| Six types of horse DRD4 gene sequence containing complete four exons and three introns were firstly cloned and sequenced using the PCR technique. The sequence of SH horse was submitted to GenBank. Its accession is EF561289. The compositions of nucleotide and amino acid encoded by the gene were analyzed. The protein structures were predicted by bioinformation. The polymorphisms of DRD4 gene of different horse groups were analyzed by PCR-SSCP, PCR-RFLP and directly sequencing of PCR products. The results were as follows:1,The sequence of amplified horse DRD4 gene included four exons and three introns. Sizes of intron1, exon2, exon3 of the DRD4 gene were different in different horse types.2,The analysis of nucleotide composition showed that G/C content was abundant in horse DRD4 gene, and the G/C contents in exon regions were significant higher than in intron regions and full sequence. In encoding regions of different species, the bases of biased were all G and C at the three sites of codon.3,The analysis of codon usage indicated that the usage of synonymous codon was very biased, and the biased types were same, but biased degrees were different in different species.4,The composition analysis of amino acid showed that the contents of 20 kinds of amino acid were exetremely unbalanced. The most was alanine (A, 15.65%) and the least was lysine (K, 0.88%). The contents of nonpolar amino acid (57.34%) were higher than contents of polar amino acid (42.66%) in DRD4 gene of six horse types.5,The result of DRD4 protein structure predicted showed that the horse DRD4 protein was composed of the hydroxy terminu of 30 amino acids which was located extracellularly, the carboxy terminu of 17 amino acids which was located intracellularly, seven stretches of hydrophobic amino acids that could span the membrane, three loops which were located intracellularly and three loops which were located extracellularly. In addition, the results of secondary structure predicted of horse DRD4 protein showed there are at least 12α-helixs, 8β-sheets and 40 loops. And the protein contained eighteen function sites, seven LCRs, one NOR and five disulfide bonds. Its tertiary structure is tubbiness.6,The analysis of PCR-SSCP showed that P4, P6 and P8 sites including exon1,4 and intron 2,3 didn't existed any variations.7,The analysis of PCR-RFLP showed: Insert/delete of 17bp and transition of A→G at intron1 led to produce three alleles containing A, B and C at StuI site. The dominant allele and genetype of TB were B and AB, and the dominant allele and genetype of other horse types were A and AA. The site was in Hardy-Weinberg equilibrium in BH horse, WZ horse and WS horse (P>0.05). Insert/delete of G at exon2 and transition of T→C at intron1 led to producting three alleles containing D, E and F at StyI site. The mutation of T→C led to variation of amino acid. The dominant allele and genetype of TB are D and DD, and others were E and EE. The site was in Hardy-Weinberg equilibrium in BH horse and WS horse (P>0.05).8,Analysis result of the genetic polymorphism parameters of different horse groups at StuI site and StyI site showed: Polymorphism of TB, SH, BH was very abundant at the two sites.9,The analysis result of directly sequencing of PCR products showed that there were 40 polymorphic sites including 26 singleton sites and 14 Parsim-Info sites in DRD4 gene exon3 region of 44 horse types. The region included twenty-four haplotypes and twenty amino acid sequences. Variation types of the region included transitionsion, transversion and VNTR. In addition, part variations of amino acid led to few changes of secondary structure of the protein.
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