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Dissection of miRNA Pathways Using Arabidopsis Mesophyll protoplasts

Molecular Plant 2015年第2期8卷 261-275页

作者： Claudia Martinho Ana Confraria Carlos Alexandre Elias Pierre Crozet Ignacio Rubio-Somoza Detlef Weigel Elena Baena-Gonzalez Plant Stress Signaling Instituto Gulbenkian de Ciencia Rua da Quinta Grande - 6 2780-156 Oeiras Portugal Department of Molecular Biology Max Planck Institute for Developmental Biology D-72076 Tubingen Germany These authors contributed equally to this article. Present address: Sainsbury Laboratory Cambridge University Bateman Street Cambridge CB2 1LR UK

MicroRNAs （miRNAs） control gene expression mostly post-transcriptionally by guiding transcript cleavage and/or translational repression of complementary mRNA targets, thereby regulating developmental processes and stress responses. Despite the remarkable expansion of the field, the mechanisms underlying miRNA activity are not fully understood. In this article, we describe a transient expression system in Arabidopsis mesophyll protoplasts, which is highly amenable for the dissection of miRNA pathways. We show that by transiently overexpressing primary miRNAs and target mimics, we can manipulate miRNA levels and consequently impact on their targets. Furthermore, we developed a set of luciferase-based sensors for quantifying miRNA activity that respond specifically to both endogenous and overexpressed miRNAs and target mimics. We demonstrate that these miRNA sensors can be used to test the impact of putative components of the miRNA pathway on miRNA activity, as well as the impact of specific mutations, by either overexpression or the use of protoplasts from the corresponding mutants. We further show that our miRNA sensors can be used for investigating the effect of chemicals on miRNA activity. Our cell-based transient expression system is fast and easy to set up, and generates quantitative results, being a powerful tool for assaying miRNA activity in vivo.

关键词： miRNA miRNA activity miRNA sensor protoplasts Arabidopsis thaliana

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Formation and Growth of Bryopsis hypnoides Lamouroux Regenerated from Its protoplasts

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Journal of Integrative Plant Biology 2005年第7期47卷 856-862页

作者： Nai-HaoYE Guang-CeWANG Fa-ZuoWANG Cheng-KuiZENG KeyLaboratoryofExperimentalMarineBiology InstituteofOceanologytheChineseAcademyofSciencesQingdao266071China KeyLaboratoryofExperimentalMarineBiology InstituteofOceanologytheChineseAcademyofSciencesQingdao266071China／／GraduateSchooltheChineseAcademyofSciencesBeijing100039China

Abstract: Tissue culture, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and spectra analysis were used for studying the aggregation mechanism of protoplasts from Bryopsis hypnoides Lamouroux and the discrepancy between the protoplast-regenerated plants and the wild type. The aggregation of protoplasts from B. hypnoides was observed in natural seawater and artificial seawater with different pH values, and the location and mechanism of the materials causing the aggregation were also studied. Results showed that the protoplasts could aggregate into some viable spheres in natural seawater and subsequently grow into mature individuals. Aggregation of the protoplasts depended exclusively upon the pH value (6–11), and the protoplasts aggregated best at pH 8–9. Some of the extruded protoplasts were separated into two parts by centrifugation: the pellet (PO) and the supernatant (PL). The PO could aggregate in artificial seawater (pH 8.3) but not in PL. No aggregation was found in PO cultured in natural seawater containing nigericin, which can dissipate the proton gradients across the membrane. These experiments suggest that the aggregation of protoplasts is proton-gradient dependent and the materials causing the aggregation were not in the vacuolar sap, but located on the surface or inside the organelles. Furthermore, the transfer of the materials across the membrane was similar to z\pH-based translocation (δpH/TAT) pathway that occurs in the chloroplasts of higher plants and bacteria. Obvious discrepancies in both the total soluble proteins and the ratio of chlorophyll a to chlorophyll b between the regenerated B. hypnoides and the wild type were found, which may be related to the exchange of genetic material during aggregation of the organelles. In the process of development, diatom Amphora coffeaeformis Agardh attached to the protoplast aggregations, retarding their further development, and once they were removed, the aggregations immediately germinated, which showed that diatoms can affect the development of other

关键词： aggregation Bryopsis hypnoides diatoms protoplasts

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EFFECT OF T-2 TOXIN ON WHEAT LEAF protoplasts

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Chinese Science Bulletin 1990年第5期35卷 431-434页

作者：常迺滔薛应龙 Department of Biochemistry Fudan University Shanghai 200031 PRC Immunology rescarch LaboratoryShenyang Agricultural University Department of Biochemistry Fudan University Shanghai 200031 PRC

Ⅰ. INTRODUCTIONCooking was the first who succeeded in getting a large number of protoplasts by using the enzymatic method in the 1960s. Now protoplasts have become an important tool in the research of plant physiology, plant genetics, phytopathology and membrane physiology. In the research of inter-relationship between protoplasts and pathotoxin,

关键词： T-2 toxin activity of protoplasts fluorescence disease resistance.

