Molecular Characterization Of Porcine TPO, TG, GNAS, TTF-1 And TTF-2 Genes

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.