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
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
[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
High-molecular-weight glutenin subunits （HMW-GSs）, one class of seed storage proteins in wheat, play an important role in determining bread-making quality of flour. More and more proves support that HMW-GS- 1Bx 14 a...High-molecular-weight glutenin subunits （HMW-GSs）, one class of seed storage proteins in wheat, play an important role in determining bread-making quality of flour. More and more proves support that HMW-GS- 1Bx 14 and - 1Bx 15 subunits are strongly positively associated with good bread-making and excellent noodle-making quality. The two subunits are encoded by two genes, Glu-1Bx14 and Glu-1Bx15, which are tightly linked and located on the 1BL. Protein assay by SDS-PAGE indicated that the expression of Glu-1Bx14 gene was always much stronger than that of Glu-1By15 in the same variety. But, variation of expression level for Glu-1By15 gene existed among varieties, such as in Xiaoyan 54, Xiaoyan 6, Yanzhan 1 and Shanyou 225. We also investigated the transcription difference of Glu-1By14 and Glu-1By15 genes in Xiaoyan 54 and Shanyou 225 by semi-quantitative RT-PCR method. The Glu-1By14 always transcripts much more than the Glu-1By15. This was basically consistent with the translation difference between the two genes. Promoters of 1Bx14, 1By15, 1By8, 1Dx2 and 1Dy12 were cloned from Xiaoyan 54, Chinese Spring and Aegilops tauschii. Sequence analysis indicated that the HMW-GS genes had high homology at their promoter regions. However, significant difference existed between sequence of 1Bx14 promoter and those of other HMW-GS genes. The transient expression experiment showed that the promoter of 1By15 has lower activity than that of 1Bx14, which was consistent with their transcription level of the two genes in varieties. In addition, transient expression of the gus driven by the promoter （P2） of HMW-GS 1Dx2 gene was higher than by other HMW-GS promoters. Therefore, we constructed 1By15 gene expression vector driven by the 1Dx2 promoter, and transformed the 1By15 gene into wheat commercial variety, Jimai 20 by pollen tube method. Of 45 independent transgenic lines identified by PCR, 3 were confirmed to contain the HMW-GS 1By15 gene via Southern hybridization. The delivered 1By15 gene expressed the expected HMW-GS protein in the seeds of transgenic plants.展开更多
In this paper, the Geometry/G/1 queueing model with inter-arrival times generated by a geometric（parameter p） distribution according to a late arrival system with delayed access and service times independently distr...In this paper, the Geometry/G/1 queueing model with inter-arrival times generated by a geometric（parameter p） distribution according to a late arrival system with delayed access and service times independently distributed with distribution {gj }, j≥ 1 is studied. By a simple method （techniques of probability decomposition, renewal process theory） that is different from the techniques used by Hunter（1983）, the transient property of the queue with initial state i（i ≥ 0） is discussed. The recursion expression for u -transform of transient queue-length distribution at any time point n^＋ is obtained, and the recursion expression of the limiting queue length distribution is also obtained.展开更多
Transient expression in Tobacco is a popular way to produce recombinant proteins in plants.The design of various expression vectors,delivered into the plant by Agrobacterium,has enabled high production levels of some ...Transient expression in Tobacco is a popular way to produce recombinant proteins in plants.The design of various expression vectors,delivered into the plant by Agrobacterium,has enabled high production levels of some proteins.To further enhance expression,researchers often adapt the coding sequence of heterologous genes to the host,but this strategy has produced mixed results in Tobacco.To study the effects of different sequence features on protein yield,we compile a dataset of the yields and coding sequences of previously published expression studies of more than 200 coding sequences.We evaluate various established gene expression models on a subset of the expression studies.We find that use of tobacco codons is only moderately predictive of protein yield as informative sequence features likely extend over multiple codons.Additionally,we show that codon usage of organisms that use tobacco as a host for expression of their proteins in a similar way as the synthetic system,like viruses and agrobacteria,can be used to predict heterologous expression.Other predictive features are related to tRNA supply and demand,the inclusion of a translational ramp of codons with lower adaptation to the tRNA pool at the beginning of the coding region,and the amino acid composition of the recombinant protein.A model based on all the features achieved a correlation of 0.57 with protein yield.We believe that our study provides a practical guideline for coding sequence design for efficient expression in tobacco.展开更多
