| The e-PCR and DNA sequence analysis techniques were applied not only to clone and predict several bovine genes but also analyze partial stuctures and functions of these genes promoters in ten bovin breeds, including Qinchuan (QC), Luxi (LX), Nanyang (NY), Jiaxian Red (JR), Xianan (XN), Brahma (BH), Brahma crossed with descendant of male murry grey and female Yunnan yellow cattle (BMY), Angus crossed with descendant of male Japanese Black cattle and female Fuzhou cattle (XL), Qinchuan improvement steers (QI) and Simmental and Luxi crossbred steers (SL). Combining biological software and bioinformatics, the characteristics of 13 body measurement traits related to these genes sequences of DNA and protein were analyzed. The tissue expression of these 13 genes were analyzed by Real-time-PCR and bioinformatics, while the polymorphism of these genes were analyzed by RT-PCR, bioinformatics, PCR-RFLP, AS-PCR, CARS-PCR-RFLP and MALDI-TOF. The frequency, equitability, total heritability and relevance of body measurement traits of 13 SNPs were analyzed by Bio-statistics.In order to produce scientific proof for marker-assisted selection of growth and devolopment traits of chinese yellow cattle and built scientific basis for high-yield breeding skills of new breed, some of the effecitve function genes and molecular marks were selected in our study reaserch. The main resules of our study are as follwed:1. The whole cDNA sequence , intron of UQCC gene and partial promoter sequences of GDF5 and CDK6 gene were separated, cloned and identified from Qinchuan cattle by e-PCR, RT-PCR and genomics.2. The characteristics and functions of gene sequences and amino acid information in 13 genes, including UQCC, GDF5, ADAMTSL3, ZBTB38, CDK6, HHIP, IHH, GHRL, PTHR1, UCP2, collA1 and collA2, were predicted and analyzed by bioinformatics. The secondary and tertiary structure, signal peptide sequence, transmembrane region and functional domain of these 13 proteins were predicted and analyzed, too. The phylogenetic trees were also built by comparison with other species and conservative analysis of corresponding proteins in other species. 3. Real-time-PCR showed the content order of UQCC in different cattle tissues and the order from high to low is spleen, heart, trachea, large intestine, fat, testis, kinney, muscle and bladder, while the content order of ADAMTSL3 is testis, heart, fat, stomach, small intestine, liver, lung, trachea, kidney, spleen, large intestine and muscle.4. Combinding abroad articles about human promter genes, bioinformatics were used to analyze the strcture and function of bovine GDF5 and CDK6 gene. Several different cattle transcription factors, named AML-LA, CdxA, GTATA-1 and MZF1, were found, which implied the transcriptional start site in GDF5 was at -359 bp position before ATG and the GT repetitive sequence between -457 bp and -423 bp could enhance gene transcription, while the samllest enhancer was between -458 bp and -377 bp and no TATA box or CAAT box was found. On the other hand, Lk-2, Ik-1, CREB and NF-E2 were found in CDK6 gene, with no TATA box or CAAT box in CDK6 either,which implied that the transcriptional start site was at -90 bp before ATG.5. PCR-RFLP technology were adopted to detect the polymorphism of three locus of UQCC gene and GDF5 gene and analysis of genotype identification in bovine population and their association with body measurement traits were conducted, the results show that the genotypes of A2691T SNP of bovine UQCC gene have a highly remarkable correlation with Rump Length (RL), Chest Depth (CD) and Pin Bone Width (PBW) in the total population (P<0.01), the genotypes of A3150G SNP have an significant association with Body Length (BL) and CD in the total population(P<0.05), joint genotypes are conspicuously relavant with BL, CD, PBW in the total population (P<0.05); the genotypes of T586C SNP of GDF5 gene have a highly remarkable correlation with BL, Hip Width (HW) in Bos Taurus population (P<0.01), they are also significantly correlated with BL in BMY population (P<0.05) and BL, Hip Height (HH) and HW in QI population (P<0.05).6. Three SNPs polymorphism of bovine ADAMTSL3 gene and ZBTB38 gene were studied using