【作者】 沙日耐；

【导师】 韩建林；

【作者基本信息】 中国农业科学院 ， 动物遗传育种与繁殖， 2012， 博士

【摘要】 牦牛是分布在以我国青藏高原为中心以及毗邻的高山和亚高山、平均海拔3000m以上地区的特有的畜种资源和宝贵的基因库,我国拥有世界牦牛总数的95%。牦牛对高寒草地生态环境条件具有很强的适应性,能在空气稀薄、牧草生长期短、气候寒冷的恶劣环境条件下生活自如、繁衍后代,这不仅与它能通过独特的毛被结构来适应严寒和高辐射有密切关系,而且还与它拥有能适应空气稀薄的心肺系统以及免疫系统密不可分。本研究主要对牦牛绒毛结构关键基因-角蛋白关联蛋白KAP3-1、KAP7、KAP13-1和KAPl家族4个成员,以及多态性非常丰富的免疫相关基因-DRB3第二外显子进行研究,首次获得了牦牛7个KAP基因的完整CDS序列,并对这些基因的遗传多样性、单倍型构建及其功能等作了深入探讨,主要研究结果如下：在KAP3-1基因的扩增产物大小为525bp,包含297bp的全长CDS；存在4个SNPs,决定4个基因型和单倍型,其中AABB为主要基因型,主要单倍型的频率在6个牦牛群体中均超过0.81；其中两个单倍型离牦牛的主要单倍型较远,而与普通牛的主要单倍型较近,且均以较低频率出现在5个牦牛群体,说明它们应该是通过长期的种间杂交从普通牛导入到牦牛基因组中的；有3个SNPs位于CDS区,均未引起氨基酸变化。KAP7基因的扩增产物大小375bp,包含264bp的全长CDS；牦牛和普通牛KAP7基因的序列完全一致,牦牛群体中没有发现遗传多样性；在普通牛中发现了2个SNPs,其中一个为错义突变,可能会导致蛋白结构或功能的变化。KAP13-1基因的扩增产物大小为607bp,包含495bp的全长CDS；牦牛和普通牛KAP13-1基因的单倍型有3个SNPs的差异；牦牛中存在3个SNPs,全部位于CDS区域,且均为错义突变；这3个SNPs决定7个基因型和4个单倍型,其中TTTTGG为主要基因型,BOSGR-KRTAP13-1*A为优势单倍型。在KAP1家族基因中,kAP1-1基因的扩增产物大小为617bp,其中CDS序列长度为471bp；存在14个SNPs,决定11种基因型和7个单倍型,ABCD基因型频率最高,在5个牦牛群体中都有出现,BOSGR-KRTAP1-1*A和BOSGR-KRTAP1-1*B为主要单倍型。牦牛KAPl-2基因的扩增产物大小为624bp,其中CDS序列长度为501bp,存在3个SNPs,决定5种基因型和4个单倍型,AACCAA为主要基因型,BOSGR-KRTAP1-2*A为主要单倍型。本研究第一次获得了普通牛和牦牛KAP1-3基因的全长CDS序列,并填补了普通牛基因组中这段序列的空白；牦牛和普通牛KAP1-3基因的扩增产物大小为525bp,其中CDS序列长度为429bp；牦牛中存在5个SNPs,决定5种基因型和5个单倍型,其中AABB基因型为优势基因型, BOSGR-KRTAP1-3*A为主要单倍型。牦牛KAP1-4基因的CDS区域存在30bp插入/缺失的长度多态现象,长度分别为：471bp、441bp和411bp；在471bp和411bp的片段中没有发现变异,在441bp的片段中存在2个SNPs;长度多态和SNPs共决定9种基因型和5个单倍型,虽然ABDD基因型总频率不是最高,但存在于5个牦牛群体中,AADD基因型虽然总频率最高,但仅在GT、DQK和AD牦牛中出现。结果显示在基因组结构中紧密连锁的KAP1家族4个基因的单倍型数不尽相同,说明其中可能存在重组现象,但重组热点及方式还需进一步验证。本研究除采用直接测序和克隆测序以外,还结合了等位基因特异引物扩增法,对牦牛DRB3第二外显子的多态性及其单倍型进行了分析,实验结果如下：在DRB3第二外显子中,共检测到66个SNPs和1个氨基酸密码子的插入缺失,其中只有3个SNPs可能会导致蛋白质的功能和结构的变化,它们分别是位于157bp处的G/T突变,导致缬氨酸替换为亮氨酸,220bp处的G/A突变,导致甘氨酸替换为丝氨酸,259bp处的G/A突变,导致丙氨酸替换为苏氨酸；天祝白牦牛和甘南牦牛82份样品中存在40个单倍型；对单倍型进行中性检验,发现天祝白牦牛DRB3第二外显子的遗传多样性可能受到人工选择的影响。更多还原