A nucleus-encoded MinE gene, designated PpMinE, from Physcomitrella patens was identified using RT-PCR. The presence of both N- and C-terminal extensions in PpMinE protein suggested its cyanobacterial origin. The tran...A nucleus-encoded MinE gene, designated PpMinE, from Physcomitrella patens was identified using RT-PCR. The presence of both N- and C-terminal extensions in PpMinE protein suggested its cyanobacterial origin. The transient expression of PpMinE using green fluorescent protein fusion in tobacco （Nicotiana tabacum L.） indicated that the PpMinE was a chloroplast-targeted protein. Overexpression of PpMinE in Escherichia coli caused division site misplacement and minicell formation, suggesting evolutionary functional conservation of MinE during plant phylogenesis. According to the phylogenetic tree, PpMinE protein has a close relationship with the highland plants, which suggests that the transfer events of MinE gene from plastid to nucleus might have occurred before the origin of the land plants.展开更多
The complete open reading frame of OsPINla was amplified through reverse transcriptase- polymerase chain reaction （RT-PCR） based on the sequence deposited in GenBank to explore the relationship between the auxin eff...The complete open reading frame of OsPINla was amplified through reverse transcriptase- polymerase chain reaction （RT-PCR） based on the sequence deposited in GenBank to explore the relationship between the auxin efflux protein OsPINla and the negative phototropism of rice roots. Sequencing results showed that the GC content of OsPINla was 65.49%. The fusion expression vector pCAMBIA-1301-OsP/N1a：：GFP containing the OsPINla gene and a coding green fluorescent protein （gfp） gene was constructed. The fusion vector was transferred into onion epidermal cells by Agrobacterium tumefaciens transformation. The transient expression of OsPINla-GFP was mainly located in the nucleus and cell membrane. Moreover, the transgenic plants were obtained by Agrobacterium-mediated genetic transformation. Molecular detection performed by using PCR and β-glucuronidase staining showed that the target construct was integrated into the genome of rice. The negative phototropic curvatures of the transgenic rice roots were higher than those of the wild type. Similarly, the expression levels of OsPINla in the transgenic plants were considerably higher than those in the wild-type plants. These results suggest that OsPINla is crucial in the negative phototropic curvature of rice roots.展开更多
Sugarcane is a prominent source of sugar and ethanol production.Genetic analysis for trait improvement of sugarcane is greatly hindered by its complex genome,long breeding cycle,and recalcitrance to genetic transforma...Sugarcane is a prominent source of sugar and ethanol production.Genetic analysis for trait improvement of sugarcane is greatly hindered by its complex genome,long breeding cycle,and recalcitrance to genetic transformation.The protoplast-based transient transformation system is a versatile and convenient tool for in vivo functional gene analysis;however,quick and effective transformation systems are still lacking for sugarcane.Here,we developed an efficient protoplast-based transformation system by optimizing conditions of protoplasts isolation and PEG-mediated transformation in S.spontaneum.The yield of viable protoplasts was approximately 1.26×107 per gram of leaf material,and the transformation efficiency of 80.19%could be achieved under the optimized condition.Furthermore,using this approach,the nuclear localization of an ABI5-like bZIPs transcription factor was validated,and the promoter activity of several putative DNase I hypersensitive sites(DHSs)was assessed.The results indicated this system can be conveniently applied to protein subcellular localization and promoter activity assays.A highly efficient S.spontaneum mesophyll cell protoplast isolation and transient transformation method was developed,and it shall be suitable for in vivo functional gene analysis in sugarcane.展开更多
Although the use of stable transformation technology has led to great insight into gene function,its application in high-throughput studies remains arduous.Agro-infiltration have been widely used in species such as Ni...Although the use of stable transformation technology has led to great insight into gene function,its application in high-throughput studies remains arduous.Agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis,but this technique does not work efficiently in other plant species,including Arabidopsis thaliana.As an efficient high-throughput transient expression system is currently lacking in the model plant species A.thaliana,we developed a method that is characterized by high efficiency,reproducibility,and suitability for transient expression of a variety of functional proteins in A.thaliana and 7 other plant species,including Brassica oleracea,Capsella rubella,Thellungiella salsuginea,Thellungiella halophila,Solanum tuberosum,Capsicum annuum,and N.benthamiana.Efficiency of this method was independently verified in three independent research facilities,pointing to the robustness of this technique.Furthermore,in addition to demonstrating the utility of this technique in a range of species,we also present a case study employing this method to assess protein–protein interactions in the sucrose biosynthesis pathway in Arabidopsis.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.
