Cloning, SNPs Detection Of NPM1 And SREBP1c Genes And Association With Growth Traits In Cattle

In this study, 1035 blood samples from 4 cattle populations- Nanyang, Qinchuan, Jiaxian Red, and Chinese Holstein were collected to extract genomic DNA, and some tissues samples from Qinchuan were taken to extract total RNA.The clone, identification and sequence analysis to the CDS of bovine Nucleophosmin 1 (NPM1) gene by reverse transcription PCR, DNA sequencing and DNA sequence analysis.Genetic variation of NPM1 and SREBP1c genes were detected by DNA sequencing, PCR-SSCP, PCR-RFLP and Forced PCR-RFLP techniques 22 exons and 20 introns in the 2 candidate genes, and association analysis were carried out to evaluate the effects of genotypes of candidate genes on growth traits of three Chinese cattle (Nanyang, Qinchuan and Jiaxian Red). The objects were to discovery the hereditary characteristics and to explore molecular markers with significant effects on economic important traits for efficient selection and improvement of Chinese cattle, and to provide genetic information for foundation of molecular marker database, protection and usage of breed resource of Chinese cattle. The results were as follows:1. The clone identification and bioinformatics analysis of bovine NPM1 geneBovine NPM1 gene cDNA by RT-PCR, the product was cloned in pGM-T vector. The sequencing result showed that the fragment sequence coincided with the prediction, which is 885 bp long. The analysis of amino acid sequence indicated that cattle NPM1 gene consisted 1 exon and coded 295 amino acids. Homologous comparison with some animals indicated that cattle NPM1 cDNA shared 92.1%,91.9%,92.0%,90.7%,89.1%,88.5%,72.2%,52.8% similarity in nucleic acid sequence with Sus scrofa, Canis familiaris, Homo sapiens, Macaca mulatta, Rattus norvegicus, Mus musculus, Gallus gallus and danio rerio. Bovine NPM1 gene cDNA was found in kidny, muscle, liver, small intestine, spleen and lung tissues by the analysis of its expression in various tissues.2. SNPs detection of NPM1 gene and their assosiation between growth traits in Nanyang, Qinchuan and Jiaxian Red cattle SNPs were detected in the exon from 4 cattle breeds in China. Comparison between the nucleotide sequences of the bovine NPM1 gene and the above sequences revealed 6 SNPs and 12-bp deletion in the coding region nt634bp~645bp of the NPM1 gene. Moreover, the SNPs at nt489, nt516, nt624, nt630 and nt636 are linked completely. The 236C>G and 636A>C were 2 missense mutations. While variation at the other 4 SNPs were synonymous mutations.In Nanyang cattle population, different genotypes were significantly associated with the body length of 6 month, heart girth of 6, 12 month, body weight and body length of 12 month, body weight of 12 month, of which the mutational individuals were higher than wildtypes (P<0.05). In Qinchuan cattle population, different genotypes were significantly associated with the body weight, heart girth, hip width, chest depth, withers length, body length and height at hip cross, of which the mutational individuals were higher than wildtypes (P<0.05). In Jiaxian Red cattle population, different genotypes were significantly associated with the body weight and average daily gain of 6 month, heart girth of 12 month, hucklebone width of 24 month, of which the mutational individuals were higher than wildtypes.3. SNPs detection of SREBP1c gene and their assosiation between growth traits in Nanyang, Qinchuan and Jiaxian Red cattleSNPs were detected in the SREBP1c gene from four cattle breeds in China. Comparison between the nucleotide sequences of the bovine NPM1 gene and the above sequences revealed 7 SNPs and 84-bp deletion in intron 7. Moreover, the SNPs at 9807G>A,10914G>A,12020 C>T and 13603 T>C were missense mutations, 10781C>G is synonymous mutations, 10649 T>G and 13475 T>C is nonsense mutation.In Nanyang cattle population, different genotypes were significantly associated with brith weight, the body weight and average daily gain of 6, 12 and 18 month, body weight of 24 month. of which the mutational individuals were higher than wildtypes (P<0.05). In Qinchuan cattle population, different genotypes were significantly associated with the body weight, height at sacrum, withers length, body length, heart girth, circumference of cannon bone, hucklebone width, hip width, height at hip cross, chest depth and chest width, of which the mutational individuals were higher than wildtypes (P<0.01 or P<0.05). In Jiaxian Red cattle population, different genotypes were significantly associated with the body weight, body height, average daily gain, heart girth and hucklebone width of 6 month, body weight, heart girth and hucklebone width of 6 month, body weight, body height and heart girth of 18 and 24 month, average daily gain of 18 month. of which the mutational individuals were higher than wildtypes (P<0.05).