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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选择的科学育种方法，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。多年来我们一直致力于BMI的基础遗传繁育工作，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。 为进一步从分子水平深入了解其遗传基础，反映其遗传特性，本研究利用现代分子生物学技术从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、抗病育种、性别鉴定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（RBKS），脂乙酰-CoA还原酶（FAR1），着丝粒蛋白P（CENPP）。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，并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。另外对GH和SRY基因进行原核表达分析，并对GH基因进行Western Blot验证。 结果表明各基因氨基酸序列在不同物种间相对保守，相似性较高，但也能找到BMI有别于其他猪种的核苷酸和氨基酸水平的SNP，如在BMI GH基因的编码区发现了和国内小型猪、国外大型猪以及野猪有差别的c.26T>C SNP位点，而且证明GH基因完全编码序列是无法诱导表达蛋白质的，只有去掉成熟肽才能实现蛋白表达。AQP3氨基酸在猪种间100%保守，但在核苷酸编码区发现一个c.204C>A的SNP位点。VEGF氨基酸存在一个p.A102T的SNP位点，核苷酸存在c.15G>C和c.438G>A二个SNP位点。SRY基因筛选到的一对特异性引物可以对BMI进行胚胎早期性别鉴定，并且研究发现0.5 mM的IPTG浓度就可以诱导大量SRY蛋白产生。以看家基因18 S校正的多组织qPCR分析表明基因在各组织中存在差异表达现象。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出BMI各基因在蛋白表达水平上功能和地位。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）、Nm_001245976（FAIM1）、Nm_001245976（PQBP1）、Nm_001246666（STXBP6）、JF944892（RBKS）、Nm_001252425（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（格式为Nm_********），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，填补了相关基因研究的一个空白，13个基因均已获得GenBank数据库认证，其中GH、AQP3、VEGF、TNF-α、IFN-α、IL-6基因的编码序列有别于GenBank其他猪种序列，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种相互区别开来的SNP位点；虽然SRY基因序列上不存在BMI独特的SNP位点，但我们开发出了一对特异性引物可以作为BMI胚胎早期性别鉴定的分子标记；FAIM1、PQBP1、STXBP6、RBKS、FAR1、CENPP等6个基因是首次分离出的猪的新基因，这些基因是与机体免疫、抗病、代谢等多种生命活动相关的功能基因。本研究结果不仅可为版纳微型猪近交系进一步选育利用提供基础数据资料，为其在分子水平方面独特种质遗传特性的研究提供方法和思路，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。 |
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huojinlong8610: 金币+500, 翻译EPI+1, ★★★★★最佳答案, 不错，速度快，翻译的行 2012-04-13 00:55:55
Mally89: 金币+3, 感谢应助！~欢迎常来！·好运！~\(^o^)/~ 2012-04-14 13:39:16
huojinlong8610: 金币+500, 翻译EPI+1, ★★★★★最佳答案, 不错，速度快，翻译的行 2012-04-13 00:55:55
Mally89: 金币+3, 感谢应助！~欢迎常来！·好运！~\(^o^)/~ 2012-04-14 13:39:16
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BMI is the use of our unique resources of native pig breeds using strict scientific breeding methods to select consecutive height of nearly Cross, to cultivate the success of the world's first large-scale mammalian laboratory animals inbred for bio-medicine, xenotransplantation research large inbred experimental animals. Over the years we have been committed to the BMI in the basis of genetic breeding work, but the genetic level to explore the genetic features of a relatively small, especially the genetic characteristics of genes related to the level of the important features of its growth, metabolism, immune have not been systematic research. Further insight into the genetic basis of the molecular level, to reflect its genetic characteristics, this study uses modern molecular biology techniques to start with the RNA or DNA, the level of gene structure and expression of BMI has not been studied with its growth and development, metabolism, immune regulation, transplant rejection, pathophysiology, breeding for disease resistance, and sex identification of germplasm genetic characteristics of 13 candidate functional genes in-depth study. These genes include: growth hormone (GH), aquaporin 3 (of AQP3 in), vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNF-alpha), alpha interferon (IFN-alpha), interleukin-6 ( IL-6), the Y chromosome sex-determining gene (the SRY), the Fas apoptosis inhibitory molecule (FAIM1), the polyglutamine binding protein 1 (PQBP1), synaptotagmin protein 6 (STXBP6) ribosylation enzymes (RBKS ), fat acetyl-CoA reductase (FAR1), centromere protein of P (CENPP). Access to the complete coding