【作者】 应三成；

【导师】 张义正；

【作者基本信息】 四川大学 ， 遗传学， 2007， 博士

【摘要】 藏猪(Sus scrofa，Tibetan swine)主要产于我国西藏、四川西部、云南西北部以及甘肃南部的农区和半农半牧区，是世界上分布在海拔最高地区(2500～4300m)的稀有放牧猪种之一。藏猪具有抗逆性强、肌肉纤维细、肌间脂肪含量高和肉质风味好等优点；但同时存在产仔数低、生长缓慢和胴体瘦肉率低等不足。由于藏猪的生产性能低下，为了提高其生产性能，因而长期以来引进其他猪种与之进行杂交，导致其数量急剧减少，若不采取有效的措施，藏猪也会和许多其他地方猪种一样濒临灭绝，其所携带的优良基因，在我们还没有认识其价值以前就永远从基因库中消失。因此，有针对性开展藏猪遗传特性方面的研究，对猪种资源的保护和开发利用具有重大的价值，对今后的新品种培育和品种复壮、实现养猪生产的可持续发展具有十分重要的战略意义。由于藏猪分布在偏远高海拔山区和处于放牧的半野生状态，因此采样和测定研究工作难度大，目前国内外对这一宝贵的猪种资源的研究很少，并且主要是集中在生长发育性能和血液蛋白多态性研究方面，有关藏猪抗病力、肌肉品质和产仔数等特色性状基因方面的研究尚属空白。天然抗性巨嗜细胞蛋白基因(natural resistance associated-macrophage protein 1，NRAMP1)、心脏脂肪酸结合蛋白基因(heart fatty acid binding protein，H-FABP)和雌激素受体基因(estrogen receptor，ESR)是与抗病力、肌肉品质和产仔数相关的侯选基因，藏猪的这3个基因尚未有人研究。因此，本论文选择藏猪的NRAMP1、H-FABP和ESR基因进行研究，以发掘藏猪的优势特色，为藏猪资源的保护和开发利用奠定基础。首先，我们克隆了藏猪NRAMP1基因。从脾脏和血液白细胞样品制备RNA后，用RT-PCR的方法克隆了藏猪NRAMP1基因，序列分析发现其开放阅读框长度为1614bp，编码538个氨基酸残基，其蛋白质分子量为58．8kD，等电点为8．01，疏水氨基酸占52．8％，亲水氨基酸占31．4％，碱性氨基酸占5．8％，酸性氨基酸占5．4％，该蛋白质不含信号肽序列。预测的藏猪NRAMP1蛋白质具有其他物种保守的所有结构区域，包括1个富含脯氨酸和精氨酸的SH3结合域(SH3-binding domain)，4个PKC磷酸化位点(S／T-X-R／K)，2个N型糖基化位点(N-X-S／T)，1个转运功能区(transport motif)和12个转膜区域(transmembrane，TM)。将藏猪的NRAMP1cDNA序列与家猪相应序列比较，有13个位点发生了突变，其同源性达99．2％，但编码的氨基酸序列完全相同。藏猪与狗、绵羊、牛、鹿、马、人和鼠的NRAMP1cDNA之间的同源性分别为91．2％、88．7％、88．1％、86．8％、85．8％、83．2％和71．6％。其次，采用半定量和定量PCR方法，对藏猪的10种组织中NRAMP1基因的表达模式和表达水平进行研究。结果表明，该基因在藏猪的10个组织以及白细胞中均获得表达，不存在组织表达特异性。该基因在藏猪中表达比西方家猪更加广泛，说明NRAMP1基因在不同的品种间可能存在差异。定量和半定量PCR结果进一步证实了NRAMP1基因在藏猪中没有组织表达特异性。该基因表达拷贝数平均为3233拷贝／μL cDNA，但各组织间表达丰度存在极大差异，表达量最高为脾脏，最低为肝脏，两者相差77倍以上。各组织表达丰度从高到低的顺序是脾脏、大脑、肺、回肠、肾脏、淋巴结、结肠、心脏、肌肉和肝脏。上述测试结果为藏猪较其他猪种具有更强的抵抗力提供了间接证据。再其次，采用PCR-RFLP技术(限制内切酶采用MspⅠ、HaeⅢ和HinfⅠ酶)检测了96头藏猪在H-FABP基因位点的遗传变异。结果表明：藏猪H-FABP基因在HinfⅠ和MspⅠ酶切位点均具有多态性，分别表现为HH、Hh、hh和AA、Aa和aa基因型，其中，H和A等位基因的频率分别为0．521和0．406；在HaeⅢ位点无变异，均表现为DD型。最后，采用PCR-RFLP技术检测了96头藏猪在ESR基因位点的遗传变异，并分析了不同基因型对产仔数的影响。结果表明：在藏猪中只有AA和AB 2种基因型，没有高产仔的优势基因型BB，其中，AA和AB基因型分布频率分别为0．75和0．25，A、B等位基因的频率分别为0．875和0．125，研究结果较好地解释了藏猪产仔数不高的现象。藏猪不同ESR基因型产仔数和产活仔数之间存在明显差异，其中AB基因型的产仔数较AA型高2．03头。这些发现对藏猪的产仔数性状的改进具有重要意义。对藏猪最具特色的3个性状相关基因进行的初步研究和所获得结果，为认识、保护和利用藏猪的优良基因资源奠定了基础。更多还原