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Factors affecting the production and regeneration of protoplasts: the case of the mycorrhizal fungus Tulasnella calospora from roots of orchids

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Forestry Studies in China 2013年第3期15卷 186-195页

作者： Li-Kun WANG Hong-Xia LIU College of Forestry Beijing Forestry University

Protoplast isolation is relevant for many different applications and has been principally used in proceduresnvolving genetic manipulation. In this study, the age of mycelium, osmotic stabilizers, enzyme, incubation temperature and incubation time were evaluated in terms of their effects on protoplast yield. The young mycelia （3 d） of Tulasnella calospora were digested for 6 h at 30℃ in a mixture of 1.2 mol·L-1 MgSO_4 ＋ 10 mmoI·L-1 K2HPO4 as the osmotic stabilizer, with a 1.0% lysing enzyme and 1.5% driselase： more than 106 protoplasts mL-1 were obtained. When collected 3y density gradient centrifugation, the concentration of protoplasts can reach 107-108 protoplasts mL-1, an amount suitable enough for experiments of transformation in fungi. For every 10_5 protoplasts, about 15-25 protoplasts can egenerate after 24-36 h cultivation in a liquid medium and after 8-10 d in an agar medium. This study produced an efficient method for protoplast production, reverting them into a typical mycelia morphology using a Tulasnella calospora solate.

关键词： mycorrhizal fungi Tulasnella calospora protoplasts osmotic stabilizer regeneration

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The protoplasts isolation,culture and shoots regeneration of broccoli(Brassica oleracea var.italica)

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华北农学报 1993年第S2期8卷 28-34页

作者：王怀名 A.Schfer-Menuhr G.Mix-Wagner

Eight F1-hybrid cultivars of broccoli were studied.We obtained cell division,celled colonies and p-calli in 5 cultivars,roots and shoots regeneration in one cultivar.The leavesof propagated plantlets in vitro were cut into 1—2mm pieces,isolated with an enzyme solutioncontaining 2% cellulase and 1%macerase on a rotary shaker（50 rpm,21℃,3h,2500 lux light）,and purified with a 0.5M sucrose solution.The purified protoplasts were placed on a drop of 1%agarose.2—3 ml liquid medium was added around the agarose drops,and all of the cultures wereincubated at 25℃ under light（4000 lux）for 16 hours.3—5 days after isolation the cell divisionwas found.About 7 days after incubation 4 multicellular colonies were formed.After 3—5 wksome p-calli were developed.When the p-calli were 2—3 mm in diameter it was transferred to asolidified medium.Once they were developed to 1 cm in diameter they were transferred on a re-generation medium.About 5 months after incubation some roots and shoots grown from the calliwere

关键词： protoplasts culture shoots regeneration broccoli( Brassica oleracea var. italica )

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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)

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Induction of Apoptosis in protoplasts and Suspension Cultures of Plant Cells

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Tsinghua Science and Technology 2000年第4期5卷 428-431页

作者：颜长辉周军陈浩明戴尧仁 Department of Biological Sciences and Biotechnology Tsinghua University Beijing 100084 China

Many studies have showed that apoptosis exists in plants. Our study shows that (1) menadione(VK3) induces apoptosis in suspension cultures of carrot cells; (2) heat shock induces apoptosis in suspension cultures of tobacco cells; and (3) ethrel induces apoptosis in carrot protoplasts. Some important indications of apoptosis were observed, including DNA laddering, TUNEL-positive reaction, condensation and degradation of nuclei.[

关键词： apoptosis protoplasts suspension culture carrot tobacco menadione ethrel heat shock

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Escherichia coli Protoplast Fusion and Screening of High L-Lysine-Producing Strain

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Agricultural Biotechnology 2022年第3期11卷 127-132,135页

作者： Zirui WANG Junqing WANG College of Biological Engineering Qilu University of Technology(Shandong Academy of Sciences)Jinan 250300China

