Site_specific mutagenesis has been widely used in molecular biology and biochemistry. The authors have developed a simple and easy method for site_specific mutagenesis of any genes on plasmids using long distance inve...Site_specific mutagenesis has been widely used in molecular biology and biochemistry. The authors have developed a simple and easy method for site_specific mutagenesis of any genes on plasmids using long distance inverse PCR in the presence of Pfu_DNA polymerase. The efficiency of this method is higher than 90% and the entire procedure can be performed just in one tube. No subcloning is needed. This method is especially useful for obtaining mutant genes on large plasmids such as Ti plasmids used for plant transformation.展开更多
Thousands of long-distance mobile mRNAs were identified from different grafting systems,based on high-throughput sequencing technology.Moreover,the long-distance delivery of RNAs was proved to involve multiple mechani...Thousands of long-distance mobile mRNAs were identified from different grafting systems,based on high-throughput sequencing technology.Moreover,the long-distance delivery of RNAs was proved to involve multiple mechanisms.Here,we analyzed the homology,motif,and tRNA-like structure(TLS)of long-distance mobile mRNAs identified by RNA-seq as well as the RNA-binding protein(RBP)in nine grafting combinations including Arabidopsis thaliana,Vitis vinifera,Cucumis sativus,Citrullus lanatus,Nicotiana benthamiana,Malus domestica,Pyrus spp.,Glycine max and Phaseolus vulgaris.Although several mRNAs were found to be shared in herbaceous,woody,and related species,the vast majority of long-distance mobile mRNAs were species-specific.Four non-specific movement-related motifs were identified,while the TLS was not necessary for mRNA long distance mobility.In addition,we found that RBPs were conserved among herbaceous and woody plants as well as related species.This paper reports a further in-depth analysis of the endogenous mechanisms by which the species-specific transportable m RNAs were selected by bioinformatics,in order to provide insights for future research on long-distance mobile mRNAs.展开更多
Gas flexible pipes are critical multi-layered equipment for offshore oil and gas development.Under high pressure conditions,small molecular components of natural gas dissolve into the polymer inner liner of the flexib...Gas flexible pipes are critical multi-layered equipment for offshore oil and gas development.Under high pressure conditions,small molecular components of natural gas dissolve into the polymer inner liner of the flexible pipes and further diffuse into the annular space,incurring annular pressure build-up and/or production of acidic environment,which poses serious challenges to the structure and integrity of the flexible pipes.Gas permeation in pipes is a complex phenomenon governed by various factors such as internal pressure and temperature,annular structure,external temperature.In a long-distance gas flexible pipe,moreover,gas permeation exhibits non-uniform features,and the gas permeated into the annular space flows along the metal gap.To assess the complex gas transport behavior in long-distance gas flexible pipes,a mathematical model is established in this paper considering the multiphase flow phenomena inside the flexible pipes,the diffusion of gas in the inner liner,and the gas seepage in the annular space under varying permeable properties of the annulus.In addition,the effect of a variable temperature is accounted.A numerical calculation method is accordingly constructed to solve the coupling mathematical equations.The annular permeability was shown to significantly influence the distribution of annular pressure.As permeability increases,the annular pressure tends to become more uniform,and the annular pressure at the wellhead rises more rapidly.After annular pressure relief followed by shut-in,the pressure increase follows a convex function.By simulating the pressure recovery pattern after pressure relief and comparing it with test results,we deduce that the annular permeability lies between 123 and 512 m D.The results help shed light upon assessing the annular pressure in long distance gas flexible pipes and thus ensure the security of gas transport in the emerging development of offshore resources.展开更多
