【作者】 王颖；

【导师】 徐宁迎；

【作者基本信息】 浙江大学 ， 动物遗传育种， 2010， 博士

【摘要】 甲状腺激素的作用遍及机体各处,几乎机体的每个细胞都是甲状腺激素作用的靶细胞,其可以促进组织分化、生长和成熟,促进物质与能量代谢。甲状腺过氧化物酶(TPO)是一种糖基化血红素蛋白,是催化甲状腺激素合成的关键酶。它参与了两个重要的反应：甲状腺球蛋白(TG)酪氨酸残基的碘化和碘化酪氨酸的偶联作用。TG是甲状腺激素合成的基本原料和场所,甲状腺素正常的生物合成途径依赖于TG结构的完整性。甲状腺转录因子-1,-2(TTF-1,-2)通过结合到TG和TPO基因的启动子特异性区域来调控其转录,另外TTF-1,-2在甲状腺胚胎发育的过程中起至关重要的作用。刺激性G蛋白α亚基(GNAS)基因上的失活性突变可导致假性甲状旁腺功能减退症,影响甲状腺的功能,继而对机体的生长发育造成影响。本研究克隆了猪TG、GNAS、TTF-1和TTF-2完整CDS的mRNA序列;分别对TPO、TTF-1和TTF-2基因进行了染色体定位；通过荧光定量PCR研究了在猪十六个组织中的组织表达谱和在初生、20、45、60、90、120和150日龄甲状腺组织内的发育表达谱；寻找了TPO基因的SNP座位和转录剪切本：分析了TG基因的启动子区域；并探讨了阉割对GNAS基因启动子区甲基化的影响。结果如下：(1)基于EST序列的电子克隆技术及人和小鼠同源性较高区域对目的基因设计引物,以金华猪甲状腺cDNA为模板扩增、克隆并测序,拼接后克隆获得了包含完整CDS的猪TG (GenBank GQ261999)、GNAS (GenBank GU126691)以及TTF-1和TTF-2基因的部分CDS序列。并利用Clustal W、ORF Finder、Signal P2.0、PSORTⅡ等软件对TG、GNAS、TTF-1和TTF-2基因的结构、编码蛋白结构、功能等特征进行了预测和分析。(2)根据猪目的基因相应的内含子序列设计引物,利用猪辐射杂种克隆板(ImpRH)将猪TPO基因定位于SSC3q22-27,连锁标记为SWR201(LOD值3.59)和SW590(LOD值2.33)；TTF-1基因定位于SSC7q22,连锁标记为NFKB (LOD值11.89)和SW1959(LOD值5.65)；TTF-2基因定位于SSClq28,连锁标记为SSC11E11 (LOD值4.44)。(3)应用real-time Q-PCR方法研究了猪TPO、TG、GNAS、TTF-1和TTF-2在金华猪16种组织中的表达谱,结果表明：GNAS呈广泛表达；TTF-1基因在甲状腺中表达量最高,肺其次、垂体次之,其他组织基本不表达；TPO、TG和TTF-2基因仅在甲状腺组织中特异表达。在甲状腺组织不同生长阶段的发育表达谱表明TPO、TTF-1和GNAS基因在不同生长阶段表达的差异不显著；而TG和TTF-2在不同生长阶段表达的差异较明显。(4)通过对基因池的"PCR-测序”,我们发现了猪TPO基因的多态座位：C/T63,G/T156,G/C268,C/T333,A/G624,A/G642以及C/T705,在TPO基因的3’非翻译区未发现多态座位。应用PCR-RFLP方法,在猪A/G642座位对国内外猪种多态性进行分析,结果表明国内猪种(金华猪、岔路黑猪、嘉兴黑猪)A基因的频率高于国外猪种(大白、长白、杜洛克)；遗传效应分析表明,在JPF2代猪中,AA基因型个体的后腿重显著高于AG和GG基因型。另外,我们还发现金华猪TPO基因存在两种新的转录本：转录本TPO-2缺少外显子8；而TPO-3缺少外显子8、14、15和16。(5)利用生物信息学软件对TG基因的启动子序列进行预测,并成功克隆了长度为938 bp的TG基因的启动子序列。将其插入到载体PGL3-Basic报告基因荧光素酶蛋白的上游,利用脂质体将所构建的TG promoter-PGL3-Basic载体转入兔胎儿成纤维细胞。结果表明我们所构建的TG启动子载体能高效特异地启动下游基因的表达。(6)阉割对金华猪GNAS基因启动子区域的甲基化状态影响表明阉割猪的甲基化水平高于非阉割猪,但差异不显著。更多还原

