The Agrobacterium-mediated transient expression system with conventional binary vectors is well established in tobacco leaves,while the same system applied to tomato leaflets has relatively low expression efficiency.H...The Agrobacterium-mediated transient expression system with conventional binary vectors is well established in tobacco leaves,while the same system applied to tomato leaflets has relatively low expression efficiency.However,impacts of the leaf age,inoculation method and incubation condition after Agrobacterium infiltration on transient protein expression efficiency are seldom investigated.In this study,we optimize Agrobacterium-mediated transient expression system using conventional binary vectors to achieve the high efficiency of target gene expression in tomato leaflets.We transiently express GFP and a nucleus-localized gene SlUVI4 fused with GFP in detached 10-,20-,and 30-day-old leaflets.The cutting points of leaflets are embedded in MS medium after the Agrobacterium-mediated vacuum infiltration,and all leaflets are kept in the dark before use.The 10-and 30-day-old leaflets have more damage than 20-day-old leaflets after the infiltration.展开更多
The genomic DNA sequence encoding soybean 24 kDa oleosin and its promoter were cloned andanalyzed for investigation of the potentials of the oleosin acted as a carrier forproduction of recombinant proteins in plant. T...The genomic DNA sequence encoding soybean 24 kDa oleosin and its promoter were cloned andanalyzed for investigation of the potentials of the oleosin acted as a carrier forproduction of recombinant proteins in plant. The -300 box, GA-rich, G-box, SEF-3, SEF-4, RY box, ABA box, CAn and TATA box were found in the upstream region of the soybeanoleosin gene, which shows the functional oleosin promoter available. Homology comparisonreveals that the soybean 24 kDa oleosin shares the highest identity with the soybeanoleosin isoform A (U09118, GenBank), reaching to 98.4% in nucleotide. A soybean oleosin-hirudin fusion gene driven by the oleosin promoter was constructed and inserted intoplant binary expression vector. The intact tobacco plantlets were transformed by meansof vacuum infiltration approach, with the Agrobacterium tumefaciens harboring the abovevector. The transient correct expression of oleosin-hirudin fusion gene was identifiedby SDS/PAGE, western blotting and enterokinase treatment.展开更多
This study was carried out to investigate the transfection effect of exogenous gene into plant protoplast cell mediated by polyethylenimine （PEI） nanovector, based on PEI gene delivery system in the field of medical...This study was carried out to investigate the transfection effect of exogenous gene into plant protoplast cell mediated by polyethylenimine （PEI） nanovector, based on PEI gene delivery system in the field of medical science. PEI/DNA complexes were prepared by using PEI polymer to bind the plant expression plasmid, pCMl205-GFPn. The ability of PEI combining and protecting DNA was investigated by agarose gel electrophoresis retardation assay. The surface characteristics of PEI/DNA complexes were observed with transmission electron microscope. The transfection efficiency of Arabidopsis thaliana protoplasts mediated by PEI/DNA complexes at different N/P ratios was analyzed based on observation of transient expression of green fluorescent protein with confocal laser scanning microscope. PEI could bind and condense DNA, and form stable 100-200 nm PEI/DNA complexes when the proportion of PEl and DNA is in the range of 5：1-1：4. Transfection efficiency of PEI/DNA complexes increased with N/P ratios in range of N/P〈5 and reached the highest at N/P=5, and began to decrease beyond N/P〉5 as higher toxicity to cells. The transfection efficiency of PEI/DNA complexes at N/P=5 was higher than PEG. This study confirmed that PEI nanovector could effectively mediate foreign gene entering into A. thaliana protoplast cell to obtain transient expression, which may be developed as a hopeful and novel transgenic method combined with plant protoplast regeneration.展开更多
Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodoph...Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are unknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were con-structed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5’- and 3’-flanking regions (Tub5’ and Tub3’) up- and down-stream of β-glucuronidase (GUS) gene (gusA), respectively, into pA, a derivative of pCAT?3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3’. These con-structs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5’-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when com-pared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35 S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.展开更多
