Study On Five Key Genes Of Porcine Hypothalamo-pituitary-thyroid Axis

Candidate gene approach is one of the important methods in quantitative trait analysis. In the present study, five candidate genes of hypothalamo-pituitary-thyroid axis which plays important roles in the regulation of animals' growth, development and many metabolic and physical processes:thyrotropin releasing hormone gene (TRH), thyrotropin releasing hormone receptor gene (TRHR), thyrotropin alpha subunit gene (CGA) and beta subunit gene (TSHB), and thyrotropin receptor gene (TSHR) were investigated using bioinformatis, radiation hybrid mapping,3'RACE, real-time quantitative RT-PCR, PCR-RFLP, tetra-primers ARMS-PCR technolgies and so on to map the genes, analyse the QTLs located on them, clone the cDNA, study the genes' tissue expression profile, seach for the genetic polymorphisms and investigate their associations with porcine economical important traits. The objectives of these studies were to characterize the five porcine key genes of HPT axis, develop and evaluate new effective genetic markers for the improvements of porcine economical important traits. The results obtained were as follows:(1) With bioinformatics method and Radiation Hybrid mapping technology, the five key genes of HPT axis were mapped to Sus Scorfa chormosome 13,4,1,1 and 7, respectively. According to the information from pig QTL datebase, all the five genes lie in the daily gain, meat quality and fat related QTLs, which suggesting that genetic polymorphisms of HPT asis might associate with individuals' growth rate, meat quality and fat traits' variation, and could be the positional and functional candidate genes of respective QTLs.(2) mRNA sequence of porcine TRHR gene was obtained at the first time. Porcine TRHR gene was near to the human TRHR gene in the evolution. Except the functional transcript variant (TV1), two additional transcript variants (TV2 and TV3) were also found in this study. TV2 was truncated in the nucleotide sequence that encodes the putative third intracellular loop and retained a part of the intron, while TV3 was the purduct of a 661-bp alternative spliced intron existed in the 2nd exon. The splicing of the alternative spliced intron interrupts the open reading frame, thus TV3 might code a new protein or use a different translation start site. The meaning of the existence of more than one transcript variant remains investigation.(3) Using real-time quantitative RT-PCR technology, tissue distributions of the five genes in brain, hypothalamus, pituitary, heart, liver, spleen, lung, kidney, thyroid, pancreas, stomach, small intestine, large intestine, muscle and fat were investigated. Co-expression of the hormones and their receptors in the same tissues, e.g. in fat tissue, indicating that regulations of the respective hormones on the tissues might partly via paracrine or autocrine effects.(4) To scan for the nucleotide polymorphisms in the genes of HPT axis, re-sequencing 11,340 bp DNA sequence of each founder animal of the Jinhua and Pietrain resource family with 17 pairs of primers were performed.37 polymorphisms in total were identified, among which 8 loci in TRH gene,7 loci in TRHR gene,14 loci in CGA gene,5 loci in TSHB gene, and 3 loci in TSHR gene. All the polymorphisms identified in this study has been submitted to the international SNP database (dbSNP) and gotten the SNP accession number (ss#). Based on the distance between each locus,the flanking sequence, and their potential functions,20 loci were selected and genotyping methods using PCR-RFLP or tetra-primers ARMS PCR technology were designed and verified.(5) The 20 SNP loci of HTP axis were genotyped in 49 Jinhua pigs and 24 Pieterain pigs, and the haplotype block analysis and Tag SNP selection were performed with HapBlock software. All the loci of the same gene located in one block, except the TRH gene in Jinhua population which were separated into two blocks. In combination of the haploblock and Tag SNP information in the two breeds, 3 TRH tag SNP,3 TRHR tag SNP,5 CGA tag SNP,3 TSHB tag SNP and 1 TSHR tag SNP were selected to genotype in Jinhua and Pietrain resource family to analyze the genetic effects of HPT axis.(6) Association analysis between the genetic polymorphisms of HPT axis and 30 porcine growth, carcass and meat quality traits were carried out in the Jinhua and Pietrain resource family (463 individuals in total), and verified in purebreds Yorkshire, Duroc, Landrace and Jiaxing Black pigs. The results showed that: 1) TRH ss325994933 had effects on the growth in later stage, especially significant (P<0.05) on the weight gain between 150 to 180 days old. And opposite dominant alleles appeared in the Chinese and Western breeds. However, the insertion allele which was dominant in the slowly growing Jinhua pigs showed growth advatage in the resource family. The results suggested that TRH might play important roles in the growth rate difference between breeds and could be the candidate gene of daily gain, though its genetic effects remains further investigation.2) Considerable population diversity of TRHR gene existed. TRHR polymorphisms had high significant effects on heat weight and carcass length, and significant effects on the conductivity, pH value, water holding capacity, and intramuscular water and fat content of loin muscle. Both TRHR's expression in muscle and its associations with the meat quality traits suggested that further investigation on roles of TRHR in musle tissue was necessary.3) CGA polymorphisms had significant effect on intramuscular water content, however no such QTL had been located on the CGA gene previously. The existence of intragenic microsatellite in CGA gene might be the reasons of its instable genetic effects. Allele distributions of all the five CGA SNPs showed no apparent differences in the pig breeds. Thus, CGA gene might do not play important roles in the forming of breed specific characteristics.4) Considerable population diversity of TSHB gene was also observed. TSHB polymorphisms had significant effects on weight gain between 90-120 days old and ham muscle weight, and high significant effects on carcass weight, carcass length and average backfat thickness. In the resource family, homozygotes of G-G-A which was from the Jinhua pigs significantly grew slower than the homozygotes of A-G-G which was from the Pietrain, in accordance with the truth that Jinhua pigs had slower growth rate. And BFT of individuals with A-G-G was significantly thinner than those with G-G-A. Futher, TSHB ss181129015 showed significant association with BFT in Duroc and its allele A which was mainly observed in Western breeds was associated with thinner BFT, supporting the results obtained from the resource family. TSHB gene, especially the ss181129015, could be utilized as genetic marker for BFT improvement in pig breeding. 5) Allele A of TSHR had low frequencies in all pig breeds. TSHR polymorphism had significant effects on weight gain between 120-180 days old and intramuscular pH value. Association between TSHR and ADG was also observed in Landrace. However, Allele A was associated with faster growth rate in the resource family but with slower growth rate in the Lancrace.