【作者】 霍金龙；

【导师】 肖蘅；

【作者基本信息】 云南大学 ， 动物学， 2012， 博士

【摘要】 版纳微型猪近交系（BMI）是利用我国特有地方猪种资源,采用连续高度近交加严格人工选择的育种方法,培育成功的世界上首个大型哺乳类实验动物近交系,可为生物医学、异种器官移植等研究提供大型纯系实验动物。虽然BMI经历了长期的遗传选育工作,形成了稳定的形态特征,但从基因水平探索其种质遗传特性的研究还相对较少,特别是与其生长、代谢、免疫等重要特性相关的基因水平的种质遗传特性还未进行过系统研究。为从分子水平深入解析BMI的种质遗传特性并提供其进一步选育利用的遗传背景资料,本研究利用分子生物学技术从RNA或DNA入手,在基因结构和表达水平上对BMI尚未研究过的与其生长发育、新陈代谢、免疫调节、移植排斥、病理生理、抗病性、性别分化等种质遗传特性相关的13个候选功能基因进行深入研究。这些基因包括：生长激素（GH）、水通道蛋白3（AQP3）、血管内皮生长因子（VEGF）、肿瘤坏死因子α（TNF-α）、α干扰素（IFN-α）^白细胞介素6（IL-6）、Y染色体性别决定基因（SRY）、Fas细胞凋亡抑制分子（FAIM1）、多聚谷氨酰胺结合蛋白1（PQBP1）、突触结合蛋白6（STXBP6）、核糖基酶（RBK）、脂乙酰-CoA还原酶（FAR1）和着丝粒蛋白P （CENPP）。首先分离并获得这些基因的完全编码区序列信息,并对这些基因进行比较基因组学、生物信息学分析,同时根据各基因不同的遗传特性,有针对性的进行多组织样品荧光定量（qPCR）表达分析,进一步对基因编码的蛋白质进行多种理化特性及功能预测和分析。另外,还对GH和SRY基因进行了原核表达分析,对GH基因进行了Western Blot验证。结果表明各基因在不同物种间相对保守,相似性较高（如BMI的STXBP6和牛的同一基因相似性高达100%）,是比较保守的功能基因,但也找到了BMI有别于其他猪品种的核苷酸和氨基酸水平的差异位点,如在BMI GH基因的编码区发现了和国内外其他12个猪种有差别的SNP26位点（c.26T>C）,而且证明GH基因完全编码序列是无法诱导表达蛋白质的,只有去掉信号肽才能实现蛋白质表达。AQP3氨基酸序列在猪品种间100%保守,但在编码区的核苷酸序列发现一个c.204C>A的颠换位点。VEGF氨基酸序列中存在一个p.A102T的替换,编码序列存在c.15G>C和c.438G>A两个核苷酸替换。IFN-a在核酸和蛋白水平上均存在有别于其他猪品种的独特序列,并且具有多个SNP位点。BMI的SRY基因具有独特的单倍型,而且筛选到一对SRY特异性引物可以对BMI进行胚胎早期性别鉴定,并且研究发现0.5mM的IPTG就可以诱导原核表达系统产生大量的SRY蛋白。除GH和SRY外,以持家基因18S校正的qPCR分析表明,其他11个基因在脾和肺中均有表达,在其他组织中分别显现强、中、弱、无等不同表达丰度,揭示所研究的基因存在同一基因在不同组织中和不同基因在相同组织中差异表达的现象。预测的蛋白质的分子量、等电点、二级结构、三级结构、跨膜结构、信号肽等理化特性和结构均反映了BMI各基因在蛋白表达水平上的功能和作用。13个基因均已获得GenBank数据库认证,登录号（accession number）分别为：JQ177096（GH）、 HQ888860（AQP3）、JF831364（VEGF）、JF831365（TNF-α）、JQ839262（IFN-α）、 JQ839263（IL-6）、GU991615（SRY）、Nm₀01245976（FAIM1）、Nm₀01246668（PQBP1）、Nm₀01246666（STXBP6）、JF944892（RBKS）、Nm₀01252425（FAR1）、JF944894（CENPP）,其中FAIM1、PQBP1、STXBP6和FAR1基因已经被GenBank数据库确定为猪的标准序列（Nm_********格式）,并分别定位在猪的13、X、7和2号染色体上,这些新基因序列的注释以及对其功能的初步研究可以为深入研究这些基因的表达调控、功能鉴定等奠定分子生物学基础。本文所克隆的13个基因在BMI中属首次研究,填补了该猪种相关基因研究的空白,13个基因均已获得GenBank数据库认证,其中GH、AQP3、IFN-α和SRY基因的编码序列有别于GenBank其他猪品种序列,为BMI独有单倍型,而且在这些基因编码区还发现了多个可以和其他猪品种相互区别开来的SNP位点；开发出了一对特异性的SRY基因引物,可以作为BMI胚胎早期性别鉴定的分子标记。本研究建立了利用近缘物种基因序列以及拼接猪EST序列电子克隆BMI新基因并快速实验验证、利用qPCR技术检测BMI基因多组织表达水平及利用原核表达系统和Western Blot印迹研究BMI功能基因的技术方法。FAIM1、 PQBP1、STXBP6、RBKS. FAR1和CENPP是首次分离出的猪的新基因,与机体免疫、抗病、代谢等多种生命活动相关。本研究结果不仅可为版纳微型猪近交系的进一步选育利用提供基础数据资料,同时为从分子水平上研究其种质遗传特性提供方法和思路,而且也可为其知识产权保护、开发和实验动物化利用提供科学依据。更多还原

