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transient expression of Exogenous Gene into Plant Cell Mediated by PEI Nanovector

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Agricultural Sciences in China 2011年第6期10卷 820-826页

作者： LI Ying CUI Hai-xin SONG Yu LI Yao HUANG Jin-li Institute of Agricultural Environment and Sustainable Development Chinese Academy of Agricultural Sciences Beijing 100081 P.R. China

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.

关键词： polyethylenimine gene nanovector plant cell transient expression Arabidopsis thaliana protoplast

A Highly Efficient Agrobacterium-Mediated Method for transient Gene expression and Functional Studies in Multiple Plant Species

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Plant Communications 2020年第5期1卷 129-140页

作者： Youjun Zhang Moxian Chen Beata Siemiatkowska Mitchell Rey Toleco Yue Jing Vivien Strotmann Jianghua Zhang Yvonne Stahl Alisdair R.Fernie Center of Plant Systems Biology and Biotechnology 4000 PlovdivBulgaria Max-Planck-Institut fur Molekulare Pflanzenphysiologie Am Muhlenberg 114476 Potsdam-GolmGermany Department of Biology Hong Kong Baptist Universityand State Key Laboratory of AgrobiotechnologyThe Chinese University of Hong KongShatinHong Kong Institute for Developmental Genetics Heinrich Heine UniversityDusseldorfGermany

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.

关键词： transient expression agro-infiltration subcellular localization protein-protein interaction

Efficient gusA transient expression in Porphyra yezoensis Protoplasts Mediated by Endogenous Beta-tubulin Flanking Sequences

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Journal of Ocean University of China 2007年第1期6卷 21-25页

作者： GONG Qianhong YU Wengong DAI Jixun LIU Hongquan XU Rifu GUAN Huashi and PAN Kehou College of Medicine and Pharmaceutics Ocean University of ChinaQingdao 266003P.R.China College of Marine Life Sciences Ocean University of ChinaQingdao 266003P.R.China College of Fisheries Ocean University of ChinaQingdao 266003P.R.China

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.

关键词： Porphyra yezoensis transient expression protoplasts beta-tubulin promoter β-glucuronidase(GUS)

Cloning of Soybean 24 kDa Oleosin Gene and Its transient expression as a Carrier for Foreign Protein

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Agricultural Sciences in China 2004年第5期3卷 321-329页

作者： XUMiao-yun LIUDe-hu LIGang-qiang BiotechnologyResearchInstitute ChineseAcademyofAgriculturalSciencesBeijing100081P.R.China

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.

关键词： Oleosin Carrier for foreign protein transient expression Promoter

transient expression of organophosphorus hydrolase to enhance the degrading activity of tomato fruit on coumaphos

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Transient expression of organophosphorus hydrolase to enhanc...

作者：赵杰宏赵德刚 MOE Key Laboratory of Green Pesticide and Agricultural Bioengineering Guizhou Key Laboratory of Agricultural Bioengineering Guizhou University Guiyang 550025China

We constructed an expression cassette of the organophosphorus pesticide degrading(opd) 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 fluores- cent 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 U/mg 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.

关键词： Bioremediation E8 promoter Organophosphorus hydrolase（OPH） transient expression

transient expression一词应如何翻译?

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中国细胞生物学学报 1998年第1期 51-51页

作者：刘定干中国科学院上海生物化学研究所研究员

在有关细胞基因表达调控的国外文献中,transientexpression一词是经常出现的。通常此词表示暂时性的基因表达,如几分钟、几小时、几天等。这一词的汉语译法,目前尚未统一,有“瞬时表达”(金冬雁等翻译、侯云德教授校的《分子克隆实验指... 详细信息

在有关细胞基因表达调控的国外文献中,transientexpression一词是经常出现的。通常此词表示暂时性的基因表达,如几分钟、几小时、几天等。这一词的汉语译法,目前尚未统一,有“瞬时表达”(金冬雁等翻译、侯云德教授校的《分子克隆实验指南》即用此译法)“一过性表达”、“暂时性表达”等。而“瞬时表达”一词用得最广。

关键词：译法拉丁文 transient expression 一过性翻译

expression of restriction endonuclease Pac I using tobacco transient expression system

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Expression of restriction endonuclease Pac I using tobacco t...