We constructed an expression cassette of the organophosphorus pesticide degrading(opal)gene under the control of the E8 promoter.Then opd was transformed into tomato fruit using an agroinfiltration transient expressio...We constructed an expression cassette of the organophosphorus pesticide degrading(opal)gene under the control of the E8 promoter.Then opd was transformed into tomato fruit using an agroinfiltration transient expression system.β-Glucuronidase(GUS)staining,reverse transcription-polymerase chain reaction(RT-PCR),wavelength scanning,and fluorescent reaction were performed to examine the expression of the opd gene and the hydrolysis activity on coumaphos of organo-phosphorus hydrolase(OPH)in tomato fruit.The results show that the agroinfiltrated tomato fruit-expressed OPH had the maximum hydrolysis activity of about 11.59 Uhng total soluble protein.These results will allow us to focus on breeding transgenic plants that could not only enhance the degrading capability of fruit and but also hold no negative effects on pest control when spraying organophosphorus pesticides onto the seedlings in fields.展开更多
Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple,easy to perform,and achieve gene expression rapidly.This study investigated the factors affe...Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple,easy to perform,and achieve gene expression rapidly.This study investigated the factors affecting transient gene expression efficiency in citrus by observing the cryo-sectioning of leaf samples under a laser confocal microscope.These factors included the composition of the infiltration buffer,the Agrobacterium cell density,the leaf development stage,the incubation temperature,and plant genotype.The highest transient expression level of yellow fluorescent protein（YFP）was detected in Mexican lime（Citrus aurantifolia）on the third day after the intermediate-aged leaves were infiltrated with the improved infiltration buffer 1（15 mmol L^-1 2-（N-morpholino）ethanesulfonic acid,10 mmol L^-1 MgCl2,and 200μmol L^-1acetosyringone）,which had an optical density of 0.8 and was incubated at 22°C.Additionally,this transient expression assay was applied to other citrus genotypes.Of note,trifoliate orange（Poncirus trifoliata）and kumquat（Fortunella obovate）had higher expression efficiency than other six genotypes of the Citrus genus.Our study provides research basis for the selection of optimization strategies in transient gene expression and improves the method for available genome investigation in citrus.展开更多
The utility of artificial microRNAs（amiRNAs） to induce loss of gene function has been reported for many plant species,but expression efficiency of the different amiRNA constructs in different transgenic plants was l...The utility of artificial microRNAs（amiRNAs） to induce loss of gene function has been reported for many plant species,but expression efficiency of the different amiRNA constructs in different transgenic plants was less predictable,In this study,expressions of amiRNAs through the gene backbone of Arabidopsis miR168a were examined by both Agrobacterium-mediated transient expression and stable plant genetic transformation.A corresponding trend in expression of amiRNAs by the same amiRNA constructs between the transient and the stable expression systems was observed in the experiments.Plant genetic transformation of the constructs that were highly expressible in amiRNAs in the transient agro-infiltration assays resulted in generation of transgenic lines with high level of amiRNAs.This provides a simple method for rapid and effective selection of amiRNA constructs used for a time-consuming genetic transformation in plants.展开更多
DNA expression constructs containing hGH gene directed by bovine αsl-casein gene regulatory sequences were injected into mouse heart or mammary glands in vivo. hGH expression was readily detected by radioimmunoassay ...DNA expression constructs containing hGH gene directed by bovine αsl-casein gene regulatory sequences were injected into mouse heart or mammary glands in vivo. hGH expression was readily detected by radioimmunoassay in milk of the nursing mice throughout lactation. Our results demonstrated the value of this simple and rapid approach in screening gene constructs and identifying transcription regulation sequences in vivo prior to the generation of transgenic animals. For the First time we report the successful expression in mammary gland via intracardial injection.展开更多
The principle and the basic steps of the transient assay system for the functional assessment of early response genes on the powdery mildew infected barley or wheat leaves were summarized in brief. The development of ...The principle and the basic steps of the transient assay system for the functional assessment of early response genes on the powdery mildew infected barley or wheat leaves were summarized in brief. The development of this technology and its extensive application were reviewed. Future studies on this approach were recommended in this paper.展开更多
Study on the transient expression of GUS gene at different growing stage of Chlorellaellipsoidea using high velocity microprojectiles, the effects of osmosis, the distance between nicroprojec-tile and target cell, bom...Study on the transient expression of GUS gene at different growing stage of Chlorellaellipsoidea using high velocity microprojectiles, the effects of osmosis, the distance between nicroprojec-tile and target cell, bombardment times, are reported in this paper. The results showed that C. ellip-soidea in exponential phase has higer level of transient expression and that treatment with osmosis can im-prove the GUS transient expression notably. The effect of distance or bombardment times was not ob-served.展开更多