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.
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.
文摘High-molecular-weight glutenin subunits （HMW-GSs）, one class of seed storage proteins in wheat, play an important role in determining bread-making quality of flour. More and more proves support that HMW-GS- 1Bx 14 and - 1Bx 15 subunits are strongly positively associated with good bread-making and excellent noodle-making quality. The two subunits are encoded by two genes, Glu-1Bx14 and Glu-1Bx15, which are tightly linked and located on the 1BL. Protein assay by SDS-PAGE indicated that the expression of Glu-1Bx14 gene was always much stronger than that of Glu-1By15 in the same variety. But, variation of expression level for Glu-1By15 gene existed among varieties, such as in Xiaoyan 54, Xiaoyan 6, Yanzhan 1 and Shanyou 225. We also investigated the transcription difference of Glu-1By14 and Glu-1By15 genes in Xiaoyan 54 and Shanyou 225 by semi-quantitative RT-PCR method. The Glu-1By14 always transcripts much more than the Glu-1By15. This was basically consistent with the translation difference between the two genes. Promoters of 1Bx14, 1By15, 1By8, 1Dx2 and 1Dy12 were cloned from Xiaoyan 54, Chinese Spring and Aegilops tauschii. Sequence analysis indicated that the HMW-GS genes had high homology at their promoter regions. However, significant difference existed between sequence of 1Bx14 promoter and those of other HMW-GS genes. The transient expression experiment showed that the promoter of 1By15 has lower activity than that of 1Bx14, which was consistent with their transcription level of the two genes in varieties. In addition, transient expression of the gus driven by the promoter （P2） of HMW-GS 1Dx2 gene was higher than by other HMW-GS promoters. Therefore, we constructed 1By15 gene expression vector driven by the 1Dx2 promoter, and transformed the 1By15 gene into wheat commercial variety, Jimai 20 by pollen tube method. Of 45 independent transgenic lines identified by PCR, 3 were confirmed to contain the HMW-GS 1By15 gene via Southern hybridization. The delivered 1By15 gene expressed the expected HMW-GS protein in the seeds of transgenic plants.
基金This work was supported by the Scientific Research Fund of Southwestern University of Finance and Economics and the Science Foundation of Sichuan Normal University.
文摘In this paper, the Geometry/G/1 queueing model with inter-arrival times generated by a geometric（parameter p） distribution according to a late arrival system with delayed access and service times independently distributed with distribution {gj }, j≥ 1 is studied. By a simple method （techniques of probability decomposition, renewal process theory） that is different from the techniques used by Hunter（1983）, the transient property of the queue with initial state i（i ≥ 0） is discussed. The recursion expression for u -transform of transient queue-length distribution at any time point n^＋ is obtained, and the recursion expression of the limiting queue length distribution is also obtained.
基金This study was supported in part by a fellowship from the Edmond J.Safra Center for Bioinformatics at Tel-Aviv University and the Israeli cultivated meat consortium.
文摘Transient expression in Tobacco is a popular way to produce recombinant proteins in plants.The design of various expression vectors,delivered into the plant by Agrobacterium,has enabled high production levels of some proteins.To further enhance expression,researchers often adapt the coding sequence of heterologous genes to the host,but this strategy has produced mixed results in Tobacco.To study the effects of different sequence features on protein yield,we compile a dataset of the yields and coding sequences of previously published expression studies of more than 200 coding sequences.We evaluate various established gene expression models on a subset of the expression studies.We find that use of tobacco codons is only moderately predictive of protein yield as informative sequence features likely extend over multiple codons.Additionally,we show that codon usage of organisms that use tobacco as a host for expression of their proteins in a similar way as the synthetic system,like viruses and agrobacteria,can be used to predict heterologous expression.Other predictive features are related to tRNA supply and demand,the inclusion of a translational ramp of codons with lower adaptation to the tRNA pool at the beginning of the coding region,and the amino acid composition of the recombinant protein.A model based on all the features achieved a correlation of 0.57 with protein yield.We believe that our study provides a practical guideline for coding sequence design for efficient expression in tobacco.
基金This work was supported by the National Natural Science Foundation of China (No. 30470879).
