Study On Sequences, Molecular Evolution And Polymorphism Of Horse Growth Hormone Gene

The different breeds of horses were studied. Using the PCR technique, the full sequences of horse growth hormone(hsGH) gene of six breeds were firstly cloned and sequenced. The length of this gene was 1923bp, including all the exons and all the introns (GenBankAccession:AY837571). And the composition of nucleotide and amino acid encoded by the gene were analyzed. The molecular evolution trees were established by DNAMAN, DNAstar, Mega2.1 and PAUP4.0. The PCR-SSCP and PCR-RFLP analysis of the full sequence of hsGH gene in different horse breeds were studied. The results were as follows: 1,The sequence of amplified horse GH gene was 1923bp, including five exons and four introns. The length of five exons were 10bp, 161bp, 117bp, 162bp and 201bp. The length of introns were 253bp, 213bp, 199bp and 272bp long. The number, length and locus of exons showed high conservative compared with other mammals. 2,The analysis of nucleotide composition showed that G/C content (over 60%) is abundant in horse GH gene, and the contents (61%) of exons were significant higher than those of noncoding regions in 5'and 3'ends. In encoding regions, the G/C contents were abundant at the first (60.3%) and third (84.1%) sites. A/T content was rich in the second site(58.7%). The codon composition were variable in different species, which were specific in each species. 3,The analysis of codon usage indicated that the usage of synonymous codon was very biased, and the biased kinds were different among species. 4,The composition analysis of amino acid showed that the contents of 20 kinds of amino acid were unbalanced. The most was leucine(L,14.28%) and the least was tryptophan(W,0.93%). The contents of polar amino acid(54.99%) were higher than those of nonpolar amino acid(45.01%) in growth hormone of all hores. The amounts of cysteine were five, same with other mammals. The location of cysteine was conservative in different species. This indicated that the cysteine was very important in supporting the structure of GH. 5,Only 0-5 amino acids were different in 6 horse breeds GH. Compared to other mammals, the evolution distance from near to far was pig, dog, cow, sheep, and human. 6,From the molecular evolution trees constructed by different tree-building methods, we got the conclusion that the evolution speed of horse GH gene was different in different regions. Exon regions were more conservative than intron regions. So, the evolution speed of intron regions was more quickly. Sanhe horse, Wuzhumuqin horse and Xinihe horse lay in a same evolution branch, it was different from other branches of horse. 7,The polymorphisms of PCR-SSCP existed only in the fifth exon, but not in the 5'flanking region, first intron, and second exon. This polymorphism of the fifth exon was controlledd by two alleles: A and B.The mutation of T→C and G→A at 1719bp and 1743bp converted allele B into A. Allele B was dominant in Chunxue horse, while allele A was dominant in other horse breeds. The gene reached Hardy-Weinberg equilibrium in Wuzhumuqin horse, Wushen horse and Baerhu horse. 8,The analysis of PCR-RFLP showed that the polymorphism sites with SmaⅠand StuⅠwere inspected, but not with BstXⅠand HindⅢ. There were two alleles A and B at SmaⅠsite. The mutation of T→C at 1256bp provided a SmaⅠsite(CCC↓GGG), which lead to the change of genotype from AA to BB. The frequency of allele B of Wushen horse was the highest (0.667) and the frequency of allele A was the highest for Chunxue horse(0.643).The gene were in Hardy-Weinberg equilibrium in Wushen horse, Wuzhumuqin horse and Baerhu horse. There were two alleles C and D at StuⅠsite.The mutation of C→T provided a StuⅠsite(AGG↓CCT) and lead to the change of genotype from CC to DD. C and D were prevailing alleles for Xinihe horse and Chunxue horse .The gene frequencies were 0.63 and 0.74 respectively. The gene only in Wushen horse was in Hardy-Weinberg equilibrium . 9,Our research based on horse'GH gene sequences will geatly benefit the utilization, development and protection of genetic resource...