【Abstract】 Banna Mini-pig Inbred Line （BMI） is the first successful cultivated large mammal inbred line in the world by highly consecutive inbreeding and strictly scientific breeding methods using unique resources of aboriginal pig breed in China. BMI is optimal large pure experimental animal models for research and application in the field of bio-medicine, xenotransplantation. Over the years, the BMI had been committed the genetic breeding work and formed stable morphological characteristics, but it was relatively little to explore its genetic features of germplasm from the molecular level, especially the genetic characteristics of genes related to its growth, metabolism, immune have not been systematically researched.To reflect its genetic characteristics of germplasm and further insight into the genetic bankground at the molecular level, structure and expression level of13BMI candidate functional genes which are related with its growth and development, metabolism, immune regulation, transplant rejection, pathophysiology, breeding for disease resistance, and sex identification of germplasm genetic characteristics were studied deeply using molecular biology techniques to start with the RNA or DNA. These genes include growth hormone （GH）, aquaporin3（AQP3）, vascular endothelial growth factor （VEGF）, tumor necrosis factor alpha （TNF-alpha）, alpha interferon （IFN-alpha）, interleukin-6（IL-6）, Y chromosome sex-determining gene （SRY）, Fas apoptosis inhibitory molecule （FAIM1）, polyglutamine binding protein1（PQBP1）, synaptotagmin protein6（STXBP6）, ribosylation enzymes （RBKS）, fat acetyl-CoA reductase （FAR1）, centromere protein of P （CENPP）. First we isolated and got the complete coding sequence of these genes, second carryed out comparative genomics and bioinformatics analysis, then targeted multi-tissues sample fluorescence quantitative （qPCR） expression analysis according to different genetic characteristics of each gene and in the last predicted and analysed a variety of physical and chemical properties of proteins. In addition, the prokaryotic expression analysis of GH and SRY gene were carried out and GH protein expression was veryfied by Western Blot.Results showed most genes were relatively conservative function genes and had very high amino acid identity among different species, such as the STXBP6identity was100%between BMI and cattle. But we also found some important SNPs, such as the c.26T>C SNP of the BMI GH gene coding region which were different from other12domestic and foreign pig breeds. In addition, we found only by removing signal peptide of GH can it be induced to express the protein. AQP3was100%conservative in amino acids level between different breeds, but had a mutation site （c.204C＞A SNP） in the coding region. VEGF exist a p.A102T amino acid mutation and had two SNPs （c.15G>C and c.438G>A） in the coding region. BMI IFN-a gene sequence was different from other breeds and had many SNPs in nucleic acid and protein levels. We found the BMI SRY owned distinctive haplotype, and screened a pair of specific primers of SRY gene which can be used as the early embryonic sex identification of BMI and found0.5mM IPTG can induce massive SRY protein to produce in prokaryotic expression system. Except GH and SRY genes, multi-tissues of the housekeeping gene18S correction qPCR analysis showed other11gene can express in spleen and lung, but differentially expressed in other tissues of BMI for strong, weak, and no express. This showed that differential express phenomenon of same gene in different tissues and different genes in the same tissue. These proteins’ physical and chemical properties and structure such as molecular weight, isoelectric point, secondary structure, tertiary structure, transmembrane structure and signal peptide reflect the special function and effect of each BMI gene.13genes have been submitted to the GenBank database and accession numbers respectively were JQ177096（GH）, HQ888860（of AQP3in）, JF831364（VEGF）, JF831365（TNF-alpha）, JQ839262（IFN-alpha）, JQ839263（IL-6）, GU991615（SRY）, Nm₀01245976（FAIM1）, Nm₀01246668（PQBP1）, Nm₀01246666（STXBP6）, JF944892（RBKS）, Nm₀01252425（FAR1）, JF944894（CENPP）. Especially, FAIM1, PQBP1, STXBP6and FAR1genes have been identified as the standard sequence （format********Nm_）, which were mapped to pig chromosome13、X、7and2respectively. These new gene sequences’annotation will be a basis for deeply study the regulation of expression of these genes and functional identification.13genes was the first study in BMI and filled a gap of the study of the related genes.13gene sequences have been submitted to the GenBank database and the coding sequences of GH, AQP3, IFN-a, SRY genes with some SNP sites were different from other breeds of the GenBank sequences, may be unique haplotypes for the BMI. We have developed a pair of specific primers which can be used as a molecular marker for BMI embryonic sex identification. This study established a series of biotechnology methods such as silico cloning BMI new genes and quickly verifying by using relative species gene sequences and splicing pig EST sequences, testing BMI gene expression level in different tissues by fluorescence quantitative PCR （qPCR）, studying BMI functional genes and protein by prokaryotic expression system and Western Blot. FAIM1, PQBP1, STXBP6, RBKS, FAR1and CENPP were the first isolated novel pig genes, which related to immune, disease resistance, metabolism and other life activities. This study not only provided a basis of data, methods and ideas for Banna mini-pig inbred line further breeding and studying its unique genetic characteristics at the molecular level, but also played an important role in its exploitation, intellectual property protection and using as experimental animal.更多还原