The Activity Analysis Of Bovine ATP5B Gene Promoter And Research For ATP5B Gene Differently Expression

The ATP5B gene encodes the catalytic subunit of mitochondrial ATP synthase complexand catalyzes the rate-limiting step of ATP formation in eukaryotic cells, is one of the mostimportant proteins in the oxidative phosphorylation pathway during the process of energymetabolism. But there is still no research about the transcription regulatory mechanism ofATP5B gene being reported. This study built the bovine ATP5B gene promoter dual luciferasereport gene recombinant plasmids, detected the activities in3T3L-1cell lines and C2C12celllines, and analyzed the bovine ATP5B gene transcription regulatory element by using thebioinformaticssoftware. In addition, the expression of ATP5B gene in QinChuan cattle wastested in different tissues, and gene sequence was analyzed by software.Bovine genome was extracted from the blood cells, and from which1898bp fragment ofthe5’ flanking region of ATP5B gene was isolated by PCR; then got7subclones by the designof primers, after that, the PCR products were purified and double digested, then directly wereinserted into pGL3-Basic vectors by homologous recombination and the vectors weretransferred into DH5α;at last, dual luciferase report gene vectors of bovine ATP5B genepromoter were constructed and transfected into the C2C12and3T3L-1cells by LPS, thenmeasured the activity of7recombined plasmids; the sequence-763bp—-85bp of ATP5B gene5’end was analyzed by online software.The RNA of16different tissuesin Qin Chuan cattle were extracted by kit。After reversetranscription, test the cDNA，then design ATP5B gene real-time quantitative primers and apair of β-actin internal primers. Finally, the proximal ATP5B gene promoter and the aminoacid sequence of different species were analyzed by software, as the research laid thefoundation for studying the function of bovine ATP5B gene.The main results were as follows:1.The7recombined plasmids of bovine ATP5B gene promoter dual luciferase reportgene vector were successfully cloned；2.Luciferase activity assay indicated that all the recombined plasmids had significantactivity, pATP5B-678and pATP5B-462had greatly significant difference compared to pGL3-Basic；3.The activities of all the recombined plasmids were higher in C2C12cell lines than3T3L-1cell lines；4.Our research confirmed the core promoter sequence of bovine ATP5B gene wasbetween-547bp—-230bp, the bioinformatics software analyzed that there are severaltranscription regulatory elements in the core promoter sequence of bovine ATP5B gene, suchas TATA box、CAAT box、 GATA box、v-Myb、deltaE；5. The analysis of ATP5B gene expression in QinChuan bull showed that, the geneexpression in the hind leg muscle and back muscle is relatively high,in testosterone fat, heart,stomach, rennet, large intestine, small intestine, back fat, is relatively moderate,in kidney andliver, the spleen, lung, caecum, rumen, is relatively low；6. According to the amino acid sequence alignment, the similarity of amino acids forATP5B coding among bovine,goat，ovis，mice and humans is as high as99%，98%，96%，96%,91%similarity with pigs, these results revealed that this is a very conservative gene；7. The software of MEGA successful build system evolutionary treewith cattle, goats,sheep, pigs, mice and human.In summary, the7dual luciferase report gene recombined plasmids of bovine ATP5Bgene promoter were successfully constructed, the analysis of activity in C2C12cells lines and3T3L-1cell lines indicated that the regions of-547bp—-230bp contain the key transcriptionregulatory elements,which lays a foundation for further research on ATP5B genetranscription regulation mechanism.In addition, the results of bovine ATP5B gene expressionspectrum analysis show that the expression of ATP5B gene is relatively highest, or it mayrelate to its function.