Background Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional ...Background Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional genes are related to agronomic traits. However, the functional validation of these genes is hindered by time-consuming and inefficient genetic transformation methods. Thus, establishing a transient transformation system of high efficiency is necessary for cotton genomics.Results To improve the efficiency of transient transformation, we used the protoplasts isolated from the etiolated cotyledon as recipient. The enzymatic digestion buffer comprised 1.5%(w/v) cellulase, 0.75%(w/v) macerozyme, and 1% hemicellulase, osmotically buffered with 0.4 mol·L^(-1) mannitol. After 5 h of dark incubation at 25℃, uniform cotton protoplasts were successfully isolated with a yield of 4.6 × 10^(6) protoplasts per gram(fresh weight) and 95% viability. We incubated 100 μL protoplasts(2.5 × 10^(5)·m L^(-1)) with 15 μg plasmid in the solution of 0.4 mol·L^(-1) mannitol and 40% PEG 4000 for 15 min, ultimately achieving an optimal transient transfection efficiency of 71.47%.Conclusions This transient system demonstrated effective utility in cellular biology research through successful applications in subcellular localization analyses, bimolecular fluorescence complementation(Bi FC) verification, and prime editing vector validation. Through systematic optimization, we established an efficient and expedited protoplast-based transient transformation system and successfully applied this platform to cotton functional genomics studies.展开更多
Genome editing is an unprecedented technological breakthrough but low plant regeneration frequencies and genotype dependence hinder its implementation for crop improvement. Here, we found that transient expression of ...Genome editing is an unprecedented technological breakthrough but low plant regeneration frequencies and genotype dependence hinder its implementation for crop improvement. Here, we found that transient expression of a complex of the growth regulators TaGRF4 and TaGIF1(TaGRF4-TaGIF1) increased regeneration and genome editing frequency in wheat. When we introduced synonymous mutation in the miR396 target site of TaGRF4, the resulting complex(mTaGRF4-TaGIF1) performed better than original TaGRF4-TaGIF1. Use of m TaGRF4-TaGIF1 together with a cytosine base editor targeting TaALS resulted in 2-9-fold increases in regeneration and transgene-free genome editing in 11 elite common wheat cultivars. Therefore, m TaGRF4-TaGIF1 will undoubtedly be of great value in crop improvement and especially in commercial applications, since it greatly increased the range of cultivars available for transformation.展开更多
A method for transient gene expression was developed for western white pine(WWP,Pinus monticola Dougl.ex D.Don)using reporter gene uidA encodingβ-glucuronidase(GUS).GUS was transiently expressed in cross sections of ...A method for transient gene expression was developed for western white pine(WWP,Pinus monticola Dougl.ex D.Don)using reporter gene uidA encodingβ-glucuronidase(GUS).GUS was transiently expressed in cross sections of primary and secondary needles,cotyledons,and current and second year stems of WWP via vacuum-infiltration with Agrobacterium tumefaciens.Histochemical assays of cross sections of secondary needles showed stronger blue color indicating GUS expression at day 1 and 2 than on other days post agroinfiltration(dpa).GUS activity expressed inside WWP cells was confirmed using light microscopy.In fluorometric assays,GUS expression was high at 1 dpa and lasted until 4 dpa in detached secondary needles,while similarly high expression levels only lasted until 2 dpa in attached secondary needles then dropped significantly.Although the length of GUS-staining zones varied among different WWP organs and between growth and dormant seasons,all tested WWP tissues using the protocol had high levels of transient GUS expression.Thus,heterologous candidate genes or endogenous silencing can be expressed in various WWP tissues or organs using this agroinfiltration approach.The current protocol for efficient transient gene expression will aid functional genomics study of WWP and its pathogens and related conifer species.展开更多
The ability to deliver nonviral DNA directly into animals would provide major practical and economic benefits. To pursue this potential, as a preliminary approach, the expression in mammary epithelial cells with nonvi...The ability to deliver nonviral DNA directly into animals would provide major practical and economic benefits. To pursue this potential, as a preliminary approach, the expression in mammary epithelial cells with nonviral DNA encoding HBsAg and hEPO was investigated by in vivo delivery directly into mammary gland. Either the bovine αs1 casein HBsAg or the WAP hEPO gene construct was introduced into mammary glands of dose by mammary artery injection. A single injection of 10 11 copies of DNA construct was implemented for a doe. Transient expression of the constructs in milk collected from milk letdown by induction up to 19 d after injection was measured with an hEPO ELISA kit from Boehringer Mannheim (Cat. No.1 693 417) for rhEPO, and a HBsAg ELISA kit (Abbott Lab, USA) for rHBsAg. The treated animals showed mean concentration of recombinant molecules at 16 μg/L for rHBsAg, and 2 mg/L for rhEPO in milk.展开更多
