Genetic Variation And Promotor Activity Regions Screening Of PNPLA3 Gene In Three Chinese Indigenous Cattle

PNPLA3(patatin-like phospholipase domain containing 3) belongs to the PNPLA family, which contains a patatin domain that participates the triglyceride metabolism and has the activity of fat acylation hydrolase. PNPLA3 has the function of both phospholipase and acyl transferase, and it is involved in the lipid metabolism, which means it is an important factor for regulating triglyceride metabolism. Because of the regulatory function in energy and fat metabolism, PNPLA3 is regarded as a candidate gene to study the meat quality, fat accumulation and growth. Meanwhile, PNPLA3 is closely related with obesity and diabetes and it becomes a potential drug target of treating obesity and non-alcoholic fatty liver disease(NAFLD). However, the association between PNPLA3 gene and growth of Chinese cattle and its function in bovine liver and fat is not quite clear, which needs further study. Hence, in this study, bovine PNPLA3 was explored to reveal the genetic variation and its relationship with growth traits. qPCR analysis was performed to determine the relative mRNA expression of PNPLA3 in different tissues of different ages. Besides, different fragments with fixed 3’ terminal of PNPLA3 promoter were cloned and recombined into pGL3-basic plasmids to screening the promoter activity regions of PNPLA3 gene. 1. Genetic variation of bovine PNPLA3 geneIn this part, 4 haplotypes involving 4 missense single nucleotide polymorphism(SNP) sites, named g.A2980G(SNP1), g.A2996T(SNP2), g.A36718G(SNP3)and g.G36850A(SNP4), in the bovine PNPLA3 gene were identified in three Chinese indigenous cattle populations(Qinchuan, Nanyang and Jiaxian) with 660 individuals through DNA pools sequencing and Forced RFLP-PCR. The results showed that Hap2(AAGA) was extremely predominant in all test populations, and SNP1, SNP3 and SNP4 were intermediate polymorphism(0.25<PIC<0.50), and 4 SNP sites were all in agreement with Hardy-Weinberg equilibrium(P>0.05). The linkage disequilibrium analysis indicated that SNP3 and SNP4 in all tested breeds were in sufficiently strong LD and others were in weak LD.The statistical analyses indicated that these 4 SNPs affected body weight and heart girth markedly(P<0.05) in QC population, specifically, individuals with genotype SNP1-A1G1, SNP2-A2A2 and SNP3-A3A3 have higher body weight and those with SNP1-A1G1 and SNP4-G4G4 could be selected to obtain bigger heart girth. The results support high potential that these SNPs may be utilized as a genetic marker in marker-assisted selection for beef cattle breeding programs. 2. The expression profile of Qinchuan PNPLA3 geneqPCR analysis was performed to reveal the relative mRNA expression of Qinchuan PNPLA3 in different tissues of different stages(fetal bovines, calves and adult cattle), which can provide fundamental data for the further study of indicating the function of PNPLA3 in lipogenesis. The results showed that the expression of PNPLA3 was affected by the tissue specificity and temporal specificity, specifically, it was highly expressed in the liver of all three stages, and it was the mos highly expressed in fat tissue for calves and adult cattle, moreover, it was also highly expressed in spleen for fetal bovines and calves. Meanwhile, the expression of PNPLA3 in the same tissue was significantly influenced by the age, especially for heart, lung, kidney and muscle tissues, which were all decreased with the increase of age. What’s more, the analysis between SNP and mRNA expression was also performed. The results showed that, in SNP1, the expression of AG is much higher than AA and GG(P<0.05), which is in accordance with the results of growth traits association analysis, while the results of SNP2 is not. Therefore, SNP1 may function as a positive regulator in growth traits while SNP2 is a negative regulator. 3. Promotor activity regions screening of PNPLA3 gene in Qinchuan cattleDifferent fragments with fixed 3’ terminal of PNPLA3 promoter were cloned and recombined into pGL3-basic plasmids by techniques, like gene clone, DNA sequencing and luciferase reporter gene analysis. After DNA sequencing, the recombined vectors were transfected into HEK293 T cells, and then the promoter activities of different fragments of 5’ flanking region were determined by quantitative assays including luciferase reporter gene analysis. The results showed that 5 luciferase reporter vectors(pGL3-Basic-P1, pGL3-Basic-P2, pGL3-Basic-P3, pGL3-Basic-P4, pGL3-Basic-P5) with different regions of PNPLA3 promoter were successfully constructed and there may be a.negative regulatory element in the middle of promoter(P2,-1696 ~-1192 bp).