【作者】 耿荣庆；

【导师】 常洪；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 运用典型群简单随机抽样的方法,分别采集牛亚科家畜普通牛(Bos taurus)、瘤牛(Bos indicus)、牦牛(Bos grunniens)、大额牛(Bos frontalis)、沼泽型亚洲水牛(Bubalus bubalis)和河流型亚洲水牛(Bubalus bubalis)共6个牛种的血液样本,避免具有相同血统来源的个体一起进入样本。结合PCR扩增技术和DNA直接测序技术,在测定6个牛种生长激素(Growth hormone,GH)基因、肌肉生长抑制素(Myostatin,MSTN)基因和线粒体细胞色素b(Cytochrome b,Cyt b)基因共3个结构基因编码区全序列的基础上,分析各基因的单核苷酸多态性(Single Nucleotide Polymorphism,SNP)和分子进化特征;引用其他牛种的同类序列资料,以非参数检验法检验Cyt b基因编码区核苷酸序列以及氨基酸序列的相对进化速率,提出肯定或否定分子钟假说的部分客观资料。结果表明:1.在GH基因和MSTN基因编码区域,在6个牛种中分别确定了16个和24个SNP位点,而且无论是在种内还是在种间,均表现为二等位基因;在Cyt b基因编码区域,在6个牛种中共确定了220个SNP位点,绝大多数位点(约占94.55%)表现为二等位基因,5%的位点表现为三等位基因,仅有0.55%的位点表现为四等位基因。2.3个结构基因编码区的单倍型多样性和核苷酸多样度在不同牛种中没有显示出完全的一致性,代表了各牛种不同基因遗传多样性程度的变化轨迹;6个牛种3个结构基因的总单倍型多样性和总核苷酸多样度却表现出一致性的变化,反映出牛亚科家畜Cyt b基因、MSTN基因和GH基因的遗传多样性水平依次降低。3.无论是在牛种内还是在牛种间,各结构基因编码区序列间的碱基组成差异都较小;碱基替换大都发生在密码子的第3位点,少部分发生在第1位点,第2位点最保守;碱基替换中存在转换偏奇,转换/颠换比在GH基因编码区为4.6,在MSTN基因编码区为2.8,在Cyt b基因编码区为5.4。4.GH基因、MSTN基因和Cyt b基因编码区都存在密码子使用偏好性,GH基因中发现了25个偏好性密码子,MSTN基因中发现了30个偏好性密码子,Cyt b基因中发现了27个偏好性密码子;在核基因GH基因和MSTN基因中,发现了11个共同的偏好性密码子;在核外mtDNA Cyt b基因中发现了与核基因共享的5个偏好性密码子,并且都是编码相同的氨基酸。5.在GH基因、MSTN基因和Cyt b基因中,大多数位点间的替代都是同义突变,非同义突变(错义突变)位点远远少于同义突变位点;同义与非同义替代发生的速率比全部小于或等于1,表明牛亚科家畜3个结构基因在进化过程中均不受达尔文正向选择的影响,少数位点受到负向选择的作用,是净化选择的结果。进一步说明3个结构基因仍然保持着非常重要的生物学功能。6.基于GH基因编码区序列构建的分子进化树分析表明,沼泽型水牛与河流型水牛聚为一类,它们与普通牛、瘤牛、牦牛以及大额牛之间的遗传分化已经十分明显。普通牛、瘤牛、牦牛、大额牛共同拥有一条相同的核苷酸序列,推测这一共享的序列可能是普通牛、瘤牛、牦牛和大额牛GH基因编码区的共同祖先序列,其它的核苷酸序列可以看作是在此基础上发生的随机突变类型(主要是遗传漂变的影响)。7.基于MSTN基因编码区序列构建的分子进化树表明,沼泽型水牛与河流型水牛之间的分化也已明显,而它们与普通牛、瘤牛、牦牛、大额牛间分化则更为明显。普通牛、瘤牛、牦牛、大额牛序列间没有显示出完全的分化,它们也没有共同拥有相同的核苷酸序列。除了个别牛种共享少数序列以外,其余的单倍型序列都是各牛种所特有的类型。8.从Cyt b基因序列揭示的牛亚科动物系统发生关系来看,牛亚科家畜可划分为家牛属、准野牛属、牦牛属和亚洲水牛属4个属,它们之间的分化十分明显;家牛属与准野牛属、牦牛属和水牛属之间的亲缘关系逐渐降低。9.从母系起源的角度来看,我国大额牛种群存在明显的分化,它可能具有十分独特的起源。虽然大额牛有部分单倍型序列与普通牛或瘤牛聚为一类,但是它不可能起源于普通牛或瘤牛,这种现象应该可以视为大额牛与普通牛及瘤牛之间曾经拥有过共同的祖先序列,并且在漫长的进化历程中仍然保留着祖先序列的基本特征。大额牛与印度野牛有着十分密切的关系,它们在较早的世代具有共同的母系起源,并且有可能是现已灭绝的某种野生牛的后代。10.从母系遗传的角度来看,雷琼牛仅起源于瘤牛应该是确信无疑的,它也是我国迄今为止发现的少数具有单一起源的瘤牛型黄牛品种。瘤牛进化网络关系分析表明,雷琼牛与亚洲其它地区瘤牛可能是在不同的地点由野生的瘤原牛独立驯化而来的,推测中国南方某些地区在史前也应该是一个独立的瘤原牛驯化中心。11.分子钟检验的结果说明,接受或者拒绝分子钟假说与所检测物种亲缘关系的远近无明显相关性,分子钟假说在一定的物种范围内是存在的;与基于氨基酸序列的检验结果相比较,基于核苷酸序列的检验结果更易于拒绝分子钟假说。12.对长期进化而言,既无基因亦无基因产物以绝对恒定速率变化,试图寻找具有通用分子钟的基因或蛋白质都是徒劳的,即分子钟只在一定的范围内存在,并且不具有通用性。更多还原

【Abstract】 Blood samples of six domestic species of Bovinae were collected from Bos taurus, Bos indicus, Bos grunniens, Bos frontalis, Swamp buffalo(Bubalus bubalis) and Water buffalo(Bubalus bubalis) using simple random sampling method in typical colony which avoiding the same bloodline into the samples. Single nucleotide polymorphisms and molecular evolution characters were analyzed basing on complete coding sequence of growth hormone gene, myostatin gene and mt DNA cytochrome b gene from six bovine species combinating PCR technology and direct DNA sequencing technology. Non-parameter test was used to testing relative evolution rate of nucleotide sequences and amino acid sequences of Cyt b gene together with cited sequences of other bovine species which provide partial evidences for molecular clock hypothesis. The conclusions indicated as follows: 1. Sixteen and twenty-four SNP sites were determined from coding region of GH gene and MSTN gene of six bovine species respectively. These SNP sites all showed two-allele model whether within species or between species. Two hundred and twenty SNP sites were determined from coding region of Cyt b gene, most sites showed two-allele model, 5% sites showed three-allele model and only 0.55% sites showed four-allele model.2. There was no complete accordance between haplotype polymorphism and nucleotide polymorphism of three structure genes in different bovine species which represented the variation track of genetic diversity extent of different gene. There was accordant variation between totall haplotype polymorphism and nucleotide polymorphism of three structure genes of six bovine species, which reflected the decreased polymorphisms gradually of Cyt b gene, MSTN gene and GH gene of domestic species of Bovinae.3. There was little difference in base composition of coding region sequences of structure gene whether within species or between species. Most base substitution arised in the third codon, few arised in the first codon and the second codon was the most conservative. Transition biased was