FADD(Fas-associated death domain protein)存在于Fas/FasL系统中,是信号传导通路的一个信号连接蛋白,FADD通过传递凋亡信号,从而介导细胞凋亡。为进一步验证FADD基因抑制细胞增殖和促进细胞凋亡的作用,从牛卵巢组织中扩增FADD基因,将F...FADD(Fas-associated death domain protein)存在于Fas/FasL系统中,是信号传导通路的一个信号连接蛋白,FADD通过传递凋亡信号,从而介导细胞凋亡。为进一步验证FADD基因抑制细胞增殖和促进细胞凋亡的作用,从牛卵巢组织中扩增FADD基因,将FADD基因连接到带有绿色荧光蛋白报告基因的真核表达载体pAcGFP-N1中,构建过表达FADD基因载体,并构建FADD基因的RNAi载体;用脂质体介导法将FADD基因RNAi载体、真核表达载体转染到牛胎儿成纤维细胞中,观察有无荧光的表达,并使用Real-Time qPCR和Western blot方法检测FADD基因mRNA、蛋白水平的表达情况。结果表明:成功构建出FADD基因RNAi载体和高表达载体,重组质粒转染牛胎儿成纤维细胞24 h后在荧光显微镜下可观察到绿色荧光,转染效率可达50%。展开更多
[Objective] The research aimed to discuss the differential expression quantity of miRNA-181b in mature（18-month-old） and immature（one-month-old） cattle＇s anterior pituitary and its regulation function.[Method] cD...[Objective] The research aimed to discuss the differential expression quantity of miRNA-181b in mature（18-month-old） and immature（one-month-old） cattle＇s anterior pituitary and its regulation function.[Method] cDNA library of miRNA in mature（18-month-old） and immature（one-month-old） cattle＇s anterior pituitary were established.After Solexa high-throughput sequencing of miRNA in the cDNA library,miRNA in anterior pituitary of bulls was identified.miRNA-181b with differential expression were selected from the sequencing results.By real-time quantitative RT-PCR,the expression laws of miRNA-181b in the anterior pituitary of Yanbian Cattle in different growth period was validated.And the target genes of miRNA-181b were forecast by using TargetScanS prediction software.[Result] The expression quantity of miRNA-181b had great difference in cattle＇s anterior pituitary different growth periods.The expression quantity of miRNA-181b in anterior pituitary of one-month-old cattle was 4.05 times as that in 18-month-old cattle.The binding of miRNA-181b with 838-844 bases in 3＇ untranslated region of FSHβ gene was specific and the binding base sites were UGAAUGUA.[Conclusion] This research provided the theoretical basis for the transcription regulation research of FSHβ.展开更多
基金Supported by National Natural Science Foundation of China(30972100)Jilin Science and Technology Development Pro-gram (201101015)~~
文摘[Objective] The research aimed to discuss the differential expression quantity of miRNA-181b in mature（18-month-old） and immature（one-month-old） cattle＇s anterior pituitary and its regulation function.[Method] cDNA library of miRNA in mature（18-month-old） and immature（one-month-old） cattle＇s anterior pituitary were established.After Solexa high-throughput sequencing of miRNA in the cDNA library,miRNA in anterior pituitary of bulls was identified.miRNA-181b with differential expression were selected from the sequencing results.By real-time quantitative RT-PCR,the expression laws of miRNA-181b in the anterior pituitary of Yanbian Cattle in different growth period was validated.And the target genes of miRNA-181b were forecast by using TargetScanS prediction software.[Result] The expression quantity of miRNA-181b had great difference in cattle＇s anterior pituitary different growth periods.The expression quantity of miRNA-181b in anterior pituitary of one-month-old cattle was 4.05 times as that in 18-month-old cattle.The binding of miRNA-181b with 838-844 bases in 3＇ untranslated region of FSHβ gene was specific and the binding base sites were UGAAUGUA.[Conclusion] This research provided the theoretical basis for the transcription regulation research of FSHβ.
