To analyze miR-139 target sites in 3＇ UTR of GHR gene in dairy cow mammary gland, a GHR 3＇ UTR- luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in dairy cow mammary g...To analyze miR-139 target sites in 3＇ UTR of GHR gene in dairy cow mammary gland, a GHR 3＇ UTR- luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in dairy cow mammary gland epithelial cells （DCMECs）. The miR-139 targeting GHR 3＇ UTR was predicted by Target Scan 5.1 software, 3＇ UTR fragment of GHR was amplified by PCR from RNA of DCMECs. PCR products were cloned into Spe Ⅰ/Hind Ⅱ modified pMIR-Report vector. The luciferase reporter vector and miRNA eukaryotic expression vector were transferred into DCMECs using lipofectamine 2000 transfection reagent. The dualluciferase reporter assay system was used to quantitiate the reporter activity. The results showed that a 107 bp 3＇ UTR fragment of GHR gene was successfully cloned into the pMIR-Report vector, which authenticated by Spe Ⅰ/Hind Ⅲ digestion and DNA sequencing. The luciferase activity of reporter construction treated with miR-139 decreased 20.87% compared with the control group. It was concluded that the GHR3＇ UTR-luciferase reporter vector had been successfully constructed. The luciferase activity of the reporter could be suppressed by miR- 139.展开更多
[Objective] The paper was to explore the changes of GHR gene expression in different tissues of mink.[Method] With American mink as the research object, the expression volumes of GHR gene in heart, liver, spleen, lung...[Objective] The paper was to explore the changes of GHR gene expression in different tissues of mink.[Method] With American mink as the research object, the expression volumes of GHR gene in heart, liver, spleen, lung, kidney, intestine and skin tissues at different growth stages(45, 90, 120, 150 and 180 days of age) were detected by real-time PCR, and comparative analysis was performed.[Result] The GHR gene expressions in heart, liver and spleen tissues at 180 days of age were extremely higher than those at other days of age( P<0.01). The GHR gene expression in lung tissue at 120 days of age was extremely higher than those at other days of age( P<0.01). The GHR gene expressions in intestine tissue at 45 and 120 days of age were extremely higher than those at other days of age(P<0.01), but no significant difference was observed between 45 and 120 days of age(P>0.05). The GHR gene expression in kidney tissue at 150 days of age was significantly higher than those at other days of age( P<0.05).The GHR gene expression in skin tissue was extremely higher than that those at other days of age( P<0.01). The GHR gene expressions in intestinal tissue at 45 and 120 days of age were extremely higher than those in other tissues(P<0.01). The GHR gene expressions in skin tissue at 90 days of age was extremely higher than those in other tissues(P<0.01). The GHR gene expressions in intestine, spleen and kidney tissues at 150 days of age were extremely higher than those in other tissues(P<0.01). The GHR gene expressions in spleen tissue at 180 days of age was extremely higher than those in other tissues(P<0.01).[Conclusion] The expression of GHR gene in mink showed obvious spatio-temporal specificity.展开更多
基金Supported by the National Key Basic Research and Development (973) Plan (2011CB100804)Northeast Agricultural University Innovation Team Project (CXT005-1-1/CXT005-1-2)
文摘To analyze miR-139 target sites in 3＇ UTR of GHR gene in dairy cow mammary gland, a GHR 3＇ UTR- luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in dairy cow mammary gland epithelial cells （DCMECs）. The miR-139 targeting GHR 3＇ UTR was predicted by Target Scan 5.1 software, 3＇ UTR fragment of GHR was amplified by PCR from RNA of DCMECs. PCR products were cloned into Spe Ⅰ/Hind Ⅱ modified pMIR-Report vector. The luciferase reporter vector and miRNA eukaryotic expression vector were transferred into DCMECs using lipofectamine 2000 transfection reagent. The dualluciferase reporter assay system was used to quantitiate the reporter activity. The results showed that a 107 bp 3＇ UTR fragment of GHR gene was successfully cloned into the pMIR-Report vector, which authenticated by Spe Ⅰ/Hind Ⅲ digestion and DNA sequencing. The luciferase activity of reporter construction treated with miR-139 decreased 20.87% compared with the control group. It was concluded that the GHR3＇ UTR-luciferase reporter vector had been successfully constructed. The luciferase activity of the reporter could be suppressed by miR- 139.
基金Supported by National Natural Science Foundation of China(31501958)Project of Jilin Provincial Department of Science and Technology(20150101113JC)Special Animal Genetic Resources Inno-vation Team Foundation of Chinese Academy of Agricultural Sciences
文摘[Objective] The paper was to explore the changes of GHR gene expression in different tissues of mink.[Method] With American mink as the research object, the expression volumes of GHR gene in heart, liver, spleen, lung, kidney, intestine and skin tissues at different growth stages(45, 90, 120, 150 and 180 days of age) were detected by real-time PCR, and comparative analysis was performed.[Result] The GHR gene expressions in heart, liver and spleen tissues at 180 days of age were extremely higher than those at other days of age( P<0.01). The GHR gene expression in lung tissue at 120 days of age was extremely higher than those at other days of age( P<0.01). The GHR gene expressions in intestine tissue at 45 and 120 days of age were extremely higher than those at other days of age(P<0.01), but no significant difference was observed between 45 and 120 days of age(P>0.05). The GHR gene expression in kidney tissue at 150 days of age was significantly higher than those at other days of age( P<0.05).The GHR gene expression in skin tissue was extremely higher than that those at other days of age( P<0.01). The GHR gene expressions in intestinal tissue at 45 and 120 days of age were extremely higher than those in other tissues(P<0.01). The GHR gene expressions in skin tissue at 90 days of age was extremely higher than those in other tissues(P<0.01). The GHR gene expressions in intestine, spleen and kidney tissues at 150 days of age were extremely higher than those in other tissues(P<0.01). The GHR gene expressions in spleen tissue at 180 days of age was extremely higher than those in other tissues(P<0.01).[Conclusion] The expression of GHR gene in mink showed obvious spatio-temporal specificity.