Three transfection reagents, Lipofectamine® 2000, TransIT-PRO® and linear 25 kDa polyethylenimine were evaluated for transient expression of enhanced green fluorescent protein in Chinese hamster ovary ce...Three transfection reagents, Lipofectamine® 2000, TransIT-PRO® and linear 25 kDa polyethylenimine were evaluated for transient expression of enhanced green fluorescent protein in Chinese hamster ovary cells. TransIT-PRO® was found to be more efficient under the examined conditions, but comes at an increased cost compared to the widely used PEI.展开更多
Foot-and-mouth disease is a highly contagious disease that produces severe economic losses in the livestock industry. This disease is being controlled by the use of an inactivated vaccine. However, the use of recombin...Foot-and-mouth disease is a highly contagious disease that produces severe economic losses in the livestock industry. This disease is being controlled by the use of an inactivated vaccine. However, the use of recombinant empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production. A plasmid containing the capsid precursor P12A and protease 3C sequences of foot-and-mouth disease virus (FMDV) was constructed and used to compare transient and stable expression in mammalian cells. When BHK-21 cells were transfected with the recombinant vector, protease 3C cleaved the capsid precursor P12A into the structural proteins VP0, VP1 and VP3. A sucrose gradient demonstrated that the structural proteins assembled into different subviral particles. Attempts to generate a stable cell line only allowed isolating low-level-expressing clones, probably due to the effect of protease 3C on the cells. Moreover, the recombinant protein yield achieved in transient expression assays was much higher than the one achieved in stable expression assays. Results indicate that mammalian cells are a good strategy to produce recombinant FMDV subviral particles. However, the alternative approach of transient gene expression in scalable systems should be used instead of the standard method that involves the generation of a stable cell line.展开更多
Polyethylene glycol (PEG)-mediated DNA transformation for transient gene expression in protoplasts and Agrobacterium tumefaciens-mediated transformation in lower epidermis of leaves are readily available in several pl...Polyethylene glycol (PEG)-mediated DNA transformation for transient gene expression in protoplasts and Agrobacterium tumefaciens-mediated transformation in lower epidermis of leaves are readily available in several plant species. In the study, these two versatile tools were used in rapeseed. A simple and efficient method was established for isolating protoplasts from rapeseed cotyledons and leaves, and found that cotyledons might be better than true leaves. Transient expression analysis showed that yellow fluorescent protein (YFP) and luciferase (LUC) could be expressed in rapeseed protoplasts. Moreover,GUS histochemical assays indicated that Agrobacterium-mediated DNA transient expression was achievable only in lower epidermis of rapeseed cotyledons and expression signal was the highest on the 5th day after injection with the bacterial suspension (OD600=0.8).These methods might provide valuable tools for rapid functional gene analysis in rapeseed.展开更多
in order to clarify the pesible role of the bcl-2 gene imolved in the cell death Program,and the relatiouship of glutamate receptors with bcl-2 gene expressin, this study examied the expression of bcl-2 gene protein...in order to clarify the pesible role of the bcl-2 gene imolved in the cell death Program,and the relatiouship of glutamate receptors with bcl-2 gene expressin, this study examied the expression of bcl-2 gene protein, the neuronal status of apoptosis and the effects of MK-801 using immunohistochemistry and in situ terminal.labelling methods after 30 min of.middle cerebral artery(MCA) occlusion and followed by 24 h of reperfusion. The presence of bcl-2 gene protein increased in the ipeilateral hemisphere of ischaemis espeially in the MCA territory MK-801 enhanced the expresion of the bcl-2 gene protein. No DNA fragmentation was detected in this experiment. In conclusion. bcl-2 gene activity increased during transient focal ischaemia, and was potentiated by MK MK801, which may be an endogenous protective mechanism .against ischaemic apoptosis. Apoptosis wasnot detected after tranient focal ischoemia. for 30 min rollowed by 24 h of reperfusiou.展开更多
