[Objective] This study aimed to increase the expression level of immun- odominant membrane protein gene （Imp） of phytoplasmas in E. coil BL21 （DE3）. [Method] On the basis of orthogonal experiment, effects of diffe...[Objective] This study aimed to increase the expression level of immun- odominant membrane protein gene （Imp） of phytoplasmas in E. coil BL21 （DE3）. [Method] On the basis of orthogonal experiment, effects of different culture conditions on recombinant bacteria E. coil BL21-pET-28a（＋）-Imp were investigated. Based on the obtained optimal culture condition, effects of different induction conditions on the ex- pression level of Imp protein were explored. The expression level of Imp fusion pro- tein was analyzed by using SDS-PAGE and Gene Tools software. [Result] The re- sults showed that the optimal conditions for culture were at 37℃, pH 7.0, with liq- uid volume of 20% and oscillation speed of 200 r/min, for induction were at 37℃ for 6 h, with initial OD600 of about 1.5 and IPTG final concentration of 0.1 mmol/L. [Conclusion] The expression level of Imp achieved 70.98 mg/L under the optimal conditions. Optimized conditions for expression of Imp fusion protein in E. coil were determined.展开更多
Genetically engineered transgenic animals and plants have proven to be extremely useful for analyzing biochemical and developmental processes.Promoters responding to chemical inducers will be powerful tools for basic ...Genetically engineered transgenic animals and plants have proven to be extremely useful for analyzing biochemical and developmental processes.Promoters responding to chemical inducers will be powerful tools for basic research in molecular biology and biotechnological applications.Various chemical inducible systems based on activation and inactivation of the target gene had been described.The transfer of regulatory elements from prokaryotes,insects,and mammals has opened new avenues to construct chemically inducible promoters that differ in their ability to regulate the temporal and spatial expression patterns,and this will dramatically increase the application of transgenic technology.This review provides an overview on regulation of gene expression,promoter activating systems,promoter inactivation systems,inducible gene over expression,and inducible anti suppression.展开更多
lsochrysis zhangfiangensis is a potential marine microalga for biodiesel production, which accumulates lipid under ni- trogen limitation conditions, but the mechanism on molecular level is veiled. Quantitative real-ti...lsochrysis zhangfiangensis is a potential marine microalga for biodiesel production, which accumulates lipid under ni- trogen limitation conditions, but the mechanism on molecular level is veiled. Quantitative real-time polymerase chain reaction （qPCR） provides the possibility to investigate the gene expression levels, and a valid reference for data normalization is an essential prerequisite for firing up the analysis. In this study, five housekeeping genes, actin （ACT）, α-tubulin （TUA）, β-tubulin （TUB）, ubiquitin （UBI）, 18S rRNA （18S） and one target gene, diacylglycerol acyltransferase （DGAT）, were used for determining the reference. By analyzing the stabilities based on calculation of the stability index and on operating the two types of software, geNorm and bestkeeper, it showed that the reference genes widely used in higher plant and microalgae, such as UBI, TUA and 18S, were not the most stable ones in nitrogen-stressed 1. zhangjiangensis, and thus are not suitable for exploring the mRNA expression levels under these experi- mental conditions. Our results show that ACT together with TUB is the most feasible internal control for investigating gene expres- sion under nitrogen-stressed conditions. Our findings will contribute not only to future qPCR studies of/. zhangfiangensis, but also to verification of comparative transcriptomics studies of the microalgae under similar conditions.展开更多
基金Supported by Science and Technology Project of Shenzhen City (JC200903180710A)~~
文摘[Objective] This study aimed to increase the expression level of immun- odominant membrane protein gene （Imp） of phytoplasmas in E. coil BL21 （DE3）. [Method] On the basis of orthogonal experiment, effects of different culture conditions on recombinant bacteria E. coil BL21-pET-28a（＋）-Imp were investigated. Based on the obtained optimal culture condition, effects of different induction conditions on the ex- pression level of Imp protein were explored. The expression level of Imp fusion pro- tein was analyzed by using SDS-PAGE and Gene Tools software. [Result] The re- sults showed that the optimal conditions for culture were at 37℃, pH 7.0, with liq- uid volume of 20% and oscillation speed of 200 r/min, for induction were at 37℃ for 6 h, with initial OD600 of about 1.5 and IPTG final concentration of 0.1 mmol/L. [Conclusion] The expression level of Imp achieved 70.98 mg/L under the optimal conditions. Optimized conditions for expression of Imp fusion protein in E. coil were determined.
文摘Genetically engineered transgenic animals and plants have proven to be extremely useful for analyzing biochemical and developmental processes.Promoters responding to chemical inducers will be powerful tools for basic research in molecular biology and biotechnological applications.Various chemical inducible systems based on activation and inactivation of the target gene had been described.The transfer of regulatory elements from prokaryotes,insects,and mammals has opened new avenues to construct chemically inducible promoters that differ in their ability to regulate the temporal and spatial expression patterns,and this will dramatically increase the application of transgenic technology.This review provides an overview on regulation of gene expression,promoter activating systems,promoter inactivation systems,inducible gene over expression,and inducible anti suppression.
基金supported by the Hundred Talent Program of the Chinese Academy of Sciences (No.A1097)National High Technology Research and Development Program ‘863’ (2012AA052101)+2 种基金Liaoning Provincial Natural Science Foundation (2012010263)Knowledge Innovation Programs of Dalian Institute of Chemical Physics,CAS (K2010A13)China Postdoctoral Science Foundation Funded Project (2014M551139)
文摘lsochrysis zhangfiangensis is a potential marine microalga for biodiesel production, which accumulates lipid under ni- trogen limitation conditions, but the mechanism on molecular level is veiled. Quantitative real-time polymerase chain reaction （qPCR） provides the possibility to investigate the gene expression levels, and a valid reference for data normalization is an essential prerequisite for firing up the analysis. In this study, five housekeeping genes, actin （ACT）, α-tubulin （TUA）, β-tubulin （TUB）, ubiquitin （UBI）, 18S rRNA （18S） and one target gene, diacylglycerol acyltransferase （DGAT）, were used for determining the reference. By analyzing the stabilities based on calculation of the stability index and on operating the two types of software, geNorm and bestkeeper, it showed that the reference genes widely used in higher plant and microalgae, such as UBI, TUA and 18S, were not the most stable ones in nitrogen-stressed 1. zhangjiangensis, and thus are not suitable for exploring the mRNA expression levels under these experi- mental conditions. Our results show that ACT together with TUB is the most feasible internal control for investigating gene expres- sion under nitrogen-stressed conditions. Our findings will contribute not only to future qPCR studies of/. zhangfiangensis, but also to verification of comparative transcriptomics studies of the microalgae under similar conditions.