sequence of these genes and these genes carry out a comparative genomics, bioinformatics analysis, according to the different genetic characteristics of each gene, targeted multi-organization sample fluorescence quantitative (qPCR) expression analysis, and a variety of physical and chemical characteristics and function prediction and analysis of the gene encoding the protein. In addition to the GH and the SRY gene in the prokaryotic expression analysis, and GH gene by Western Blot verification. The results show that the relatively conservative amino acid sequence of each gene among different species, similar to high, but can also be found BMI is different from other breeds of nucleotide and amino acid level of SNP, such as found in the BMI of GH gene coding region and miniature pigs of the domestic and foreign large-scale pig and wild boar difference c.26T> C SNP, but also to prove the GH gene complete coding sequence can not be induced to express the protein, only to remove the mature peptide to protein expression. AQP3 amino acids in pigs between 100% conservative, but found a c.204C> A SNP site in the nucleotide coding region. VEGF amino acid exists a p.A102T SNP nucleotide to exist c.15G> C and c.438G> A, two SNP loci. SRY gene screening to a pair of specific primers can be a BMI of early embryonic sex identification, and found the concentration of 0.5 mM IPTG can induce a large number of SRY protein to produce. Multi-organization of the housekeeping gene 18 S correction qPCR analysis showed that the differentially expressed genes in each tissue phenomenon. The physical and chemical properties and structure of the protein molecular weight, isoelectric point, secondary structure, tertiary structure, transmembrane helices and signal peptide reflects the function and status of the BMI of each gene in the protein expression level. 13 genes have been reached with the GenBank database, authentication, registration number (Accession Number), respectively: JQ177096 (GH), HQ888860 (of AQP3 in), JF831364 (VEGF), JF831365 (TNF-alpha), JQ839262 (IFN-alpha), JQ839263 ( IL-6), GU991615 (SRY), Nm_001245976 (FAIM1), Nm_001245976 (PQBP1), Nm_001246666 (STXBP6), JF944892 (RBKS), Nm_001252425 (FAR1), JF944894 (CENPP), which FAIM1, PQBP1, STXBP6 and FAR1 genes have been GenBank database to determine the standard sequence (format ******** Nm_) and pigs, the preliminary study of these new gene sequence annotation as well as its function can be laid for the in-depth study of the regulation of expression of these genes, functional identification the basis of molecular biology. 13 genes cloned in BMI is the first study, to fill a gap of the related genes, 13 genes have been reached with the GenBank database certification of GH, of AQP3 in, of VEGF and TNF-alpha and IFN-α, IL 6 gene coding sequence is different from other breeds of the GenBank sequence as a BMI of unique haplotypes and encoded by these genes also found that more can and other breeds with each other to distinguish SNP loci; SRY BMI unique SNP in the gene sequence does not exist, but we have developed a pair of specific primers can be used as the the BMI embryonic sex identification of molecular markers; FAIM1, PQBP1, STXBP6 RBKS, FAR1 CENPP and other six genes is the first isolated pig genes, these genes are functional genes related to immune, disease, metabolic and other life activities. In this study, results not only for the Banna minipig inbred further breeding use to provide the basis for data, methods and ideas of its unique genetic characteristics at the molecular level, but also its intellectual property protection, development and experimental animals of use to provide a scientific basis. |
2楼2012-04-13 00:32:43