[Objective] This study was conducted to investigate cloning and expression of Pun1 gene controlling pungency of pepper. [Method] With Capsicum annuum L as a material, the cDNA sequence of Capunl gene was obtained, wit...[Objective] This study was conducted to investigate cloning and expression of Pun1 gene controlling pungency of pepper. [Method] With Capsicum annuum L as a material, the cDNA sequence of Capunl gene was obtained, with a total length of 1 457 bp, coding 440 amino acids. [Result] Phylogenetic analysis showed that Capunl was closest to Pun1 of C. chinense, with a genetic distance of 0.019 3. Plant expression vector pCAM-Punl-GFP was constructed and transformed into to- bacco, and it was found that the protein coded by fusion gene Punl：：GFP was lo- cated on cell membrane. Prokaryotic expression vectors were constructed, and by SDS-PAGE and Western Blot detection, an induced protein with a molecular weight of 63 ku was obtained. It was found by real-time fluorescence quantitative expres- sion that Pun1 gene was expressed at the highest level 30 d after flowering, de- creased then, and could not be detected substantially 40 and 45 d after flowering. [Conclusion] This study provides information and reference for molecular regulation mechanism of Pun1 gene.展开更多
基金support of Taishan Scholar Foundation of Shandong Province(tsqn201909073,tsqn201812034)National Natural Science Foundation of China(31872951)。
文摘The Agrobacterium-mediated transient expression system with conventional binary vectors is well established in tobacco leaves,while the same system applied to tomato leaflets has relatively low expression efficiency.However,impacts of the leaf age,inoculation method and incubation condition after Agrobacterium infiltration on transient protein expression efficiency are seldom investigated.In this study,we optimize Agrobacterium-mediated transient expression system using conventional binary vectors to achieve the high efficiency of target gene expression in tomato leaflets.We transiently express GFP and a nucleus-localized gene SlUVI4 fused with GFP in detached 10-,20-,and 30-day-old leaflets.The cutting points of leaflets are embedded in MS medium after the Agrobacterium-mediated vacuum infiltration,and all leaflets are kept in the dark before use.The 10-and 30-day-old leaflets have more damage than 20-day-old leaflets after the infiltration.
基金supported by a grant from the National High Tech R&D Program(863 Program)of China(2001AA2121).
文摘The genomic DNA sequence encoding soybean 24 kDa oleosin and its promoter were cloned andanalyzed for investigation of the potentials of the oleosin acted as a carrier forproduction of recombinant proteins in plant. The -300 box, GA-rich, G-box, SEF-3, SEF-4, RY box, ABA box, CAn and TATA box were found in the upstream region of the soybeanoleosin gene, which shows the functional oleosin promoter available. Homology comparisonreveals that the soybean 24 kDa oleosin shares the highest identity with the soybeanoleosin isoform A (U09118, GenBank), reaching to 98.4% in nucleotide. A soybean oleosin-hirudin fusion gene driven by the oleosin promoter was constructed and inserted intoplant binary expression vector. The intact tobacco plantlets were transformed by meansof vacuum infiltration approach, with the Agrobacterium tumefaciens harboring the abovevector. The transient correct expression of oleosin-hirudin fusion gene was identifiedby SDS/PAGE, western blotting and enterokinase treatment.
基金supported by the National High Technology R&D Program of China (2006AA10A203)the Genetically Modified Organisms Breeding Major Projects, Ministry of Agriculture, China (2009ZX09010-006B)
文摘This study was carried out to investigate the transfection effect of exogenous gene into plant protoplast cell mediated by polyethylenimine （PEI） nanovector, based on PEI gene delivery system in the field of medical science. PEI/DNA complexes were prepared by using PEI polymer to bind the plant expression plasmid, pCMl205-GFPn. The ability of PEI combining and protecting DNA was investigated by agarose gel electrophoresis retardation assay. The surface characteristics of PEI/DNA complexes were observed with transmission electron microscope. The transfection efficiency of Arabidopsis thaliana protoplasts mediated by PEI/DNA complexes at different N/P ratios was analyzed based on observation of transient expression of green fluorescent protein with confocal laser scanning microscope. PEI could bind and condense DNA, and form stable 100-200 nm PEI/DNA complexes when the proportion of PEl and DNA is in the range of 5：1-1：4. Transfection efficiency of PEI/DNA complexes increased with N/P ratios in range of N/P〈5 and reached the highest at N/P=5, and began to decrease beyond N/P〉5 as higher toxicity to cells. The transfection efficiency of PEI/DNA complexes at N/P=5 was higher than PEG. This study confirmed that PEI nanovector could effectively mediate foreign gene entering into A. thaliana protoplast cell to obtain transient expression, which may be developed as a hopeful and novel transgenic method combined with plant protoplast regeneration.
文摘Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are unknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were con-structed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5’- and 3’-flanking regions (Tub5’ and Tub3’) up- and down-stream of β-glucuronidase (GUS) gene (gusA), respectively, into pA, a derivative of pCAT?3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3’. These con-structs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5’-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when com-pared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35 S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.
