Study On The FCGRT Promoter Gene Polymorphism And The Effect Of Hormone Treatment On The MRNA Level Of FcRn

This research was composed of four experiments to study the FCGRT promoter gene polymorphism and the effect of hormone treatment on the mRNA level of FcRn gene. By taking the SNP identified by PCR-SSCP as the research object, two experiments including the construction of luciferase reporter gene vectors and expression of FcRn mRNA in mammry gland were conducted to study the transcriptional activity affected by promoter gene polymorphism. Furthermore, the mRNA expression of FcRn was studied by real time PCR in Holstein cows mammary gland epithelial cell treated with hormone.EXPERIMENT 1: The PCR-SSCP polymorphisms of FCGRT promoter gene were studied in 189 Holstein cows. The results showed that PCR-SSCP polymorphisms were identified in promoter amplified fragment (P1,P2 and P3) of FCGRT gene. For the amplified fragment of P1, the genotype frequency of AA, AB and BB was 25.40%, 59.26% and 15.34%, respectively. For the amplified fragment of P2, the genotype frequency of CC,CD and DD was 20.63%, 56.61% and 22.75%, respectively. For the amplified fragment of P3, the genotype frequency of EE,EF and FF was 1.59%, 12.17% and 86.24%, respectively. The sequencing analysis indicated that the polymorphisms were seperately dued to three single point mutation C to T, C to A and T to A at amplified fragment. Except for SNP1 in Holstein cows, the SNP2 and SNP3 sites in the populations were in a state of Hardy-Weinberg equlibrium.EXPERIMENT 2: The luciferase reporter gene vectors containing three different haplotypes DNA of FCGRT gene promoter were constructed to identify the transcriptional activity of FCGRT gene promoter in 293 cells. Three haplotypes (CC, CA and TA) can be constructed based on two single nucleotide polymorphism (SNPs) sites (C-1116T and C-756A) in promoter of bovine FCGRT gene. The 1789bp DNA fragments of FCGRT promoter including the two SNPs were obtained by polymerase chain reaction (PCR) based on bovine genome DNA from the subjects with the CC/CC,CA/CA and TA/TA genotypes, which were digested by restriction endonucleases Kpn I and BgI ll. After fragment recovery, those were ligated to three PGL3-Basic Vectors respectively, which were digested by restriction endonucleases Kpn I and BgI ll as well. All recombinant plasmids were identified by sequencing. The recombinant constructs were then transiently transfected into 293 cells by an electroporation method. After 24h of transfection, the cells were lysed and the Luc activity in lysates was assayed. The results showed as follows: The expression vectors containing three different haplotypes DNA of FCGRT promoter are successfully constructed. The activities of CA-PGL3-Basic was significant higher than those of CA-PGL3-Basic and TA-PGL3-Basic, CA-PGL3-Basic was significant higher than that of TA-PGL3-Basic(P < 0. 05). There is significant difference of three haplotypes, and SNP in the promoter have effects on transcriptional activity. The successfully constructed expression vectors containing three different haplotypes lays the groundwork for the further study of promoter function and transcription regulation.EXPERIMENT 3: The mammary gland samples collected of health holstein cows were detected the SNP genotypes of FCGRT by sequencing, then the correlations between levels of FcRn mRNA and haplotypes were assessed to explore the influence of SNPs in the FCGRT promoter region on the expression of FcRn mRNA in holstein cows. The results showed as follows: The effects of SNP on the expression of FcRn mRNA in the mammary gland were significantly diferent among holstein cows with different haplotype. The haplotype C-C was associated with higher level of FcRn mRNA in the mammary gland of holstein cows, which was significantly higher than the haplotype T-A and C-A (P < 0.05). The FcRn mRNA expression of haplotype T-A was significantly higher than the haplotype C-A (P < 0.05). The SNPs of FCGRT promoter gene can modulate the FcRn expression in mammary gland,and further affect on expression of FcRn and transportation of IgG.EXPERIMENT 4: The mRNA expression of FcRn was studied by real time PCR in Holstein cows mammary gland epithelial cell treated with hormone. We determined FcRn mRNA expression in vitro cultured mammary epithelial cell treated by different concentration thyroxine hormone (0, 0.01, 0.1, 1μmol/L), Insulin (0, 0.005, 0.1, 0.5μmol/L) and glucagon (0, 0.01, 0.1, 1μmol/L) respectively. The results showed as follows: With increasing hormone supply, the treatment of thyroxine hormone and glucagon can obviously raise the level of mRNA expression of FcRn ( P < 0.05). However, the mRNA expression of FcRn increased at first and then decreased with the increase concentration of Insulin ( P < 0.05). Consequently, the treatment with thyroxine hormone, Insulin and glucagon can obviously change the level of FcRn mRNA, the results obtained provide the basis for further research of the change of FcRn and influence factors.