The Study On The Genetic Characteristics And Prokaryotic Expression Of AdPLA Gene In Chinese Cattle

In this study, genetic variation of AdPLA gene was detected by DNA pool sequencing and forced-PCR-RFLP methods in 5 breeds (Nanyang cattle, Qinchuan cattle, Luxi cattle, Jiaxian Red cattle and Caoyuan Red cattle), and association analysis was carried out to evaluate the effects of genotypes of AdPLA gene on some growth traits, including weight, height, body length, heart girth and hucklebone width. The objects were to discovery the genetic characteristics and to explore molecular markers with significant effects on growth traits for efficient selection, and to provide genetic information for protection and usage of Chinese cattle breed resources. Meanwhile, Overlap PCR technique was applied to clone the coding sequence of cattle AdPLA gene, the prokaryotic expression vector pET28a(+)-AdPLA was constructed, and the fusion protein His-AdPLA was expressed successfully in E.coli when induced by IPTG, two recombinant pEGFP-C1 vectors with different coding sequences of AdPLA gene were constructed, which would be useful for AdPLA gene function analysis.The results were as follows:1. Polymorphisms identification and association analysis with cattle growth traitsThe polymorphisms of 5 exons and flank regions of cattle AdPLA gene were detected and three novel SNPs (NC₀07330.4:g.43638506 C>T, g.43658457 T>C and g.43661404 T>C, located in intron 1, intron 2 and exon 4, named P6, P7 and P8, respectively) were revealed. P8 was located in NM₀01075280:m.381 and no amino acid exchange was caused. In order to use Forced-PCR-RFLP to detect these SNPs, EcoRII, AsuII or FbaI restriction endonuclease recognition site was introduced respectively.Three genotypes CC, TT and TC were revealed in P6 locus, association analysis demonstrated that CC genotype was significantly higher than TT genotype at body length and heart girth traits at 24 month in NY breed (P<0.05). In P7 locus, no significant association with growth traits in NY breed had been observed. In addition, in P8 locus, TT genotype was significantly higher than CC genotype at body length at 18 months and heart girth at 24 months (P<0.05). Therefore, this study contributed to evaluate them as genetic markers in bovine genetics and breeding and had potential application in breeding programs.2. Bioinformatics analysis, clone and Prokaryotic expression of cattle AdPLA gene Some Bioinformatics software, such as ProtParam, TMHMM and PredictProtein, were carried out to analyze cattle AdPLA gene and protein. The protein was comprised by 159 amino acids, molecular weight was 17.66 KDa, the theoretical pI (isolectric point) was 8.91, and formula was C791H1250N224O221S7. TMHMM analysis suggested only one tranmembance was in cattle AdPLA protein, and the result of PredictProtein analysis was similar.The coding sequence of AdPLA gene was obtained by Overlap PCR method from cattle genomic DNA and cloned into pGM-T vector, the positive clone sequencing result was same as the sequence in GenBank (NM₀01075280). The positive plasmid was digested with BamHI and HindIII, then the fragment was ligated with the expression vector pET28a(+), the recombined plasmid was transformed into E.coli BL21(DE3) and induced with IPTG. SDS-PAGE results showed that the fusion protein His-AdPLA was expressed in E.coli, which indicated the recombinant vector pET28a(+)-AdPLA was constructed successfully.3. Recombinant vectors pEGFP-C1 with different coding sequences of AdPLA gene were constructedThe stop codon of Chinese cattle AdPLA gene was different from Hereford cattle, in the position of the stop codon"TAG"in Hereford cattle, but Chinese cattle was"CAG", which caused 9 more nucleotides than Hereford cattle. Moreover, it was proved that the C terminal of AdPLA protein was important for potein location, in this study, the two coding sequences with different stop codon were cloned, and combined into vector pEGFP-C1, which will be useful for the study on function of cattle AdPLA protein, especially the C terminal.