Copy Number Variaitons And Promoter Activity Of LEPR Gene In Chinese Cattle

Genetic variations are significantly associated with animal phenotype. Until now, numerous sequence variations have been identified and investigated to be associated with economic traits of domestic animals, which provides theory basis for marker-associated selection（MAS） and transgenic breeding in animal genetics and breeding programmes. Single nucleotide polymorphisms（SNPs） and copy number variations（CNVs） are two important sources of genetic variations, and their genetic effects have been gradually evaluated.LEPR transmits leptin signaling and plays essential roles in energy expenditure, appetite regulation and fat deposition. Cattle genome re-sequencing from our laboratory showed that partial region of LEPR gene intron 3 was overlapped with one of CNV regions. However, LEPR CNV has not been reported in Chinese cattle yet. In the present study, we qualified copy numbers of LEPR gene in four Chinese cattle breeds（Qinchuan, Nanyang, Jinnan and Xianan） by quantitative PCR, and explored their impacts on LEPR gene expression and phenotypic traits in Qinchuan and Nanyang cattle. Moreover, we detected potential genetic variations in LEPR gene promoter and evaluated their genetic effects in Chinese cattle breeds. The main results are shown below: 1. The distribution of LEPR CNV in Chinese cattle breeds and its association with cattle phenotypeFirst, we selected 30 individuals from each breed, including Qinchuan, Nanyang, Jinnan and Xianan cattle, to detect the distribution of relative LEPR copy numbers in four Chinese cattle breeds（Qinchuan cattle as control）. Additionally, we analyzed the association of LEPR CNV and cattle traits in Nanyang（n=220） and Qinchuan cattle（n=223）. The results showed that Nanyang cattle displayed copy number loss compared to Qinchuan cattle, whereas Xianan cattle appeared copy number gain compared to other three cattle breeds. Meanwhile, LEPR CNV possessed a high variability in intragroup QC and NY populations. Additionally, LEPR CNV significantly associated with growth traits in Nanyang cattle, where individuals with gain copy numbers performed better phenotypic traits. 2. The expression pattern of LEPR gene and the effect of CNV on gene expressionIn this study, heart, liver, spleen, lung, kidney, muscle, intestines, brain（Fetus） and adipose（Adult） tissues were collected from three Qinchuan fetal and adult cattle, respectively, and their DNA and total RNA were extracted. qPCR was carried out to establish the expression pattern of LEPR gene in Chinese cattle and to evaluate the effect of CNV on LEPR gene expression. The results showed that LEPR expressed ubiquitously in Qinchuan cattle at different growth stages. Meanwhile, the CNV was negatively associated with gene expression, where copy number loss had higher gene expression level compared to copy number gain. 3. The genetic effects of two SNPs g.-1285C>T and g.17T>C within LEPR gene promoterTwo novel SNPs（AC₀00160.1: g.-1285C>T and g.17T>C） within LEPR gene promoter were identified by DNA pools sequencing and PCR-RFLP methods. The genetic effects of the two SNPs were evaluated in three Chinese cattle breeds（Qinchuan, Jinnan and Xianan）. The results showed the differences of genetic characteristics of two SNPs were observed among three cattle breeds. Association analysis showed that the two SNPs can significantly influence cattle traits, where TT genotype at g.-1285C>T and CC genotypeat g.17T>C appeared better phenotype compared to CC genotype and TT genotype（P<0.05）, respectively. However, TT and CC genotypes had low expression levels（P<0.05）. 4. LEPR gene core promoter selecting and the effects of two SNPs on promoter activitiesWe downloaded the promoter sequence of LEPR from UCSC database and predicted the potential core promoter and related transcription factors binding by using the online software（http://www.genomatix.de/）. Five truncated fragments（pro1⁵） of promoter were cloned into the pGL3-basic luciferase reporter vector. Additionally, mutant vectors containing different alleles at g.-1285C>T and g.17T>C loci were also constructed. Each recombinant vector was transfected in C2C12 and 293 T cell lines and relative luciferase activitis were ananlyzed using Dual-Luciferase Reporter Assay System. The results showed the second truncated fragement（pro2） had the highest luciferase activity, suggesting that this region was the core promoter region of LEPR gene. Meanwhile, at g.-1285C>T locus, homozygote CC showed higher promoter activity compared to homozygote TT in two cell lines. At g.17T>C, promoter activity had no obvious differences between two alleles.