基金Project supported by the National Key Technology R&D Program of China(No.2007BAD59B06)the International Science and Technology Cooperation Program of China(No.2007DFA31260)
文摘We constructed an expression cassette of the organophosphorus pesticide degrading(opal)gene under the control of the E8 promoter.Then opd was transformed into tomato fruit using an agroinfiltration transient expression system.β-Glucuronidase(GUS)staining,reverse transcription-polymerase chain reaction(RT-PCR),wavelength scanning,and fluorescent reaction were performed to examine the expression of the opd gene and the hydrolysis activity on coumaphos of organo-phosphorus hydrolase(OPH)in tomato fruit.The results show that the agroinfiltrated tomato fruit-expressed OPH had the maximum hydrolysis activity of about 11.59 Uhng total soluble protein.These results will allow us to focus on breeding transgenic plants that could not only enhance the degrading capability of fruit and but also hold no negative effects on pest control when spraying organophosphorus pesticides onto the seedlings in fields.
基金financed by the National Natural Science Foundation of China (30900972, 31572111)the Special Found for Agro-scientific Research in the Public Interest, China (201203076-06)the Graduate Innovative Projects of Hunan Province, China (CX2013B290)
文摘Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple,easy to perform,and achieve gene expression rapidly.This study investigated the factors affecting transient gene expression efficiency in citrus by observing the cryo-sectioning of leaf samples under a laser confocal microscope.These factors included the composition of the infiltration buffer,the Agrobacterium cell density,the leaf development stage,the incubation temperature,and plant genotype.The highest transient expression level of yellow fluorescent protein（YFP）was detected in Mexican lime（Citrus aurantifolia）on the third day after the intermediate-aged leaves were infiltrated with the improved infiltration buffer 1（15 mmol L^-1 2-（N-morpholino）ethanesulfonic acid,10 mmol L^-1 MgCl2,and 200μmol L^-1acetosyringone）,which had an optical density of 0.8 and was incubated at 22°C.Additionally,this transient expression assay was applied to other citrus genotypes.Of note,trifoliate orange（Poncirus trifoliata）and kumquat（Fortunella obovate）had higher expression efficiency than other six genotypes of the Citrus genus.Our study provides research basis for the selection of optimization strategies in transient gene expression and improves the method for available genome investigation in citrus.
基金supported by the Ministry of Education of China and Agriculture and Agri-Food Canada(MOE-AAFC) PhD student research program
文摘The utility of artificial microRNAs（amiRNAs） to induce loss of gene function has been reported for many plant species,but expression efficiency of the different amiRNA constructs in different transgenic plants was less predictable,In this study,expressions of amiRNAs through the gene backbone of Arabidopsis miR168a were examined by both Agrobacterium-mediated transient expression and stable plant genetic transformation.A corresponding trend in expression of amiRNAs by the same amiRNA constructs between the transient and the stable expression systems was observed in the experiments.Plant genetic transformation of the constructs that were highly expressible in amiRNAs in the transient agro-infiltration assays resulted in generation of transgenic lines with high level of amiRNAs.This provides a simple method for rapid and effective selection of amiRNA constructs used for a time-consuming genetic transformation in plants.
文摘DNA expression constructs containing hGH gene directed by bovine αsl-casein gene regulatory sequences were injected into mouse heart or mammary glands in vivo. hGH expression was readily detected by radioimmunoassay in milk of the nursing mice throughout lactation. Our results demonstrated the value of this simple and rapid approach in screening gene constructs and identifying transcription regulation sequences in vivo prior to the generation of transgenic animals. For the First time we report the successful expression in mammary gland via intracardial injection.
基金supported by the National High Tech Research and Development Program,China(863 Program,222092).
文摘The principle and the basic steps of the transient assay system for the functional assessment of early response genes on the powdery mildew infected barley or wheat leaves were summarized in brief. The development of this technology and its extensive application were reviewed. Future studies on this approach were recommended in this paper.
文摘Study on the transient expression of GUS gene at different growing stage of Chlorellaellipsoidea using high velocity microprojectiles, the effects of osmosis, the distance between nicroprojec-tile and target cell, bombardment times, are reported in this paper. The results showed that C. ellip-soidea in exponential phase has higer level of transient expression and that treatment with osmosis can im-prove the GUS transient expression notably. The effect of distance or bombardment times was not ob-served.
