The Study On The Genetic Characteristics Of Acrp30 Gene In Cattle And Its Expression In E.Coli

Genetic variation of Acrp30 gene was detected by PCR-SSCP and DNA sequencing techniques in five populations (Nanyang cattle, Qinchuan cattle, Jiaxian Red cattle, Jinnan cattle and Chinese Holstein), and the association analysis was carried out to evaluate the effects of genotypes of Acrp30 gene on growth traits of three Chinese cattle (Nanyang cattle, Qinchuan cattle, Jiaxian Red cattle). The objects were to discovery the hereditary characteristics and to explore molecular markers with significant effects on economic important traits for efficient selection and to provide genetic information for foundation of molecular marker database. Cloning and expression of Acrp30 gene in E.coli were also studied in Qinchuan cattle and this work was a basis for further research on its functions. The results were as follows:1. The genetic variation of Acrp30 gene and association of genetic variations with growth traits in the three populationsThe varations of five loci in Acrp30 gene were studied. Two SNPs (+822 C>T和+866 C>T,the first nucleotide of the start codon ATG was defined as +1) were detected in intron2 and one SNP in 3'flanking region for the first time. In intron2, the tested populations were in Hardy-Weinberg equilibrium while in 3'flanking region was disequilibrium. There was significant difference between tested populations in genotype distributions in intron2 (P<0.01). No significant association was found between genotypes of intron2 and 3'flanking region and the growth traits of Qinchuan, Nanyang and Jiaxian cattle (P>0.05).2. The genetic variation of the promoter in Acrp30 gene and the analysis of the sequence Among the three loci at the promoter region, 5 point mutations(-10241 G>T,-10171 C>T, -10007 G>C, -9886 C>T,-9936 C>G)and a insertion mutation(-9940--9939 ins 67 bp)were detected in P1 locus (-9862--10333)for the first time, and one point mutation(-10997 C>T)and a deletion mutation(-10744--10740 delGATTA)were detected in P3 locus(-10598--11080) for the first time. The genotype distributions of ins/del mutation in the P1 locus the Holstein population was significantly different from other tested native cattle (P<0.05) as well as the ins/del mutation in the P3 locus. A search for transcription factor binding sites in the region was done using the program MatInspector (http//www.genomatix.). There were two putative PPAR binding sites, one SREBP binding sites and a ChRE in the P1 locus. And two C/EBP and two PPAR-γputative binding sites were found in P3 locus. Through promoter mapping analysis,the deletion in the P3 locus resulted in a new putative CCAAT/enhancer binding protein (C/EBP) binding site, and the insertin in the P1 locus in fact was a copy sequence which contained a ChRE.3. Cloning and expression of Acrp30 gene in Qinchuan cattleThe coding sequence of Acrp30 gene of Qinchuan cattle was amplified by overlap-PCR and confirmed by sequencing. The recombinant expressive plasmid pET32a+-Acrp was transduced into E.coli strain BL21 (DE3). Induced by IPTG, the fusion protein, His- Acrp, was expressed with molecular weight 44 kDa in E.coli. The fusion protein was purified through Nickel-affinity chromatography column.