Study On Genetic Variation, Molecular Cloning And Prokaryotic Expression Of FGF21Gene In Chinese Cattle

Fibroblast growth factor21（FGF21）, a member of FGF family which expressed highly inliver, is a hepatic hormone that regulates peripheral glucose tolerance, energy balance andlipid metabolism. Many studies have shown a strong association between FGF21and obesity,hyperlipidemia and diabetes. Specifically, endogenous FGF21was shown to facilitateinsulin-independent uptake of glucose in adipocytes and to promote lipolysis contributing toreduction of fat storage. Exogenous FGF21administration in experimental models wasdemonstrated to lower both glucose and triglyceride plasma levels and promote weight loss.For cattle breeding, the analysis of fatty metabolism and energy balance in cattle had a greattheoretical significance.The goal of this study was to identify SNPs of bovine FGF21gene in five cattle breedsfrom China and to assess their associations with growth traits. CRS-PCR-RFLP, DNA pooland DNA sequencing methods were employed to screen the genetic variation within FGF21gene in a sample of1255Chinese indigenous bovine （NY, n=210; QC, n=224; JX, n=416; LX,n=168; CY, n=237）. Meanwhile, RT-PCR technique was applied to clone the codingsequence of FGF21gene of QC cattle, the prokaryotic expression vector pET28a-FGF21wasconstructed and the fusion protein His-FGF21was expressed successfully in E.coli BL21（DE3） when induced by IPTG. This study provides a good foundation for further research ofcattle FGF21and lays a pathway for developing the FGF21protein in the future.The results were as follows:1Four novel SNPs of the bovine FGF21gene and their associations with growth traitsBased upon the sequence of bovine FGF21gene, three pairs of PCR primers were designedto amplify of the whole length of bovine FGF21gene. Four mutations were identified:NC₀07316: F159T>C, F297C>G, F940C>T and F1151C>T, F159locus was a synonymousmutation and the other three were in the introns. Haplotype analysis, linkage disequilibrium analysis and association of genotype to phenotype were carried out in the four SNPs.Genetic diversity analysis reveals that the wild allele was obviously higher than the mutantallele at the four loci in all five breeds. Interestingly, when comparing with the fourindigenous bovine breeds （NY, JX, LX and QC）, no mutant homozygosity genotype wasdetected in crossbred bovine breed （CY）. All the indigenous bovine breeds at the four locipossessed middle genetic diversity （0.250<PIC<0.500） while the crossbred bovine breed （CY）population possessed low genetic diversity （PIC<0.250）. Moreover, the χ2-test showed thatthe genotype distributions within all five breeds were in Hardy–Weinberg equilibrium（P>0.05） except for the JX and LX cattle at F159locus, which showed that the five Chinesebovine populations were almost in a dynamic equilibrium even in artificial selection. Inaddition, seven different haplotypes were revealed and the haplotype TCCC was predominantin all five cattle breeds. While taken all breeds together, TCCC and CGTT were abundanthaplotypes that accounted for more than66%of total variability. The results of linkagedisequilibrium analysis distributed unevenly in the test breeds. But we can see that there was astrong linkage between F297locus and F940locus in all four indigenous bovine breeds （NY,JX, LX and QC） as compared with a weak linkage （r2<0.33） in CY breed.In NY cattle, at the age of18months, cattle with genotype of mutant homozygosity （GG）appeared superior in body weight as compared with those with CC and CG genotypes（P<0.05） In F297C>G locus. Interesting, for the F940C>T locus, the animals with genotypeof mutant homozygosity （TT） had significantly better body weight than those with CC and CTgenotypes too （P<0.01）, and significance also found at the age of18months. No significantassociation had been observed in QC breed and we also found that F940C>T locus of bovineFGF21are associated with hip width in JX cattle. Our result shows that F297and F940ofFGF21gene is an important candidate gene for these traits and will contribute to bovinebreeding and genetics through MAS.2. Bioinformatics analysis, cloning and prokaryotic expression of cattle FGF21geneBioinformatics analysis revealed that FGF21is a secreted protein with a membranespanning domain and a signal peptide on the position of its N terminal, and the cleavage sitewas located between pos.28and29amino acid. In this study, the coding sequence of FGF21gene was obtained by using RT-PCR. SDS-PAGE results showed that the fusion proteinHis-FGF21was expressed in E.coli BL21（Rosetta） when induced by IPTG, which indicatedthe prokaryotic expression system of recombined vector pET28a-FGF21as constructedsuccessfully.