【作者】 何俊；

【导师】 柳小春； 施启顺；

【作者基本信息】 湖南农业大学 ， 动物遗传育种与繁殖， 2012， 博士

【摘要】 本文通过在湖南湘潭沙子岭猪保种场随机选取亲缘关系相近的33头一周龄内的仔猪和30头湖南农业大学种猪场一周龄内大约克仔猪,共63头,屠宰后迅速取肝、小肠等组织样品,提取RNA、DNA;截取空肠组织,用于制备上皮细胞刷状缘,鉴定F4受体黏附表型。文章采用aCGH芯片筛选F4受体相关CNVs,针对ETEC F4ad受体,对其候选基因GPI1进行了克隆,获得了该基因的相关序列,利用生物信息学软件和工具预测了该基因的蛋白质理化性质、结构和功能,进一步对GPI1基因进行了定量表达分析,弄清楚该基因的基本表达规律,最后通过对基因的两个SNP位点与黏附特性的关联分析,期望找到有效的遗传标记,应用与育种实践。文章获得了以下主要研究结果。1.利用aCGH芯片,在猪的全基因组范围内筛选与F4受体有关的CNVs,找到了F4ab,ac,ad三种受体相关CNVs所在的染色体位置,以及CNVs产生的原因,其中F4ab、F4ac受体分别有11个和48个CNVs,而F4ad受体有66个CNVs;2.通过对获得的CNVs作进一步分析,找到了与CNVs对应的相关基因。发现糖代谢有关基因、SLC基因家族、ZNF基因家族、KIF基因家族、Toll like受体5基因、U6等基因多次出现,这些基因都与动物疾病和免疫力密切相关。尤其是GPI1基因,很可能是ETEC F4ad受体的候选基因。3.通过对GPI1基因的克隆测序,获得了GPIl基因1515bp的序列,通过与GenBank中的序列进行BLAST比对分析,发现与猪和人的GPI基因高度同源,一致性在90%以上。4.将获得的GPI1基因序列提交到了GenBank中,登录号为JX125039。5.通过对GPI1基因氨基酸预测,GPI1共出232个氨基酸组成,相对分子量为24964.5,理论等电点为9.44,其氨基酸组成中Leu的含量达19.8%,高于其它氨基酸含量8.2%—19.4%,不稳定参数为39.82,属于稳定蛋白,半衰期为30h。该蛋白脂肪系数为122.80,平均疏水度为0.512。氨基酸序列不含信号肽区域。利用TMHMM程序对猪GPI1进行跨膜区分析,该蛋白为疏水蛋白,这与GPI1作为F4ad受体的基本特征相符。在232位氨基酸间有3个跨膜区,这与GPI1作为细胞重要的信号传导成分的特征一致。SOPMA程序预测其二级结构：α—螺旋50.86%,p—转角4.74%,伸展链10.78%,无规卷曲33.62%。6. GPI1基因在黏附和不黏附的不同黏附类型中,在不黏附的小肠中表达量比黏附的表达量高,并且同一黏附类型中,沙子岭猪样品中的GPI1基因在小肠的表达量比大白猪高,且两种黏附类型和两个品种间的GPI1基因表达量差异显著(P<0.05)。在不同品种中,也是不黏附型比黏附型要高,且黏附和不黏附仔猪的表达量差异显著(P<0.05)；而在两个品种不同组织的表达量也差异显著(P<0.05),肝脏中的表达量比小肠中的表达量要高。7.通过利用生物信息学预测SNP和内切酶位点,采用PCR-RFLP进行验证,发现了exon3的25bp处存在C→T的突变,exon5的211bp处存在G→C的突变。将两个外显子的SNP分别与黏附特性进行关联,发现这两个外显子的SNP与黏附特性存在相关,可以作为腹泻抗性的潜在遗传标记。更多还原

【Abstract】 33Piglets were relative cultivars and from Shaziling breed conservation farm and30Yorkshire Piglets which from Hunan Agriculture University were used in the experiment, all of them are only a week old. After slaughtering, we collected the tissue samples of liver and small intestine as soon as possible so that we can extract DNA and RNA. To identify F4receptor adhesion phenotype, we should collect the jejunum organization, which was used to manufacture Brush Border Membrane.In this experiment, we used aCGH to screen the CNVs related with F4receptor. Especially for ETEC F4ad receptor, we obtained some related sequence of this gene and predicted protein physicochemical properties, structure and function of the gene by bioinformatics software and tools. Furthermore, we analyze the GPI1gene quantitatively so that we had figured out the expression regular of this gene. To find some effective genetic markers and make it used in breeding practice, we take association analysis between two gene locus of SNP and adhesion characteristics. The article has got the results as follows:1. The position of the related CNVs of F4ab receptor, ac receptor and ad receptor found by screening the related CNVs of F4receptor in the whole gene of pig. At the same time, we have clarified the origin of CNVs produce. In these, F4ab receptor and F4ac respectively contain11and48CNVs, but F4ad receptor contaid67CNVs;2. By further analyzing the CNVs that we have got, we have found the related gene of CNVs. We also discovered that the related gene of glucose metabolism, SLC gene-family, ZNF gene-family, KIF gene-family. Toll like receptor5gene and U6gene appear frequently. All of these genes are closely related to animal disease and immunity, especially for GPI1. It’s very likely that GPI1is the candidate gene of ETEC F4ad receptor.3. By cloning and sequencing for GPI1, we’ve got1515bp sequence of this gene. Meanwhile, we took BLAST analysis between this sequence and other sequences in GenBank. As the consequence of the analysis, GPI1of pig and human are high homology, and the consistency is more than90%.4. The GPI1gene sequence was submitted to the GenBank, and the submit accession number is JX125039. 5. By the prediction of amino acid of GPI1gene, GPI1consists of232amino acids, its relative molecular mass is24964.5, its theoretical isoelectric point is9.44. In the amino acid composition of GPI1, the leucine content of19.8%which is higher than the other amino acid content of8.2%-19.4%. its instability parameter is39.82, belongs to the stable protein. The half life of the protein period is30hours. The fat coefficient of the protein is122.80and the average hydrophobic degree is0.512. This amino acid sequence doesn’t contain signal peptide area. We took the transmembrane domain analysis with TMHMM program, and the result show us that this protein is hydrophobic protein, it coincide with that GPI1as the basic characteristic of the F4ad receptor. There are3transmembrane domain among the232amino acids, and it coincide with the characteristic that GPI1is the important intracellular signal transduction. SOPMA program predict the secondary structure as follows:a-helical is50.86%; β-turn is4.74%; extend chain is10.78%; random coil is33.62%.6. GPI1in the small intestine without adhesion is higher than GPI1in the small intestine with adhesion. And in the same types of adhesion, expression level of GPI1of piglets from Shaziling is more than Yorkshire’s. The expression levels of GPI1between two kinds of adhesive type and two two varieties are significant differences(P<0.05). In different species, the expression level of GPI1in the small intestine without adhesion is higher than others, too. And there is significant difference between adhesion and inadhension(P<0.05).The expression level of different organization in two speices has a significant difference(P<0.05).The expression level of GPI1in liver is higher than in small intestine.7. We predicted SNP and endonuclease by using bioinformatics. At the same time, we took verification with PCR-RFLP so that we have found that there is a C→T mutation at25bp of exon3and there is a G→C mutation at211bp of exon5. When taking association with SNP of two exons, we’ve found that the SNP of two exons is related to the adhesion characteristics, and it can be treated as diarrhea resistance of potential genetic marker.更多还原