旨在克隆奶牛核受体亚家族1 D组成员1(nuclear receptor subfamily 1 group D member 1,NR1D1)基因的蛋白编码序列(coding sequence, CDS),构建其真核表达载体,预测分析NR1D1基因的生物学功能,并检测该基因的组织表达谱及其在卵巢组织...旨在克隆奶牛核受体亚家族1 D组成员1(nuclear receptor subfamily 1 group D member 1,NR1D1)基因的蛋白编码序列(coding sequence, CDS),构建其真核表达载体,预测分析NR1D1基因的生物学功能,并检测该基因的组织表达谱及其在卵巢组织的表达分布。本研究以5头健康的24月龄雌性奶牛为试验动物,PCR扩增带有同源臂的奶牛NR1D1基因CDS区全长片段,利用同源重组法将目的片段连接至线性化的pcDNA3.1-Puro-N-3HA空载体,构建重组质粒,利用酶切、PCR及测序对重组质粒进行鉴定。结合质粒测序结果,利用生物信息学软件预测分析该基因的生物学功能。将鉴定正确的质粒转染至HEK293T细胞,利用实时荧光定量PCR(qPCR)与Western blotting技术检测NR1D1基因在mRNA与蛋白水平的表达。此外,利用qPCR检测奶牛NR1D1基因的组织表达谱,并利用免疫组织化学技术检测NR1D1蛋白在奶牛卵巢组织的表达分布。结果,成功获得带有同源臂的奶牛NR1D1基因的CDS区全长片段(1 884 bp),酶切、PCR及测序结果表明pcDNA3.1-3HA-cNR1D1真核表达载体构建成功。生物信息学分析结果显示,NR1D1蛋白不存在跨膜结构,为核蛋白,其核定位序列存在于第200位氨基酸附近,且NR1D1蛋白中存在86个潜在的磷酸化位点;奶牛与小鼠NR1D1蛋白三级结构较为相似,奶牛NR1D1蛋白可能在奶牛生殖、代谢与免疫等生理过程中发挥重要作用。将pcDNA3.1-3HA-cNR1D1转染至HEK293T细胞,奶牛NR1D1基因在mRNA与蛋白水平均成功过表达。qPCR结果显示,NR1D1基因在奶牛瘤胃、结肠与肝中表达量显著高于其他组织;免疫组织化学结果显示,奶牛NR1D1表达于不同发育时期卵泡的颗粒细胞中。本研究成功构建了奶牛NR1D1基因真核表达载体,且在HEK293T细胞成功实现了该基因的过表达,对奶牛NR1D1基因及其编码蛋白的理化性质与生物学特性进行了预测分析,并获得了该基因的组织表达谱及在奶牛卵巢组织的表达分布情况,为深入探究奶牛NR1D1基因的生物学功能提供了前期基础与关键材料。展开更多
The isomer ^(229m)Th is the most promising candidate for clocks based on the nuclear transition because it has the lowest excitation energy of only 8.10±0.17 eV.Various experiments and theories have focused on me...The isomer ^(229m)Th is the most promising candidate for clocks based on the nuclear transition because it has the lowest excitation energy of only 8.10±0.17 eV.Various experiments and theories have focused on methods of triggering the transition between the ground state and isomeric state,among which the electronic bridge(EB)is one of the most efficient.In this paper,we propose a new electronic bridge mechanism via two-photon excitation based on quantum optics for a two-level nuclear quantum system.The long-lived 7 s1/2 electronic shell state of^(229m)Th^(3+),with a lifetime of approximately 0.6 s,is chosen as the initial state and the atomic shells(7 s-10 s)could be achieved as virtual states in a two-photon process.When the virtual states return to the initial state 7 s1/2,there is a chance of triggering the nucleus 229Th^(3+),to its isomeric state ^(229m)Th ^(3+),via EB.Two lasers at moderate intensity((10^(10)-10^(14))W/m^(2)),with photon energies near the optical range,are expected to populate the isomer at a saturated rate of approximately 10^(9) s^(-1),which is much higher than that due to other mechanisms.We believe that this twophoton EB scheme can help in the development of nuclear clocks and deserves verification via a series of experiments with ordinary lasers in laboratories.展开更多
Almost all organisms exhibit ~24-h rhythms, or circadian rhythms, in a plentitude of biological processes.These rhythms are driven by endogenous molecular clocks consisting of a series of transcriptional and translati...Almost all organisms exhibit ~24-h rhythms, or circadian rhythms, in a plentitude of biological processes.These rhythms are driven by endogenous molecular clocks consisting of a series of transcriptional and translational feedback loops. Previously, we have shown that the inner nuclear membrane protein MAN1 regulates this clock and thus the locomotor rhythm in flies, but the mechanism remains unclear. Here, we further confirmed the previous findings and found that knocking down MAN1 in the pacemaker neurons of adult flies is sufficient to lengthen the period of the locomotor rhythm. Molecular analysis revealed that knocking down MAN1 led to reduced m RNA and protein levels of the core clock gene period(per),likely by reducing its transcription. Over-expressing per rescued the long period phenotype caused by MAN1 deficiency whereas per mutation had an epistatic effect on MAN1, indicating that MAN1 sets the pace of the clock by targeting per.展开更多