【Abstract】 The yak is distributed in the highland alpine and subalpine areas with an average altitude above3000m and centered on the Qinghai-Tibetan Plateau. It is a unique and valuable gene pool of livestock genetic resources. China accounts for about95%of the world’s total yak population. Yak has a strong adaptability to the alpine grasslands, survives freely and reproduces normally with thin air, short grass growing season, cold weather and very harsh environmental conditions. These distinctive physiological characteristics are attributed not only to its exceptional fiber and coat structures to become accustomed to cold and to prevent strong ultraviolet radiation, but also to its cardiopulmonary and immune systems to adapt to less oxygen in the air.In this study, a few key genes including seven keratin-associated proteins (KAPs)(KAP3-1, KAP7, KAP13-1and four members of KAP1family and the highly polymorphic exon2of DRB3genes associated with fiber and immunity traits were selected as candidates critical to genetic improvement of yak grown and development. The complete coding sequences of the seven KAPs genes were generated for the first time in yak. The genetic diversity, haplotype reconstruction and probable function of these genes were investigated in yak as well as taurine cattle. The major research findings are as follows:The PCR product of KAP3-1gene was525bp in length containing the297bp long complete coding sequence (CDS). There were four SNPs determining four haplotypes and genotypes each with the major yak haplotype BOSGR-KRTAP3-1*A present in all six yak populations at frequencies more than0.81and genotype AABB to be predominant in all six yak populations. Two of the four haplotypes were closely linked to the major taurine cattle haplotype and present at relatively low frequencies in five yak populations, indicating their taurine cattle origins through genetic introgression following historical hybridization events. Three out of the four SNPs were found in the CDS region and they were all synonymous. The PCR product of KAP7gene was375bp in length carrying the264bp long complete CDS. Yak only had a single haplotype which was shared by the major taurine cattle haplotype. There was a second haplotype in taurine cattle with two SNPs between the two haplotypes, of which one was non-synonymous but the other to be synonymous. The PCR product of KAP’13-1gene was607bp in length including the495bp long complete CDS. Three SNPs were found between the yak and cattle haplotypes while there were three non-synonymous SNPs present within the CDS of yak KAP13-1gene defining four haplotypes and seven genotypes, of which TTTTGG was the predominant genotype and BOSGR-KRTAP13-1*A to be the dominant haplotype.Among the KAP1gene family, the PCR product of KAP1-1gene was617bp in length including471bp long CDS. There were14SNPs defining11genotypes and seven haplotypes with ABCD genotype to be most frequent and present in all five yak populations and BOSGR-KRTAP1-1*A and BOSGR-KRTAP1-1*B to be major haplotypes. The PCR product of KAP1-2gene was624bp in length carrying501bp long complete CDS which had three SNPs determining five genotyping and four haplotypes with AACCAA genotype to be predominant and BOSGR-KRTAP1-2*A to be major haplotype. This study successfully generated the complete CDS of yak and taurine cattle KAP1-3gene which filled the gap in the current bovine genome assembly. The PCR product of KAP1-3gene was525bp including429bp long complete CDS. Five SNPs were detected in yak sequences and they defined five genotypes and haplotypes each with AABB genotype to be predominant and BOSGR-KRTAP1-3*A to be major haplotype. The KAP1-4gene in yak had length polymorphisms of30bp In/Del in its CDS with length variations in471bp,441bp and411bp. There was no SNP in471bp and411bp long fragments but two SNPs were found in441bp long fragment. The length polymorphisms and SNPs determined nine genotypes and five haplotypes, of which ABDD genotype were present in all yak populations though it was less frequent than the AADD genotype present only in GT, DQK and AD yak populations. It is evident that the numbers of haplotypes across the four members of the KAP1gene family were all different, indicating possible recombination in this closely linked genomic region which calls for further investigation.In search for polymorphisms and determining haplotypes in yak DRB3exon2, we not only used direct and cloning sequencing protocols, but also explored an allele-specific sequencing method to ensure the PCR amplifications of specific haplotypes from this highly polymorphic genomic fragment. A total of66SNPs present in bi-, tri-or tetra-alleles as well as a single amino acid codon In/Del mutations were identified within the yak DRB3exon2, of which only three SNPs leading to amino acid replacements were predicted to have probable functional and structural significance:157G/T mutation for Val>Leu,220G/A mutation for Cys>Ser, and259G/A mutation for to Arg>Thr. Forty haplotypes were reconstructed from82samples of Tianzhu White yak and Gannan yak. Fu and Li’s D*neutrality test revealed a probable impact of artificial selection on the genetic diversity of DRB3exon2in the Tianzhu White yak.更多还原