Copy Number Variation Of The CYP4A11Gene In Bovine And Its Biological Effects Investigation

As a member of the CYP4A subfamily, CYP4A11was currently known to promote theω-oxidation of arachidonic acid, which generate20-HETE, a kind of angiotonics. It wasreported that20-HETE has two roles in the regulation of blood pressure. On one hand, itcauses the blood pressure rise by contracting kidney vessel, on the other hand, it reduce bloodpressure by the inhibition of kidney tubules reabsorption of sodium. In our previous study, theCYP4A11gene was found to be copy repeated in Chinese cattle, otherwise, it was locatedwithin QTL-2541and QTL-10699which were correlated with meat quality and carcass. Butthere’s no any report about cattle CYP4A11CNV until now. In this study, moleculartechnologies including quantitative PCR, gene clone, cell culture, eukaryotic expression andso on were used, to first analyse the dosage effects of cattle CYP4A11CNV, and then theirinfluence on some individual phenotypes and adipogenesis. The purpose was to reveal thebiological function of cattle CYP4A11CNV, and supply informantion for the CNVinvestigation and cattle breeding. The contents contain: tissue expression level of cattleCYP4A11gene, the difference in gene expression level between different copy number ofCYP4A11gene, the distribution of CYP4A11gene copy number variation in cattle and theirinfluence on growth traits, the subcellular localization of cattle CYP4A11gene, the influenceof cattle CYP4A11gene over-expression on adipogenesis. The results were as follows:1. The differences in mRNA expression level of cattle CYP4A11gene in time and spaceQuantitative PCR was used to detect the expression difference of cattle CYP4A11gene indifferent stages and tissues. The results showed that the CYP4A11gene expressed in liver,kidney, heart, lung, spleen, muscle and adipose. It was similar in expression trend betweenfetal calves and calves, with the highest expression level in liver, then kidney, the others werevery low. From fetal calves to adults, the CYP4A11expression level in liver first rises andthen declines, this was related with the metabolism and growth condition at different stages,however, the CYP4A11expression level in kidney always rise from fetal calves to adults,suggesting the important role of CYP4A11in blood pressure with cattle development. TheCYP4A11expression level in adipose was higher in adults than in calves, implying the relationship between CYP4A11gene and fat deposition in adults.2. The CYP4A11copy number was positively related with its mRNA expression level30individuals were used to detect the types of the CYP4A11gene CNV, and to analyzethe mRNA expression level in four tissues (liver, kidney, muscle, adipose). The laterassociation analysis between the CNV types and mRNA levels showed that the CYP4A11copy number was positively related with the mRNA expression level.3. A similarity in the distribution of cattle CYP4A11gene CNV types were observed amongdifferent breedsQuantitative PCR was used to detect the CNV types of the CYP4A11gene in six Chinesecattle groups. It was found that three CNV types including Gain, Normal and Loss wereobtained in every population. In all populations, the frequency of Normal samples wasobviously higher than that of Gain and Loss, indicating the dominant role of Normal in the sixpopulations, also suggest the similarity of these breeds in genetics and selective breeding.Otherwise, there were differences in frequency of the three types among different populations,which revealed the difference between breeds.4. The significant influence of cattle CYP4A11gene CNV on growth traitsBy association analysis of CYP4A11gene CNV types with cattle growth traits, we foundthat the CYP4A11gene copy number were significantly associated with hucklebone width inJiaxian adults (P<0.05), and with heart girth and chest depth in Qinchuan adults (P<0.05). InNanyang, the CYP4A11gene CNV types were significantly associated with the twelve-monthbody length (P<0.05). In Jinnan, they were significantly associated with body weight andhucklebone width in adults (P<0.05). In Chinese Red Steppe, they were significantlyassociated with the six-month heart girth and cannon girth (P<0.05). There was norelationship between the CYP4A11gene CNV types and growth traits in Luxi, however, asimilar trend with other groups was observed. These data showed the significant influence ofCYP4A11gene copy numbers on growth traits, especially body measurements. The copynumber of CYP4A11gene could be used as a candidate marker in cattle breedinginvestigation.5. The CYP4A11protein was expressed in both nucleus and cytoplasmThe CDS region of cattle CYP4A11gene was cloned and constructed to pEGFP-C1vector to generate a recombined vector, pEGFP-C1-CYP4A11, which was then transfected to3T3-L1and293T cell line to execute the subcellular localization of cattle CYP4A11gene. Wefinally detected the CYP4A11fuse protein in both nucleus and cytoplasm. It showed that theCYP4A11protein functions not only in nucleus but also in cytoplasm. 6. Over-expression of cattle CYP4A11gene contribute to adipogenesis in3T3-L1cellOver-expression of cattle CYP4A11gene was carried out in3T3-L1cell to detect theexpression levels of candidate marker genes related with adipose differentiation, and toanalyze the differentiation degree of3T3-L1cells to mature adipose cells. The results showedthat after over-expressed cattle CYP4A11gene, the expression level of PPARg, FABP4, LPLand FASN gene rose to1.34,7.81,1.02and1.28times more than the control grouprespectively, with a significantly change in FABP4(P=0.047). Relying on their positive rolesin regulating adipose cell differentiation, the rise in expression level of the four genesillustrated that over-expression of cattle CYP4A11gene in3T3-L1cell promote adipose celldifferentiation and adipose deposition. At the same time, Oil red O was used to stain cells. Inboth the control and experimental group, lipid droplet gathered. This showed that3T3-L1cellhad differentiated to mature adipose cell after twenty days induction. Otherwise, thedifferentiation degree of experimental group was significantly higher than that of the controlgroup（P=0.001）. This further proved the promotion of cattle CYP4A11gene over-expressionto the differentiation of3T3-L1cell and the generation of fat droplet.