了初步研究,研究结果对今后合理利用、开发和保护我国马种的遗传资源具有一定的指导意义。更多还原

【Abstract】 The different breeds of horses were studied. Using the PCR technique, the full sequences of horse growth hormone(hsGH) gene of six breeds were firstly cloned and sequenced. The length of this gene was 1923bp, including all the exons and all the introns (GenBankAccession:AY837571). And the composition of nucleotide and amino acid encoded by the gene were analyzed. The molecular evolution trees were established by DNAMAN, DNAstar, Mega2.1 and PAUP4.0. The PCR-SSCP and PCR-RFLP analysis of the full sequence of hsGH gene in different horse breeds were studied. The results were as follows: 1、The sequence of amplified horse GH gene was 1923bp, including five exons and four introns. The length of five exons were 10bp, 161bp, 117bp, 162bp and 201bp. The length of introns were 253bp, 213bp, 199bp and 272bp long. The number, length and locus of exons showed high conservative compared with other mammals. 2、The analysis of nucleotide composition showed that G/C content (over 60%) is abundant in horse GH gene, and the contents (61%) of exons were significant higher than those of noncoding regions in 5’and 3’ends. In encoding regions, the G/C contents were abundant at the first (60.3%) and third (84.1%) sites. A/T content was rich in the second site(58.7%). The codon composition were variable in different species, which were specific in each species. 3、The analysis of codon usage indicated that the usage of synonymous codon was very biased, and the biased kinds were different among species. 4、The composition analysis of amino acid showed that the contents of 20 kinds of amino acid were unbalanced. The most was leucine(L,14.28%) and the least was tryptophan(W,0.93%). The contents of polar amino acid(54.99%) were higher than those of nonpolar amino acid(45.01%) in growth hormone of all hores. The amounts of cysteine were five, same with other mammals. The location of cysteine was conservative in different species. This indicated that the cysteine was very important in supporting the structure of GH. 5、Only 0-5 amino acids were different in 6 horse breeds GH. Compared to other mammals, the evolution distance from near to far was pig, dog, cow, sheep, and human. 6、From the molecular evolution trees constructed by different tree-building methods, we got the conclusion that the evolution speed of horse GH gene was different in different regions. Exon regions were more conservative than intron regions. So, the evolution speed of intron regions was more quickly. Sanhe horse, Wuzhumuqin horse and Xinihe horse lay in a same evolution branch, it was different from other branches of horse. 7、The polymorphisms of PCR-SSCP existed only in the fifth exon, but not in the 5’flanking region, first intron, and second exon. This polymorphism of the fifth exon was controlledd by two alleles: A and B.The mutation of T→C and G→A at 1719bp and 1743bp converted allele B into A. Allele B was dominant in Chunxue horse, while allele A was dominant in other horse breeds. The gene reached Hardy-Weinberg equilibrium in Wuzhumuqin horse, Wushen horse and Baerhu horse. 8、The analysis of PCR-RFLP showed that the polymorphism sites with SmaⅠand StuⅠwere inspected, but not with BstXⅠand HindⅢ. There were two alleles A and B at SmaⅠsite. The mutation of T→C at 1256bp provided a SmaⅠsite(CCC↓GGG), which lead to the change of genotype from AA to BB. The frequency of allele B of Wushen horse was the highest (0.667) and the frequency of allele A was the highest for Chunxue horse(0.643).The gene were in Hardy-Weinberg equilibrium in Wushen horse, Wuzhumuqin horse and Baerhu horse. There were two alleles C and D at StuⅠsite.The mutation of C→T provided a StuⅠsite(AGG↓CCT) and lead to the change of genotype from CC to DD. C and D were prevailing alleles for Xinihe horse and Chunxue horse .The gene frequencies were 0.63 and 0.74 respectively. The gene only in Wushen horse was in Hardy-Weinberg equilibrium . 9、Our research based on horse’GH gene sequences will geatly benefit the utilization, development and protection of genetic resource更多还原