【作者】 张春林；

【导师】 王加启； 赵国琦；

【作者基本信息】 扬州大学 ， 动物营养与饲料科学， 2010， 硕士

【摘要】 本试验由四部分组成,主要研究了中国荷斯坦奶牛FcRn受体α链基因(FCGRT)启动子基因多态性以及体外激素处理对FcRn mRNA表达丰度的影响。以PCR-SSCP方法发现的SNP位点为研究对象,从构建不同单倍型荧光素酶报告基因载体和分析乳腺内FcRn mRNA表达丰度两个方面研究了启动子多态性对转录活性的影响。并且,通过体外激素处理培养的奶牛乳腺上皮细胞,为进一步研究乳腺内FcRn受体变化以及影响因素提供了依据。试验一:以189头中国荷斯坦奶牛为研究对象,对FCGRT启动子采用PCR-SSCP方法进行多态性检测。结果表明:FCGRT启动子在P1、P2和P3引物扩增片段中存在PCR-SCP多态性,位于引物P1扩增产物有AA、AB、BB 3种基因型,其频率分别为25.40%、59.26%、15.34%;引物P2扩增产物有CC、CD、DD 3种基因型,其频率分别为20.63%、56.61%、22.75%;引物P3扩增产物有EE、EF、FF 3种基因型,其频率分别为1.59%、12.17%、86.24%。经克隆测序分析,在三个片段上分别发生了T→C、C→A、G→T的碱基序列突变。经χ2适合性检验,除SNP1外,中国荷斯坦奶牛在该基因位点上的SNP2和SNP3均处于Hardy-Weinberg平衡状态。试验二:构建奶牛FCGRT基因启动子3种不同单倍型荧光素酶报告载体并在293细胞中检测其转录活性。根据奶牛FCGRT基因启动子区2个单个核苷酸多态性(SNPs)位点C-1116T和C-756A,构建出3种单倍型(CC、CA和TA)。分别以CC/CC、CA/CA和TA/TA 3种基因型的奶牛基因组DNA为模板,用PCR法扩增出包含启动子两个SNP位点的长1789bp的DNA片断,利用Kpn I/BgI ll双酶切,纯化回收后分别与同样双酶切的荧光素酶报告基因载体PGL3-Basic相连接,构建CC-PGL3-Basic、CA-PGL3-Basic、TA-PGL3-Basic 3个表达载体,并测序验证其DNA序列。用电穿孔方法将报告基因载体转染至293细胞,采用双荧光素酶报告基因系统评估不同单倍型FCGRT启动子活性。结果表明,试验成功构建含有牛FCGRT基因启动子3种不同单倍型DNA序列的真核表达载体,单倍型CC-PGL3-Basic载体相对荧光检测值显著高于单倍型CA-PGL3-Basic和CA-PGL3-Basic的相对荧光值,单倍型CA-PGL3-Basic相对荧光值又显著高于单倍型TA-PGL3-Basic(P<0.05)。FCGRT启动子不同单倍型载体表达存在差异,SNP影响了FCGRT启动子的转录活性。奶牛FCGRT基因启动子不同单倍型的真核表达载体成功构建,为FCGRT基因启动子功能研究和转录调控奠定了基础。试验三:采集健康中国荷斯坦奶牛乳腺组织,通过测序检测FCGRT启动子单核苷酸多态性,分析各单倍型对表达水平影响,研究中国荷斯坦奶牛FCGRT启动子SNP对FcRn mRNA表达水平的影响。结果表明,不同单倍型中国荷斯坦奶牛乳腺内FcRn mRNA表达水平明显不同。FCGRT启动子单倍型C-C奶牛乳腺中FcRn表达最高,显著高于单倍型T-A和C-A(P<0.05)。单倍型C-A乳腺组织FcRn表达量显著高于单倍型T-A(P<0.05)。FCGRT启动子SNP对FcRn的mRNA的表达有一定的影响,可能会进一步的影响乳腺中FcRn的表达以及对IgG的转运。试验四:以中国荷斯坦奶牛乳腺上皮细胞为研究工具,采用了RT-PCR方法检测激素处理对FcRn mRNA表达的影响。本文测定了体外培养乳腺上皮细胞时,添加不同浓度甲状腺素T4(0、0.01、0.1、1μmol/L)、胰岛素(0、0.005、0.1、0.5μmol/L)、胰高血糖素(0、0.01、0.1、1μmol/L)对FcRn mRNA的表达的影响。试验结果表明:随着激素添加剂量的增加,甲状腺素和胰高血糖素能够显著的促进表达量升高(P<0.05),而胰岛素添加后FcRn基因mRNA的表达先升高后降低(P<0.05)。因此,添加外源胰岛素、胰高血糖素和甲状腺素能够影响FcRn基因mRNA的表达,为进一步研究乳腺内FcRn受体变化以及影响因素提供了依据。更多还原

【Abstract】 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.更多还原