基金supported by Biological Breeding of Early Maturing and Disease Resistant Cotton Varieties (NO.2023ZD04041)the Project of China Agriculture Research System (Grant No. CARS-15-06)+2 种基金Natural Science Foundation of Henan Province (Grant No. 232300421041 and 222300420382)National Natural Science Foundation of China (Grant No. U21 A20213)the Central Public-interest Scientific Institution Basal Research Fund (Grant No. 1610162023017 and 1610162023028)。
文摘Background Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional genes are related to agronomic traits. However, the functional validation of these genes is hindered by time-consuming and inefficient genetic transformation methods. Thus, establishing a transient transformation system of high efficiency is necessary for cotton genomics.Results To improve the efficiency of transient transformation, we used the protoplasts isolated from the etiolated cotyledon as recipient. The enzymatic digestion buffer comprised 1.5%(w/v) cellulase, 0.75%(w/v) macerozyme, and 1% hemicellulase, osmotically buffered with 0.4 mol·L^(-1) mannitol. After 5 h of dark incubation at 25℃, uniform cotton protoplasts were successfully isolated with a yield of 4.6 × 10^(6) protoplasts per gram(fresh weight) and 95% viability. We incubated 100 μL protoplasts(2.5 × 10^(5)·m L^(-1)) with 15 μg plasmid in the solution of 0.4 mol·L^(-1) mannitol and 40% PEG 4000 for 15 min, ultimately achieving an optimal transient transfection efficiency of 71.47%.Conclusions This transient system demonstrated effective utility in cellular biology research through successful applications in subcellular localization analyses, bimolecular fluorescence complementation(Bi FC) verification, and prime editing vector validation. Through systematic optimization, we established an efficient and expedited protoplast-based transient transformation system and successfully applied this platform to cotton functional genomics studies.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, XDA24020102 and XDA24010402)the National Natural Science Foundation of China (31788103 and 31971370)the Chinese Academy of Sciences (QYZDY-SSW-SMC030)
文摘Genome editing is an unprecedented technological breakthrough but low plant regeneration frequencies and genotype dependence hinder its implementation for crop improvement. Here, we found that transient expression of a complex of the growth regulators TaGRF4 and TaGIF1(TaGRF4-TaGIF1) increased regeneration and genome editing frequency in wheat. When we introduced synonymous mutation in the miR396 target site of TaGRF4, the resulting complex(mTaGRF4-TaGIF1) performed better than original TaGRF4-TaGIF1. Use of m TaGRF4-TaGIF1 together with a cytosine base editor targeting TaALS resulted in 2-9-fold increases in regeneration and transgene-free genome editing in 11 elite common wheat cultivars. Therefore, m TaGRF4-TaGIF1 will undoubtedly be of great value in crop improvement and especially in commercial applications, since it greatly increased the range of cultivars available for transformation.
文摘A method for transient gene expression was developed for western white pine(WWP,Pinus monticola Dougl.ex D.Don)using reporter gene uidA encodingβ-glucuronidase(GUS).GUS was transiently expressed in cross sections of primary and secondary needles,cotyledons,and current and second year stems of WWP via vacuum-infiltration with Agrobacterium tumefaciens.Histochemical assays of cross sections of secondary needles showed stronger blue color indicating GUS expression at day 1 and 2 than on other days post agroinfiltration(dpa).GUS activity expressed inside WWP cells was confirmed using light microscopy.In fluorometric assays,GUS expression was high at 1 dpa and lasted until 4 dpa in detached secondary needles,while similarly high expression levels only lasted until 2 dpa in attached secondary needles then dropped significantly.Although the length of GUS-staining zones varied among different WWP organs and between growth and dormant seasons,all tested WWP tissues using the protocol had high levels of transient GUS expression.Thus,heterologous candidate genes or endogenous silencing can be expressed in various WWP tissues or organs using this agroinfiltration approach.The current protocol for efficient transient gene expression will aid functional genomics study of WWP and its pathogens and related conifer species.
文摘The ability to deliver nonviral DNA directly into animals would provide major practical and economic benefits. To pursue this potential, as a preliminary approach, the expression in mammary epithelial cells with nonviral DNA encoding HBsAg and hEPO was investigated by in vivo delivery directly into mammary gland. Either the bovine αs1 casein HBsAg or the WAP hEPO gene construct was introduced into mammary glands of dose by mammary artery injection. A single injection of 10 11 copies of DNA construct was implemented for a doe. Transient expression of the constructs in milk collected from milk letdown by induction up to 19 d after injection was measured with an hEPO ELISA kit from Boehringer Mannheim (Cat. No.1 693 417) for rhEPO, and a HBsAg ELISA kit (Abbott Lab, USA) for rHBsAg. The treated animals showed mean concentration of recombinant molecules at 16 μg/L for rHBsAg, and 2 mg/L for rhEPO in milk.