文摘旨在克隆奶牛核受体亚家族1 D组成员1(nuclear receptor subfamily 1 group D member 1,NR1D1)基因的蛋白编码序列(coding sequence, CDS),构建其真核表达载体,预测分析NR1D1基因的生物学功能,并检测该基因的组织表达谱及其在卵巢组织的表达分布。本研究以5头健康的24月龄雌性奶牛为试验动物,PCR扩增带有同源臂的奶牛NR1D1基因CDS区全长片段,利用同源重组法将目的片段连接至线性化的pcDNA3.1-Puro-N-3HA空载体,构建重组质粒,利用酶切、PCR及测序对重组质粒进行鉴定。结合质粒测序结果,利用生物信息学软件预测分析该基因的生物学功能。将鉴定正确的质粒转染至HEK293T细胞,利用实时荧光定量PCR(qPCR)与Western blotting技术检测NR1D1基因在mRNA与蛋白水平的表达。此外,利用qPCR检测奶牛NR1D1基因的组织表达谱,并利用免疫组织化学技术检测NR1D1蛋白在奶牛卵巢组织的表达分布。结果,成功获得带有同源臂的奶牛NR1D1基因的CDS区全长片段(1 884 bp),酶切、PCR及测序结果表明pcDNA3.1-3HA-cNR1D1真核表达载体构建成功。生物信息学分析结果显示,NR1D1蛋白不存在跨膜结构,为核蛋白,其核定位序列存在于第200位氨基酸附近,且NR1D1蛋白中存在86个潜在的磷酸化位点;奶牛与小鼠NR1D1蛋白三级结构较为相似,奶牛NR1D1蛋白可能在奶牛生殖、代谢与免疫等生理过程中发挥重要作用。将pcDNA3.1-3HA-cNR1D1转染至HEK293T细胞,奶牛NR1D1基因在mRNA与蛋白水平均成功过表达。qPCR结果显示,NR1D1基因在奶牛瘤胃、结肠与肝中表达量显著高于其他组织;免疫组织化学结果显示,奶牛NR1D1表达于不同发育时期卵泡的颗粒细胞中。本研究成功构建了奶牛NR1D1基因真核表达载体,且在HEK293T细胞成功实现了该基因的过表达,对奶牛NR1D1基因及其编码蛋白的理化性质与生物学特性进行了预测分析,并获得了该基因的组织表达谱及在奶牛卵巢组织的表达分布情况,为深入探究奶牛NR1D1基因的生物学功能提供了前期基础与关键材料。
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(No.XDB16)the Strategic Priority Research Program of the CAS(No.XDB34030000)。
文摘The isomer ^(229m)Th is the most promising candidate for clocks based on the nuclear transition because it has the lowest excitation energy of only 8.10±0.17 eV.Various experiments and theories have focused on methods of triggering the transition between the ground state and isomeric state,among which the electronic bridge(EB)is one of the most efficient.In this paper,we propose a new electronic bridge mechanism via two-photon excitation based on quantum optics for a two-level nuclear quantum system.The long-lived 7 s1/2 electronic shell state of^(229m)Th^(3+),with a lifetime of approximately 0.6 s,is chosen as the initial state and the atomic shells(7 s-10 s)could be achieved as virtual states in a two-photon process.When the virtual states return to the initial state 7 s1/2,there is a chance of triggering the nucleus 229Th^(3+),to its isomeric state ^(229m)Th ^(3+),via EB.Two lasers at moderate intensity((10^(10)-10^(14))W/m^(2)),with photon energies near the optical range,are expected to populate the isomer at a saturated rate of approximately 10^(9) s^(-1),which is much higher than that due to other mechanisms.We believe that this twophoton EB scheme can help in the development of nuclear clocks and deserves verification via a series of experiments with ordinary lasers in laboratories.
基金supported by grants from the National Natural Science Foundation of China (31471125 and 31671215)‘‘1000 Talents’’ Program of China
文摘Almost all organisms exhibit ~24-h rhythms, or circadian rhythms, in a plentitude of biological processes.These rhythms are driven by endogenous molecular clocks consisting of a series of transcriptional and translational feedback loops. Previously, we have shown that the inner nuclear membrane protein MAN1 regulates this clock and thus the locomotor rhythm in flies, but the mechanism remains unclear. Here, we further confirmed the previous findings and found that knocking down MAN1 in the pacemaker neurons of adult flies is sufficient to lengthen the period of the locomotor rhythm. Molecular analysis revealed that knocking down MAN1 led to reduced m RNA and protein levels of the core clock gene period(per),likely by reducing its transcription. Over-expressing per rescued the long period phenotype caused by MAN1 deficiency whereas per mutation had an epistatic effect on MAN1, indicating that MAN1 sets the pace of the clock by targeting per.