2018中国遗传学会第十次全国会员代表大会暨学术讨论会

作者： Shuang Huang Weining Jian Peng Zuo Guozhen Zhang Jieying Fan Zhengyi Wei Shaochen Xing Institute of Agricultural Biotechnology Jilin Academy of Agricultural Sciences School of life science Jilin Normal Uninversity

Restriction endonucleases are indispensable tool enzymes in genetic engineering and molecular biology research. Most of the restriction endonucleases on the market are recombinant enzymes expressed, isolated and purified in Escherichia coli, but some of them cannot be expressed in E. coli and therefore are directly extracted and purified from the original host bacterial cells where these they were discovered. Purchase of these enzymes usually requires a customization, which lead to a long time for delivery and high price. Plant transient expression system is an efficient expression platform for foreign proteins with a variety of modification and secondary（tertiary） structures to ensure the biological activity. In this study, expression parameters were optimized from four aspects, namely, plant growth condition, time point for infection, infection method and parameters of infection, in two tobacco species, Nicotiana babacum and N. benthamiana with tobacco mosaic virus（TMV） used as the transient expression vector and green fluorescent protein gene（GFP） used as the reporter. With the optimal parameters, we expressed a gene encoding a restriction endonuclease Pac I, PacIR, which from Candidatus Uhrbacteria and sequence optimized according to tobacco codon usage bias. SDS-PAGE and Western analysis showed that the target protein was expressed and preliminary activity analysis showed that the recombinant Pac I had restriction DNase activity. Further isolation and purification of recombinant Pac I and activity analysis are in progress.

关键词： transient expression DNA endonuclease tobacco mosaic virus（TMV） Pac I

In vitro and in vivo transient expression in Insect Cells Mediated by the Cationic Liposome DDAB/DOPE

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In vitro and in vivo Transient Expression in Insect Cells Me...

中国蚕学会第三届青年学术研讨会暨浙江省第二届青年学术论...

作者： Qing Li Xiao, Ya Jing Zhou, Zhi Fang Zhang, Jia Lu HeKey Laboratory of Silkworm Biotechnology, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangsu, 212018, P. R. China

＜正＞Cationlc liposomes complexed with DNA have been extensively utilized for the delivery of reporter ortherapeutic genes both in culture and Jn vivo.We investigated and determined the optimum conditions of a cationic liposome,composed of dimethyldioctadecylammonium bromide（DDAB）and deoleoyl phosphatidylethanolamine（DOPE）,mediated areporter plasmld expressing luciferase into insect cell lines（Sf-21 and Bm-N）and silkworm larvae.Together the datademonstrated that Bombyx mori nuclear polyhedrosis virus（BmNPV）genomic DNA（128 kb）was successfully transfectedinto Bm-5 cells using this liposome.These results suggest that DDAB/DOPE liposome will be useful as delivery agents forgene transfer to insecet cells both in vitro and in vivo.

关键词： Cationic liposome transient expression insect cells silkworm larvae

aFGF expressed in plants by transient expression system

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aFGF expressed in plants by transient expression system

东北三省及内蒙古地区遗传学研究进展学术研讨会

作者： WANG Xing-zhi,LIU Jing-ying,YANG Li-ping,FAN Ya-jun,LI Liang,WANG Hong-wei,LI Wei,WANG Lei, LUAN Ming,ZHU Xiao-juan(Northeast Normal University,Changchun 130024,China) 东北师范大学

Acidic fibroblast growth factor(aFGF) plays an important role in morphogenesis,angiogenesis and wound healing and is therefore of potential medical interest.Three technologies have been applied in transient expression... 详细信息

关键词： aFGF plant as bioreactor transient expression