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BMI是利用我國(guó)特有地方豬種資源，采用連續(xù)高度近交加嚴(yán)格選擇的科學(xué)育種方法，培育成功的世界上首個(gè)大型哺乳類實(shí)驗(yàn)動(dòng)物近交系，可為生物醫(yī)學(xué)、異種器官移植等研究提供大型純系實(shí)驗(yàn)動(dòng)物。多年來我們一直致力于BMI的基礎(chǔ)遺傳繁育工作，但從基因水平探索其種質(zhì)遺傳特性的研究還相對(duì)較少，特別是與其生長(zhǎng)、代謝、免疫等重要特性相關(guān)的基因水平的種質(zhì)遺傳特性還未進(jìn)行過系統(tǒng)研究。
為進(jìn)一步從分子水平深入了解其遺傳基礎(chǔ)，反映其遺傳特性，本研究利用現(xiàn)代分子生物學(xué)技術(shù)從RNA或DNA入手，在基因結(jié)構(gòu)和表達(dá)水平上對(duì)BMI尚未研究過的與其生長(zhǎng)發(fā)育、新陳代謝、免疫調(diào)節(jié)、移植排斥、病理生理、抗病育種、性別鑒定等種質(zhì)遺傳特性相關(guān)的13個(gè)候選功能基因進(jìn)行深入研究。這些基因包括：生長(zhǎng)激素（GH），水通道蛋白3（AQP3），血管內(nèi)皮生長(zhǎng)因子（VEGF），腫瘤壞死因子α（TNF-α），α干擾素（IFN-α），白細(xì)胞介素6（IL-6），Y染色體性別決定基因（SRY），F(xiàn)as細(xì)胞凋亡抑制分子（FAIM1），多聚谷氨酰胺結(jié)合蛋白1（PQBP1），突觸結(jié)合蛋白6（STXBP6），核糖基酶（RBKS），脂乙酰-CoA還原酶（FAR1），著絲粒蛋白P（CENPP）。獲得這些基因的完全編碼區(qū)序列信息，并對(duì)這些基因進(jìn)行比較基因組學(xué)、生物信息學(xué)分析，同時(shí)根據(jù)各基因不同的遺傳特性，有針對(duì)性的進(jìn)行多組織樣品熒光定量（qPCR）表達(dá)分析，并對(duì)基因編碼的蛋白質(zhì)進(jìn)行多種理化特性及功能預(yù)測(cè)和分析。另外對(duì)GH和SRY基因進(jìn)行原核表達(dá)分析，并對(duì)GH基因進(jìn)行Western Blot驗(yàn)證。
結(jié)果表明各基因氨基酸序列在不同物種間相對(duì)保守，相似性較高，但也能找到BMI有別于其他豬種的核苷酸和氨基酸水平的SNP，如在BMI GH基因的編碼區(qū)發(fā)現(xiàn)了和國(guó)內(nèi)小型豬、國(guó)外大型豬以及野豬有差別的c.26T>C SNP位點(diǎn)，而且證明GH基因完全編碼序列是無法誘導(dǎo)表達(dá)蛋白質(zhì)的，只有去掉成熟肽才能實(shí)現(xiàn)蛋白表達(dá)。AQP3氨基酸在豬種間100%保守，但在核苷酸編碼區(qū)發(fā)現(xiàn)一個(gè)c.204C>A的SNP位點(diǎn)。VEGF氨基酸存在一個(gè)p.A102T的SNP位點(diǎn)，核苷酸存在c.15G>C和c.438G>A二個(gè)SNP位點(diǎn)。SRY基因篩選到的一對(duì)特異性引物可以對(duì)BMI進(jìn)行胚胎早期性別鑒定，并且研究發(fā)現(xiàn)0.5 mM的IPTG濃度就可以誘導(dǎo)大量SRY蛋白產(chǎn)生。以看家基因18 S校正的多組織qPCR分析表明基因在各組織中存在差異表達(dá)現(xiàn)象。蛋白質(zhì)的分子量、等電點(diǎn)、二級(jí)結(jié)構(gòu)、三級(jí)結(jié)構(gòu)、跨膜結(jié)構(gòu)、信號(hào)肽等理化特性和結(jié)構(gòu)均反映出BMI各基因在蛋白表達(dá)水平上功能和地位。13個(gè)基因均已獲得GenBank數(shù)據(jù)庫(kù)認(rèn)證，登錄號(hào)（Accession Number）分別為：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基因已經(jīng)被GenBank數(shù)據(jù)庫(kù)確定為豬的標(biāo)準(zhǔn)序列（格式為Nm_********），這些新基因序列的注釋以及對(duì)其功能的初步研究可以為深入研究這些基因的表達(dá)調(diào)控、功能鑒定等奠定分子生物學(xué)基礎(chǔ)。
本文所克隆的13個(gè)基因在BMI中屬首次研究，填補(bǔ)了相關(guān)基因研究的一個(gè)空白，13個(gè)基因均已獲得GenBank數(shù)據(jù)庫(kù)認(rèn)證，其中GH、AQP3、VEGF、TNF-α、IFN-α、IL-6基因的編碼序列有別于GenBank其他豬種序列，為BMI獨(dú)有單倍型，而且在這些基因編碼區(qū)還發(fā)現(xiàn)了多個(gè)可以和其他豬種相互區(qū)別開來的SNP位點(diǎn)；雖然SRY基因序列上不存在BMI獨(dú)特的SNP位點(diǎn)，但我們開發(fā)出了一對(duì)特異性引物可以作為BMI胚胎早期性別鑒定的分子標(biāo)記；FAIM1、PQBP1、STXBP6、RBKS、FAR1、CENPP等6個(gè)基因是首次分離出的豬的新基因，這些基因是與機(jī)體免疫、抗病、代謝等多種生命活動(dòng)相關(guān)的功能基因。本研究結(jié)果不僅可為版納微型豬近交系進(jìn)一步選育利用提供基礎(chǔ)數(shù)據(jù)資料，為其在分子水平方面獨(dú)特種質(zhì)遺傳特性的研究提供方法和思路，而且也可為其知識(shí)產(chǎn)權(quán)保護(hù)、開發(fā)和實(shí)驗(yàn)動(dòng)物化利用提供科學(xué)依據(jù)。

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1樓 2012-04-12 23:55:42

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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.

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2樓2012-04-13 00:32:43

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