A 2.3 kb DNA fragment containing Pfu DNA polA gene was amplified by PCR from total DNA of Pyrococcus furiosus and cloned into a pGEM-T vector. The recombinant clone pT-pfu was digested with Nco I and Xho I and the fra...A 2.3 kb DNA fragment containing Pfu DNA polA gene was amplified by PCR from total DNA of Pyrococcus furiosus and cloned into a pGEM-T vector. The recombinant clone pT-pfu was digested with Nco I and Xho I and the fragment was inserted into an expression vector pET3d-X. The Pfu polA gene was expressed in Escherichia coli BL21 (DE3). The gene product (Pfu) was purified with heat denaturation, polyethylenemine (PEI) precipitation and Bio-rex 70 ion-exchange chromatography. The recombinant Pfu was verified by protein N-terminal sequencing. With the recombinant Pfu, large λ DNA fragments were successfully amplified in long-distance PCR.展开更多
本研究根据公开发表的烟草K326基因组和烟草430K SNP固相芯片检测数据,以7份种质两两组合之间每条染色体上20个多态标记为目标,基于多重PCR扩增的精准定位测序分型技术(mGPS,Genotyping by Pinpoint Sequencing of multiplex PCR produc...本研究根据公开发表的烟草K326基因组和烟草430K SNP固相芯片检测数据,以7份种质两两组合之间每条染色体上20个多态标记为目标,基于多重PCR扩增的精准定位测序分型技术(mGPS,Genotyping by Pinpoint Sequencing of multiplex PCR products)开发出烟草1.8K育种液相芯片(YT1.8K.1)。利用该芯片对上述7份种质两两之间杂交的21个杂交组合进行基因分型检测,每个杂交组合之间的平均差异位点数为650个,能同时满足每个组合定向改良筛选高遗传背景回复率单株的需要。利用该芯片对23个烟草品种进行基因型分型检测和聚类分析,聚类分类结果与品种系谱基本吻合;利用该芯片从367个BC2F1群体中筛选出5个背景回复率高于94.96%的单株,高于理论均值87.5%,表明该育种芯片可应用于烟草种质资源聚类分析、定向改良育种的遗传背景筛选。展开更多
为了建立水禽细小病毒(WPV)快速检测方法,根据序列比对结果在水禽细小病毒NS基因SF3保守区域内设计特异性引物,建立SYBR Green Ⅰ荧光定量PCR通用检测方法。该方法的扩增效率(E)为90.0%,相关系数(R~2)=0.99,标准曲线方程为y=-3.607x+38....为了建立水禽细小病毒(WPV)快速检测方法,根据序列比对结果在水禽细小病毒NS基因SF3保守区域内设计特异性引物,建立SYBR Green Ⅰ荧光定量PCR通用检测方法。该方法的扩增效率(E)为90.0%,相关系数(R~2)=0.99,标准曲线方程为y=-3.607x+38.77;除WPV出现S形扩增曲线外,新城疫病毒(NDV)、H9亚型禽流感病毒(H9 AIV)、鸭坦布苏病毒(DTMUV)、鸭肝炎病毒(DHAV)、鸭肠炎病毒(DEV)、鸭呼肠孤病毒(DRV)样品均未出现S形阳性扩增曲线;批内变异系数(CV)为0.15%~0.23%,批间变异系数为0.09%~0.28%。结果表明,SYBR Green Ⅰ荧光定量PCR检测方法重复性好、灵敏度高和特异性强。临床样品检测结果表明,SYBR Green Ⅰ荧光定量PCR与普通PCR的符合率达98.4%,灵敏度是普通PCR的1 000倍。SYBR Green Ⅰ荧光定量PCR检测方法不仅能定性检测WPV,还可以进行定量检测,可用于种鸭场、种鹅场的WPV净化检测,也可用于WPV临床大量样品的快速检测。展开更多
文摘Site_specific mutagenesis has been widely used in molecular biology and biochemistry. The authors have developed a simple and easy method for site_specific mutagenesis of any genes on plasmids using long distance inverse PCR in the presence of Pfu_DNA polymerase. The efficiency of this method is higher than 90% and the entire procedure can be performed just in one tube. No subcloning is needed. This method is especially useful for obtaining mutant genes on large plasmids such as Ti plasmids used for plant transformation.
基金supported by the 111 Project(Grant No.B17043)the 2115 Talent Development Program of China Agricultural University。
文摘Thousands of long-distance mobile mRNAs were identified from different grafting systems,based on high-throughput sequencing technology.Moreover,the long-distance delivery of RNAs was proved to involve multiple mechanisms.Here,we analyzed the homology,motif,and tRNA-like structure(TLS)of long-distance mobile mRNAs identified by RNA-seq as well as the RNA-binding protein(RBP)in nine grafting combinations including Arabidopsis thaliana,Vitis vinifera,Cucumis sativus,Citrullus lanatus,Nicotiana benthamiana,Malus domestica,Pyrus spp.,Glycine max and Phaseolus vulgaris.Although several mRNAs were found to be shared in herbaceous,woody,and related species,the vast majority of long-distance mobile mRNAs were species-specific.Four non-specific movement-related motifs were identified,while the TLS was not necessary for mRNA long distance mobility.In addition,we found that RBPs were conserved among herbaceous and woody plants as well as related species.This paper reports a further in-depth analysis of the endogenous mechanisms by which the species-specific transportable m RNAs were selected by bioinformatics,in order to provide insights for future research on long-distance mobile mRNAs.