【Abstract】 Tibetan Pig (Sus scrofa, Tibetan pig) is indigenous to vast areas of Tibet, West of Sichuan, North of Gansu and Northernwest of Yunnan Province. It is the representative of the grazing breeds, which has unique resistance to harsh climate, poor feeding and veterinary preventation compared with other pig breeds. It has advantages of better resistance to stressful environment, more fine muscle fibre, more intramuscle fat and better flavour, and the disadvantages of low litter size, slow growth and low lean meat percentage. Due to its low productive performances, many exotic breeds have been introduced to cross with them sine 1950s. After 60 years’ crossbreeding, the quantity of pure Tibetan Pigs is decreased dramatically. Nowadays, even in their provenance, it is difficult to find pure Tibetan Pigs. The breed is at risk of extinction. It is important to understand the rare breed resource before it becomes extinction.So far, a few papers have been reported about Tibetan pigs, and the researches related to resistance, meat quality and litter size candidate genes are even less. In addition, natural resistance-associated macrophage protein (NRAMP1), heart fatty-binding protein (H-FABP) and estrogen receptor (ESR) have been documented well in other breeds of swine. Herein, genes encoding above three proteins were chosen in Tibanten pigs in the present study.First of all, full cDNA of NRAMP1 was cloned with RT-PCR.The length of cDNA fragment is 1614 bp, encoding a peptide of 538 amino acid residues with a molecular weight of 58.8 Kda and isoelectric point 8.01. The predicted NRAMP1 protein is a typical NRAMP1, which is composed of a putative N-terminal proline- and serine-rich Src homology 3 (SH3)-binding domain, four phosphorylation sites for protein kinase C (PKC) , two N-linked glycosylation sites and twelve transmembrane (TM) domains. Sequence homology analysis showed that there are 13 nucleotides different between Tibetan pig and domestic pig but which is not resulted in the amino acid difference. By blasting the homologous sequences in GenBank database with demostic pig, dog, sheep, american bison red deer, horse, human and norway rat, their nucleotide sequence identities are 99.2%, 91.2, 88.7%, 88.1%, 86.8%, 85.8%, 83.2% and 71.6%, respectively.Secondly, NRAMP1 gene expression in 10 tissues of Tibetan Pig were analyzed by using semi-quantitative and real-time quantitative PCR methods. The gene expression was observed in all the tissues and no tissue-specific expression model was found in Tibetan Pig. Real-time quantitative PCR proved that the gene expression abundance was greatly different in various tissues. The maximal expression was observed in spleen and the minimum in liver. The average copy number is 3233 per ug cDNA. The difference between the maximal and the minimum is more than 77 times. The abundance of NRAMP1 expression in Tibetan Pig from high to low is in the following order: spleen, brain, lung, ileum, kidney, lymph node, colon, heart, muscle and liver. The finding suggests that there likely exists interspecies variation between Tibetan Pigs and demostic pig breeds.Thirdly, the genetic variation of H-FABP gene in 96 Tibetan Pigs were detected by PCR-RFLP with MspⅠ, HaeⅢ, HinfⅠ. The results showed as follows: there are polymorphisms in HinfⅠand MspⅠlocus, with the genotypes of HH, Hh, hh and AA, Aa, aa, among which the gene frequencies of H and A are 0.521 and 0.406. However, there is no variation in HaeⅢ locus, with the genotype of DD only.Last, the genetic variation of ESR gene in 96 Tibetan Pigs were detected by PCR-RFLP with MspⅠ, HaeⅢ, HinfⅠ, meanwhile the relationship between ESR genotype and their litter size was analyzed. The results showed that there are 2 genotypes (AA and AB). The distribution of AA and AB is 0.75 and 0.25, respectively. The gene frequencies for A and B are 0.875 and 0.125, respectively. The litter size of AB type is 2.3 piglets more than that of AA.These findings are significant importance for realizing, preservation and utilization of the prescious resource in the future.更多还原