Study On Genetic Variation,Its Effects And Clone Of TPO Gene In Chickens

Thyroid peroxidase, which located on the apical membrane surface of thyrocytes,is the key enzyme involved in thyroid hormone synthesis, mainly catalyses the iodination oftyrosine residues and the coupling of iodotyrosines on thyroglobulin to form thyroxine (T4) andtriiodothyronine (T3). The objectives of this study were to identify genetic polymorphisms of thechicken TPO gene and to analyze potential association between single nucleotide polymorphisms(SNPs) and growth and carcass traits in chicken. Through polymerase chain reaction-restrictionfragment length polymorphism (PCR-RFLP) and DNA sequencing methods, three novel mutationsof the chicken TPO gene were detected in the F2resource population from Gushi chickens andAnka broilers. The Gushi chicken TPO (cTPO) gene was cloned using clone and RT-PCRmethods，sequence analyses were done using boinformatics tools，and expression profile in heart,liver, spleen, lung, kidney, breast and leg muscle, brain, cerebellum, hypothalamus and thyroid wasanalyzed by semiquantitative PCR．The main results were summarized as below:1. Three SNPs were found in chicken TPO gene with the location of exon2(c.111G>A),exon4(c.430G>A), intron13(g.29996C>T). The association analysis indicated that the c.111G>Apolymorphisms was significantly associated with shank girth at12weeks, chest depth at4weeks,breast bone length at8,12weeks, breast weight and leg weight (P <0.05), body bone weight andpectoral angle at12weeks (P <0.01) and chest depth at4weeks and pectoral angle at12weeks (P <0.05) with significant additive effects. The c.430G>A polymorphism was significantly associatedwith shank length at8weeks and pelvis breadth at4weeks (P <0.05), chest width and bodyslanting length at8weeks (P <0.01) and chest width at8weeks (P <0.05) with significant additiveeffects. The g.29996C>T polymorphisms was significantly associated with body weight, breast bonelength, pectoral angle at12weeks, heart weight, gizzard weight, claw weight, and leg muscleweight (P <0.05) and claw weight (P <0.05) with significant additive effects.2. The partial coding region sequence of TPO gene in Gushi chicken was obtained. Ourresults suggested that the coding sequence was968bp, with a ORF of960bp which encodes435amino acids．Comparison to predicted mRNA of chicken, there is a G481A mutation inthe coding sequences, lead Ala to Thr. The phylogeny analysis results showed that cTPOexhibited high homology to bos.3. Semiquantitative PCR analyses showed that TPO was highly expressed in thyroidgland, then in hypothalamus and kidney.Conclusions:The mutations of TPO gene effect the growth of chicken in different stages. The c.111G>A mutation was significantly associated with chest depth at4weeks and pectoral angle at12weekswith significant additive effects, the c.430G>A mutation was associated with chest width at8weekswith significant additive effects, which might be used in pure breeding to improve the growth traitof chickens.