AS-PCR technology, at the same time, genotypes identification in bovine population and their association with body measurement traits were analyzed. The result show that the genotypes of T1532C SNP of bovine ADAMTSL3 gene have no significant correlation with body measurement in total population (P>0.05), and the genotypes of C1899T SNP there is a remarkable correlation with PBW(P<0.05), joint genotypes is significantly relavant with BL, CD, Heart Girth (HG) and PBW in the total population(P<0.05); the genotypes of A841G SNP of bovine ZBTB38 gene have a conspicuous association with BL in total population (P<0.05), HH and HG in the QI population (P<0.05) and Withers Height (WH) in XL population.7. ACRS-PCR-RFLP technology were adopted to study a SNP polymorphism of bovine CDK6 gene, genotype identification in bovine population and their association with body measurement were analyzed, the results show that the genotypes of T-1075C SNP of CDK 6 gene have a remarkable association with BL and HG in total population (P<0.05).8. The polymorphism of 9 locus of bovine ZBTB38 gene were studied through DNA sequencing. The analysis results of haplotype, linkage disequilibrium, genotype identification in bovine population and their association with body measurement traits show that, from the nine SNPs, four SNPs haplotype frequencies, including TTGCTGCGG (0.643), TTATTGCGG (0.137), CCGCTCTAA (0.130) and CCGCCGCGA (0.041), are higher than 0.03; SNPs of T2145C, T2286C, G2457C, C2583T, G2643A, G2676A could be seen as an integrity (r2>0.33), G2323A and C2325T could be seen as an integrity (r2>0.33), SNPs of T2145C, T2286C, G2457C, C2583T, G2643A, G2676A have no correlation with boty measurement traits in total bovine population (P>0.05), only SNPs of G2323A and C2325T have an significant association with body measurement traits of BL,WH and RL in the total population.9. Thirty one locus were selected from bovine 65 SNPs to anyalze their polymorphism using MALDI-TOF and multi-PCR technology. Haplotype, linkage disequilibrium, genotyope identification in bovine population and their association with body measurement traits were analyzed. The results show that 8 locus of SNP are faulse, 23 SNPs are effective and located on the chromosome of 2, 19, 21, 29, 4 (4 SNPs), 13 (4 SNPs), 15 (6 SNPs), 17 (2 SNPs) and 22 (3 SNPs) respectively. From all these effective SNPs, three SNPs on chromosome 15, including SNP 58, SNP 60 and SNP 63, could be seen as an integrity (r2>0.33); SNP22 and SNP25 of chromosome 22 could be seen as an integrity (r2>0.33); eleven SNPs, including SNP9 of GDF5 gene, SNP36 and SNP37 of col1A2 gene, SNP46 of ZBTB38 gene, SNP49 and SNP50 of UQCC gene, SNP55, SNP58, SNP 60, SNP61 and SNP 63 of UCP2 gene, have no significant correlation with body measurement traits in total bovine population (P>0.05); the genotypes of SNP4 on CDK6 gene are significantly associated with HH and HG in the total population (P<0.05), the genotypes of SNP5 on CDK6 gene have prominent association with PBW in the total pouplation (P<0.05), genotypes of SNP12 on HHIP gene are significantly relavant with WH in the total population (P<0.05), genotypes of SNP13 on HHIP gene are significantly relavant with BL and WH in the total population (P<0.05), genotypes of SNP14 on IHH gene have highly significant difference with CD in the total population (P<0.01), genotypes of SNP16 on GHPL gene are have a remarkable association with WH in the total population (P<0.05), genotypes of SNP22 on PTHR1 gene are significantly associated with BL, WH, PBW, HG and HW in the total population (P<0.05), genotypes of SNP25 on PTHR1 gene are significantly relavant with BL, HW, HG and PBW in the total population (P<0.05), genotypes of SNP30 on col1A1 gene are significantly associated with PBW in the total population (P<0.05), genotypes of SNP41 on INS gene have a distinct association with RL in the total population (P<0.05), the genotypes of SNP52 on GDF5 gene have a remarkable correlation with BL and WH in the total population (P<0.05), genotypes of SNP64 on UCP2 gene have significant correlation with HW and HG in the total population (P<0.05). |
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