文摘Three transfection reagents, Lipofectamine® 2000, TransIT-PRO® and linear 25 kDa polyethylenimine were evaluated for transient expression of enhanced green fluorescent protein in Chinese hamster ovary cells. TransIT-PRO® was found to be more efficient under the examined conditions, but comes at an increased cost compared to the widely used PEI.
文摘Foot-and-mouth disease is a highly contagious disease that produces severe economic losses in the livestock industry. This disease is being controlled by the use of an inactivated vaccine. However, the use of recombinant empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production. A plasmid containing the capsid precursor P12A and protease 3C sequences of foot-and-mouth disease virus (FMDV) was constructed and used to compare transient and stable expression in mammalian cells. When BHK-21 cells were transfected with the recombinant vector, protease 3C cleaved the capsid precursor P12A into the structural proteins VP0, VP1 and VP3. A sucrose gradient demonstrated that the structural proteins assembled into different subviral particles. Attempts to generate a stable cell line only allowed isolating low-level-expressing clones, probably due to the effect of protease 3C on the cells. Moreover, the recombinant protein yield achieved in transient expression assays was much higher than the one achieved in stable expression assays. Results indicate that mammalian cells are a good strategy to produce recombinant FMDV subviral particles. However, the alternative approach of transient gene expression in scalable systems should be used instead of the standard method that involves the generation of a stable cell line.
基金supported by the National KeyBasic Research Program of China (2015CB150200)the Natural Science Foundation of Hubei Province (2016CFB286)
文摘Polyethylene glycol (PEG)-mediated DNA transformation for transient gene expression in protoplasts and Agrobacterium tumefaciens-mediated transformation in lower epidermis of leaves are readily available in several plant species. In the study, these two versatile tools were used in rapeseed. A simple and efficient method was established for isolating protoplasts from rapeseed cotyledons and leaves, and found that cotyledons might be better than true leaves. Transient expression analysis showed that yellow fluorescent protein (YFP) and luciferase (LUC) could be expressed in rapeseed protoplasts. Moreover,GUS histochemical assays indicated that Agrobacterium-mediated DNA transient expression was achievable only in lower epidermis of rapeseed cotyledons and expression signal was the highest on the 5th day after injection with the bacterial suspension (OD600=0.8).These methods might provide valuable tools for rapid functional gene analysis in rapeseed.
文摘in order to clarify the pesible role of the bcl-2 gene imolved in the cell death Program,and the relatiouship of glutamate receptors with bcl-2 gene expressin, this study examied the expression of bcl-2 gene protein, the neuronal status of apoptosis and the effects of MK-801 using immunohistochemistry and in situ terminal.labelling methods after 30 min of.middle cerebral artery(MCA) occlusion and followed by 24 h of reperfusion. The presence of bcl-2 gene protein increased in the ipeilateral hemisphere of ischaemis espeially in the MCA territory MK-801 enhanced the expresion of the bcl-2 gene protein. No DNA fragmentation was detected in this experiment. In conclusion. bcl-2 gene activity increased during transient focal ischaemia, and was potentiated by MK MK801, which may be an endogenous protective mechanism .against ischaemic apoptosis. Apoptosis wasnot detected after tranient focal ischoemia. for 30 min rollowed by 24 h of reperfusiou.
基金Supported by College Student Innovation Fund Project of Jilin University(2015821243)~~
文摘[Objective] This study was conducted to investigate cloning and expression of Pun1 gene controlling pungency of pepper. [Method] With Capsicum annuum L as a material, the cDNA sequence of Capunl gene was obtained, with a total length of 1 457 bp, coding 440 amino acids. [Result] Phylogenetic analysis showed that Capunl was closest to Pun1 of C. chinense, with a genetic distance of 0.019 3. Plant expression vector pCAM-Punl-GFP was constructed and transformed into to- bacco, and it was found that the protein coded by fusion gene Punl：：GFP was lo- cated on cell membrane. Prokaryotic expression vectors were constructed, and by SDS-PAGE and Western Blot detection, an induced protein with a molecular weight of 63 ku was obtained. It was found by real-time fluorescence quantitative expres- sion that Pun1 gene was expressed at the highest level 30 d after flowering, de- creased then, and could not be detected substantially 40 and 45 d after flowering. [Conclusion] This study provides information and reference for molecular regulation mechanism of Pun1 gene.