基金supported by the Natural Science Research Project of Guangling College of Yangzhou University,China (ZKZD18004)General Program of Natural Science Research in Higher Education Institutions of Jiangsu Province,China (20KJD430006)。
文摘Gas flexible pipes are critical multi-layered equipment for offshore oil and gas development.Under high pressure conditions,small molecular components of natural gas dissolve into the polymer inner liner of the flexible pipes and further diffuse into the annular space,incurring annular pressure build-up and/or production of acidic environment,which poses serious challenges to the structure and integrity of the flexible pipes.Gas permeation in pipes is a complex phenomenon governed by various factors such as internal pressure and temperature,annular structure,external temperature.In a long-distance gas flexible pipe,moreover,gas permeation exhibits non-uniform features,and the gas permeated into the annular space flows along the metal gap.To assess the complex gas transport behavior in long-distance gas flexible pipes,a mathematical model is established in this paper considering the multiphase flow phenomena inside the flexible pipes,the diffusion of gas in the inner liner,and the gas seepage in the annular space under varying permeable properties of the annulus.In addition,the effect of a variable temperature is accounted.A numerical calculation method is accordingly constructed to solve the coupling mathematical equations.The annular permeability was shown to significantly influence the distribution of annular pressure.As permeability increases,the annular pressure tends to become more uniform,and the annular pressure at the wellhead rises more rapidly.After annular pressure relief followed by shut-in,the pressure increase follows a convex function.By simulating the pressure recovery pattern after pressure relief and comparing it with test results,we deduce that the annular permeability lies between 123 and 512 m D.The results help shed light upon assessing the annular pressure in long distance gas flexible pipes and thus ensure the security of gas transport in the emerging development of offshore resources.
文摘A 2.3 kb DNA fragment containing Pfu DNA polA gene was amplified by PCR from total DNA of Pyrococcus furiosus and cloned into a pGEM-T vector. The recombinant clone pT-pfu was digested with Nco I and Xho I and the fragment was inserted into an expression vector pET3d-X. The Pfu polA gene was expressed in Escherichia coli BL21 (DE3). The gene product (Pfu) was purified with heat denaturation, polyethylenemine (PEI) precipitation and Bio-rex 70 ion-exchange chromatography. The recombinant Pfu was verified by protein N-terminal sequencing. With the recombinant Pfu, large λ DNA fragments were successfully amplified in long-distance PCR.
文摘本研究根据公开发表的烟草K326基因组和烟草430K SNP固相芯片检测数据,以7份种质两两组合之间每条染色体上20个多态标记为目标,基于多重PCR扩增的精准定位测序分型技术(mGPS,Genotyping by Pinpoint Sequencing of multiplex PCR products)开发出烟草1.8K育种液相芯片(YT1.8K.1)。利用该芯片对上述7份种质两两之间杂交的21个杂交组合进行基因分型检测,每个杂交组合之间的平均差异位点数为650个,能同时满足每个组合定向改良筛选高遗传背景回复率单株的需要。利用该芯片对23个烟草品种进行基因型分型检测和聚类分析,聚类分类结果与品种系谱基本吻合;利用该芯片从367个BC2F1群体中筛选出5个背景回复率高于94.96%的单株,高于理论均值87.5%,表明该育种芯片可应用于烟草种质资源聚类分析、定向改良育种的遗传背景筛选。
文摘为了建立水禽细小病毒(WPV)快速检测方法,根据序列比对结果在水禽细小病毒NS基因SF3保守区域内设计特异性引物,建立SYBR Green Ⅰ荧光定量PCR通用检测方法。该方法的扩增效率(E)为90.0%,相关系数(R~2)=0.99,标准曲线方程为y=-3.607x+38.77;除WPV出现S形扩增曲线外,新城疫病毒(NDV)、H9亚型禽流感病毒(H9 AIV)、鸭坦布苏病毒(DTMUV)、鸭肝炎病毒(DHAV)、鸭肠炎病毒(DEV)、鸭呼肠孤病毒(DRV)样品均未出现S形阳性扩增曲线;批内变异系数(CV)为0.15%~0.23%,批间变异系数为0.09%~0.28%。结果表明,SYBR Green Ⅰ荧光定量PCR检测方法重复性好、灵敏度高和特异性强。临床样品检测结果表明,SYBR Green Ⅰ荧光定量PCR与普通PCR的符合率达98.4%,灵敏度是普通PCR的1 000倍。SYBR Green Ⅰ荧光定量PCR检测方法不仅能定性检测WPV,还可以进行定量检测,可用于种鸭场、种鹅场的WPV净化检测,也可用于WPV临床大量样品的快速检测。