[Objectives]Protoplast fusion of two parental strains with weak L-lysine production ability was carried out to obtain new fusion strains with strong L-lysine production ability.[Methods]The effects of bacterial age,lysozyme concentration,enzymolysis temperature and time on the protoplast formation rate and regeneration rate were investigated by single factor experiments.On this basis,with the protoplast formation rate as an index,the protoplast preparation process was optimized by an orthogonal experiment.[Results]Bacterial age and enzymolysis time had a greater impact on the protoplast formation rate,followed by enzymolysis temperature and lysozyme concentration.The optimal process for the preparation of L-lysine-producing Escherichia coli protoplasts was to prepare parental protoplasts from bacterial cells cultured for 15 h in the late logarithmic growth phase by enzymolysis with 0.8 mg/ml lysozyme at 37℃for 180 min and promote fusion with PEG6000.In order to facilitate the screening of fusion protoplasts,the empty plasmids p ET-28a and p ET-Duet were transformed into L-lysine-producing E.coli,respectively,and strains p ET-28a-lys01 and p ET-Duet-lys01 were obtained.Fusion strains were then obtained through protoplast fusion.Double-resistance KA1-10were screened on plates containing kanamycin and ampicillin,and a high-yielding fusion strain KA8,which produced L-lysine,was screened by fermentation experiments finally.[Conclusions]The results of this study provide a reference method for further improving the yield of L-lysine and other amino acid strains.

关键词： Escherichia coli L-lysine protoplasts Cell fusion Microbial breeding

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Menadione-induced apoptosis and its mechanism in plants

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Chinese Science Bulletin 2000年第4期45卷 350-354页

作者： SUN Yingli ZHOU Jun DAI Yaoren ZHAI Zhonghe College of Life Sciences Peking UniversityBeijing 100871China Biology Department Tsinghua UniversityBeijing 100084China

Menadione can induce apoptosis in tobacco protoplasts, Typical characteristics are detected including the condensation of chromatin, the formation of apoptotic bodies and the degradation of genomic DNA into 'DNA ladder'. Specific DNase is activated during this process. Ca2+ and Mg2+ are necessary for its activation, while 2n2+ and EDTA (Ethylenediaminetetraacetic acid) can inhibit its activation. The fragmentation of DNA and lamin can be inhibited by DEVD (Ac-Asp -Glu- Val- Asp-aldehyde). The fragmentation of lamin can also be inhibited by PMSF (Phenylmethylsulfonyl fluoride) and CH (Cyclohexinide). These results show that activation of specific DNase and proteases is involved in menadion-induced apoptosis in plants.

关键词： tobacco protoplasts menadione apoptosis DNA fragmentation lamin protease.

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Construction of PEG-mediated Genetic Transformation and Gene Knockout System in Fusarium oxysporum f. sp.cubense Tropic Race 4

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Agricultural Biotechnology 2020年第1期9卷 15-17,21页

作者： Lei ZHANG Yan GUO Yunyue WANG Weihua TANG Sijun ZHENG State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan Ministry of Education Key Laboratory of Agriculture Biodiversity for Plant Disease ManagementYunnan Agricultural UniversityKunming 650201China Agricultural Environment and Resources Institute Yunnan Academy of Agricultural SciencesKunming 650205China Institute of Plant Physiology and Ecology Shanghai Institutes for Biological SciencesChinese Academy of SciencesShanghai 200032China Bioversity International Kunming 650205China

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense tropic race 4(Foc TR4), is a typical vascular and soil-borne disease which has significantly threatened the sustainable development of banana industry. In order to reveal the infection process and pathogenesis of Foc TR4, the young mycelia(66.7 mg/ml) of wild-type strain of Foc TR4(WT-Foc TR4) cultured for 18-20 h were lysed with enzyme mixture for protoplast formation, which consisted of 25 mg/ml driselase, 0.4 mg/ml chitinase, 15 mg/ml lysing enzyme and 1.2 mol/L potassium chloride. The resulted protoplasts of 2×10~7 cells/ml were used to test the efficiency of transformation mediated by polyethylene glycol, and up to 9 transformants per microgram of DNA were obtained. AmCyan, RFP and YFP genes were stably transferred into the WT-Foc TR4, separately, using the protoplast transformation system. The gene FoOCH1 encoding α-1, 6-mannosyltransferase in the WT-Foc TR4 was knocked out using the split-marker recombination technology. The genetic transformation and gene knockout system in this pathogen lays a foundation for the study of functional genomics and plant-pathogen interactions.

关键词： Fusarium wilt of banana Fusarium oxysporum f.sp.cubense protoplasts Transformation Split-marker recombination

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