文摘A nucleus-encoded MinE gene, designated PpMinE, from Physcomitrella patens was identified using RT-PCR. The presence of both N- and C-terminal extensions in PpMinE protein suggested its cyanobacterial origin. The transient expression of PpMinE using green fluorescent protein fusion in tobacco （Nicotiana tabacum L.） indicated that the PpMinE was a chloroplast-targeted protein. Overexpression of PpMinE in Escherichia coli caused division site misplacement and minicell formation, suggesting evolutionary functional conservation of MinE during plant phylogenesis. According to the phylogenetic tree, PpMinE protein has a close relationship with the highland plants, which suggests that the transfer events of MinE gene from plastid to nucleus might have occurred before the origin of the land plants.
基金supported by the grants from the National Natural Science Foundations of China(Grant Nos.31071353 and 31100197)the Anhui Provincial Natural Science Fund of Youth,China(Grant No.1308085QC50)the Fund of Provincial Excellent Young Talents in Universities and Colleges,China(Grant No.2012SQRL057)
文摘The complete open reading frame of OsPINla was amplified through reverse transcriptase- polymerase chain reaction （RT-PCR） based on the sequence deposited in GenBank to explore the relationship between the auxin efflux protein OsPINla and the negative phototropism of rice roots. Sequencing results showed that the GC content of OsPINla was 65.49%. The fusion expression vector pCAMBIA-1301-OsP/N1a：：GFP containing the OsPINla gene and a coding green fluorescent protein （gfp） gene was constructed. The fusion vector was transferred into onion epidermal cells by Agrobacterium tumefaciens transformation. The transient expression of OsPINla-GFP was mainly located in the nucleus and cell membrane. Moreover, the transgenic plants were obtained by Agrobacterium-mediated genetic transformation. Molecular detection performed by using PCR and β-glucuronidase staining showed that the target construct was integrated into the genome of rice. The negative phototropic curvatures of the transgenic rice roots were higher than those of the wild type. Similarly, the expression levels of OsPINla in the transgenic plants were considerably higher than those in the wild-type plants. These results suggest that OsPINla is crucial in the negative phototropic curvature of rice roots.
基金funded by the National Natural Science Foundation of China(3190020451 and 31771862)。
文摘Sugarcane is a prominent source of sugar and ethanol production.Genetic analysis for trait improvement of sugarcane is greatly hindered by its complex genome,long breeding cycle,and recalcitrance to genetic transformation.The protoplast-based transient transformation system is a versatile and convenient tool for in vivo functional gene analysis;however,quick and effective transformation systems are still lacking for sugarcane.Here,we developed an efficient protoplast-based transformation system by optimizing conditions of protoplasts isolation and PEG-mediated transformation in S.spontaneum.The yield of viable protoplasts was approximately 1.26×107 per gram of leaf material,and the transformation efficiency of 80.19%could be achieved under the optimized condition.Furthermore,using this approach,the nuclear localization of an ABI5-like bZIPs transcription factor was validated,and the promoter activity of several putative DNase I hypersensitive sites(DHSs)was assessed.The results indicated this system can be conveniently applied to protein subcellular localization and promoter activity assays.A highly efficient S.spontaneum mesophyll cell protoplast isolation and transient transformation method was developed,and it shall be suitable for in vivo functional gene analysis in sugarcane.
基金supported by funding from the Max Planck Society(A.R.F.and Y.Z.)the European Union’s Horizon 2020 project PlantaSYST SGA-CSA no.739582 under FPA no.664620(A.R.F.and Y.Z.).
文摘Although the use of stable transformation technology has led to great insight into gene function,its application in high-throughput studies remains arduous.Agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis,but this technique does not work efficiently in other plant species,including Arabidopsis thaliana.As an efficient high-throughput transient expression system is currently lacking in the model plant species A.thaliana,we developed a method that is characterized by high efficiency,reproducibility,and suitability for transient expression of a variety of functional proteins in A.thaliana and 7 other plant species,including Brassica oleracea,Capsella rubella,Thellungiella salsuginea,Thellungiella halophila,Solanum tuberosum,Capsicum annuum,and N.benthamiana.Efficiency of this method was independently verified in three independent research facilities,pointing to the robustness of this technique.Furthermore,in addition to demonstrating the utility of this technique in a range of species,we also present a case study employing this method to assess protein–protein interactions in the sucrose biosynthesis pathway in Arabidopsis.