observed and the ratio of transition to transversion were 4.6, 2.8 and 5.4 in GH gene, MSTNgene and Cyt b gene respectively.4. Codon usage biased arised in the coding region of GH gene, MSTN gene and Cyt b gene. There were twenty-five, thirty and twenty-seven biased codons in GH gene, MSTN gene and Cyt b gene respectively. Eleven biased codons were found in GH gene and MSTN gene. Five biased codons were shared between exo-nucleus mtDNA Cyt b gene and nucleus gene, which coding the same amino acid.5. Most substitutions were synonymous and nonsynonymous (missense mutation) was greatly less than synonymous in the coding region of GH gene, MSTN gene and Cyt b gene. The ratio of synonymous and nonsynonymous substitution was all less or equal to one, which manifesting that three structure genes of domestic species of Bovinae were not affected by Darwin positive selection in the process of evolution and few site was affected by negative selection which was the result of purifying selection. These results further demonstrated that three structure genes still retain important biological functions.6. Molecular tree basing on GH gene coding sequence showed that swamp buffalo and water buffalo clustered and there was obvious differentiation between these two buffalo together with very obvious differentiation between these two buffalos and Bos taurus, Bos indicus, Bos grunniens, Bos frontalis. These four bovine species shared an identical sequence, which maybe an ancestral sequence of them and other sequences could be regarded as the random mutation type deriving from it (mainly affected by genetic drift).7. Molecular tree based on coding sequence of MSTN gene showed that there was not only obvious differentiation between swamp buffalo and water buffalo but also more obvious differentiation between these two buffalos and Bos taurus, Bos indicus, Bos grunniens, Bos frontalis. No complete differentiation appeared among Bos taurus, Bos indicus, Bos grunniens, Bos frontalis and no shared nucleotide sequence among them. Except few shared sequence between few bovine species, the rest haplotypes were specific types of different bovine species. 8. The phylogenetic relationship revealed by Cyt b gene showed that domestic species of Bovinae could be divided into four genus with obvious differentiation which including Bos, Bibos, Poephagus and Bubalus. The phylogenetic relationship between Bos and Bibos, Poephagus, Bubalus decreased gradually.9. There was obvious differentiation of gayal population in China from maternal origin and it maybe possess a special origin. Although part haplotypes of Bos frontalis clustered with haplotypes of Bos taurus and Bos indicus, it couldn’t origin from Bos taurus or Bos indicus. This phenomenon could be regarded as that Bos frontalis, Bos taurus and Bos indicus once shared identical ancestral sequences. The basic characters of these ancestral sequences were still retained in Bos frontalis in the process of long evolution. There was very close relationship between Bos frontalis and Bos gaurus, which showed that they might share the same maternal origin in an anterior time and maybe the progeny of a kind of extinct wild bovine species.10. It was sure that Leiqiong cattle origined from Bos indicus from maternal genetics. Leiqiong cattle was also few cattle breed of zebu type possessing single origin. Network relationships of Bos indicus showed that Leiqiong cattle and other Asian zebu might be domesticated independently from wild Bos nanadicus in different locality. It was deduced that there was an independent domesticated center of Bos nanadicus in antiquity in the south of China.11. The result from molecular clock testing showed that there was no obvious relevance between accepting or refusing molecular clock hypothesis and phylogenetic relationship of tested species. Molecular clock hypothesis existed in some extent of species. It was easy to refusing molecular clock hypothesis basing on the tested result from nucleotide sequence compared with the result from amino acid sequence.12. There was no gene or the product of gene variing in a stable rate absolutely in long evolution. Searching of popular molecular clock of gene or protein was in vain, that was to say, molecular clock only existed in some extent and possessed no generality.更多还原