【Abstract】 Almost every cell in our body is the target cell of thyroid hormone, which can accelerate the differentiation, growth and maturity, increase the material and energy metabolism. Thyroid peroxidase (TPO) is a thyroid specific glycosylated hemoprotein that plays a key role in the thyroid hormone synthesis by catalyzing the iodination and coupling of iodotyrosine residues in thyroglobulin (TG) to produce thyroid hormone. TG functions as the matrix for thyroid hormone synthesis and in the storage of the inactive form of thyroid hormone. Synthesis of T3 and T4 follows a metabolic pathway that depends on the integrity of the TG structure. Transcription of the TG and TPO genes are regulated by thyroid-specific transcription factors (TTF-1 and TTF-2). TTF-1,-2 have important roles in the development and differentiation of thyroid. Inactivating mutations in the human GNAS (Guanine nucleotide-binding protein alpha-stimulating activity polypeptide) gene are associated with the inherited disorder "pseudohypoparathyroidism", which can affect the function of thyroid and lead to somatic and developmental abnormalities.In this study, the complete CDS of TG, GNAS, TTF-1 and TTF-2 were cloned; the TPO, TTF-1 and TTF-2 genes were mapped; their expression level were conducted in sixteen tissues and the development pattern were determined in thyroid grand at the age of 1,20,45, 60,90,120 and 150 days; the single nucleotide polymorphism and alternative splicing form of TPO gene were searched; the TG promoter region was studied as well as the effect of castration on methylation of GNAS promoter.(1) The complete CDS of TG, GNAS and partial CDS of TTF-1, TTF-2 were obtained by electronic cloning and comparative genomic technology. The sequences of TG and GNAS were deposited in GenBank (GenBank accession NO.GQ261999 and GU126691). Additionally we analyzed the gene structure, protein structure, conserved motifs by using Clustal W and some related bioinformatics softwares.(2) Using the ImpRH panel, we determined that pig TPO was closely linked with microsatellite markers SWR201 and SW590 on SSC3q22-27; TTF-1 was closely linked with NFKB and SW1959 on SSC7q22; while TTF-2 was closely linked with SSC11E11 on SSC1q28. (3) Real-time Q-PCR was performed to analyze the tissue and development expression pattern of porcine TPO, TG, GNAS, TTF-1 and TTF-2. The results showed that the GNAS gene was expressed ubiquitously; TTF-1 gene expression was highest in the thyroid, lung followed, then the pituitary, no expression in other tissues; while TPO, TG and TTF-2 were only expressed at thyroid. Developmental expression pattern showed that TPO, TTF-1 and GNAS gene expression were relatively stable; while the TG and TTF-2 expression fluctuated more significantly at different growth stages.(4) Gene-pool and sequencing revealed seven SNPs in porcine TPO, C/T63, G/T156, G/C268, C/T333, A/G624, A/G642, C/T705, while none were found in the 3’-untranslated region. Genotyping results of one SNP (A/G642) in the fourteenth exon of TPO gene showed great variation in allele frequency between Chinese indigenous and introduced commercial breeds. The ham weight trait of pigs with AA genotype was significantly higher than that of AG and GG genotype (P<0.05). Two novel transcript variants in porcine TPO gene were found:the splicing variant TPO-2 lacked exon 8, while TPO-3 lacked exon 8 and exon 14,15,16.(5) After prediction the promoter of TG by bioinformatics software, we get the proximal promoter region of 938 bp. The promoter region was ligated into PGL3-Basic vector, and then the recombinant TG promoter-PGL3-Basic was transiently transfected into rabbit embryonic fibroblasts by liposome transfection. The results showed that TG promoter vector we constructed can originate the expression of the downstream target gene efficiently and specially.(6) The methylation status analysis of GNAS gene promoter region at different growth stages in castrated and uncastrated Jinhua pigs demonstrated that the methylation degree of GNAS promoter in castrated pigs was higher than that of uncastrated pigs, but the difference was not significant.更多还原