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Copper—Controllable,Site—Specific DNA Excision in transgenic plants

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Agricultural Sciences in China 2003年第6期2卷 597-601页

作者： PENG Xiang-lei, LIANG Bin, CHEN Ming, HU Yuan-lei and LIN Zhong-ping(National Key Laboratory of Protein Engineering and Plant Genetic Engineering , Peking University , Beijing 100871 , P. R. China) National Key Laboratory of Protein Engineering and Plant Genetic Engineering Peking University Beijing 100871 P. R. China

A copper-inducible, Cre-loxP recombination-mediated DNA excision system has been developed in transgenic tobacco plants. The copper inducible system derived from yeast was used for the control of the expression of the Cre recombinase. Upon copper induction, the GVS reporter gene expression unit flanked by two direct lox sites was excised from the transgenic tobacco genome. Quantitative fluorometric GUS assays, Northern blot and PCR analyses showed a high-efficient, copper-dependent and Cre-loxP mediated DNA recombination in all the tested transgenic lines. The copper inducible foreign gene excision might be of great potential in genetic control of transgenic crops.

关键词： transgenic plants Copper-inducible system MRE Cre-loxP DNA recombination

Production and identification of salt-tolerant transgenic rice plants

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Chinese Rice Research Newsletter 2001年第3期9卷 14-15页

作者： WANGHuizhong LUDezhao YanMeixian CHENShouyi PENGXuexian HUAZhihua GAOZhenyu QIANQian HUANGDanian CNRRI Hangzhou310006 LabofPlantGeneticsInst theChineseAcademyofSciencesBiejing100081China

To relieve the damage of salt to the cell, many plants accumulated some low_molecular weight organic compounds, which were called osmoprotectant, such as betaine, mannitol, and sorbitol. Therefore if the gene_encoding enzyme responsible for synthesizing the osmoprotectant was transformed into rice plants, the salt_tolerance capacity of the transgenic plants would be increased, and the enormous loss caused by salinization of soil would be reduced.

关键词： transgenic rice transgenic plants organic compounds

Transfer of CrylAc and Cry2Ab proteins from genetically engineered Bt cotton to herbivores and predators

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Insect Science 2018年第5期25卷 823-832页

作者： Michael Meissle Jorg Romeis Agroscope Research Division Agroecology and Environment Zurich Switzerland

With the cukivation of Bt cotton, the produced insecticidal Cry proteins are ingested by herbivores and potentially transferred along the food chain to natural enemies, such as predators. In laboratory experiments with Bollgard II cotton, concentrations of CrylAc and Cry2Ab were measured in Lepidoptera larvae （Spodoptera littoralis, Hello- this virescens）, plant bugs （Euschistus heros）, aphids （Aphis gossypii）, whiteflies （Bemisia tabaci）, thrips （ Thrips tabaci, Frankliniella occidentalis）, and spider mites （Tetranychus urticae）. Tritrophic experiments were conducted with caterpillars ofS. littoralis as prey and larvae of ladybird beetles （Harmonia axyridis, Adalia bipunctata） and lacewings （Chrysop- erla carnea） as predators. Immunological measurements （ELISA） indicated that herbivores feeding on Bt cotton contained 5%-50% of the Bt protein concentrations in leaves except whiteflies and aphids, which contained no or only traces of Bt protein, and spider mites, which contained 7 times more Cry 1Ac than leaves. Similarly, predators contained 1%-30% of the Cry protein concentration in prey. For the nontarget risk assessment, this indicates that Bt protein concentrations decrease considerably from one trophic level to the next in the food web, except for spider mites that contain Bt protein concentrations higher than those measured in the leaves. Exposure of phloem sucking hemipterans is negligible.

关键词： arthropod food web Bacillus thuringiensis Bt cotton Cry protein ELISA transgenic plants

Kunitz Trypsin Inhibitor： An Antagonist of Cell Death Triggered by Phytopathogens and Fumonisin B1 in Arabidopsis

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Molecular Plant 2008年第3期1卷 482-495页

作者： Jing Li Günter Brader E. Tapio Palva Viikki Biocenter Department of Biological and Environmental Sciences Division of Genetics University of Helsinki POB 56 FIN-00014 Helsinki Finland Current address: Sainsbury Laboratory John Innes Centre Colney Norwich NR4 7UH United Kingdom

Programmed cell death （PCD） is a central regulatory process in both plant development and in plant responses to pathogens. PCD requires a coordinate activation of pro-apoptotic factors such as proteases and suppressors inhibiting and modulating these processes. In plants, various caspase-like cysteine proteases as well as serine proteases have been implicated in PCD. Here, we show that a serine protease （Kunitz trypsin） inhibitor （KTI1） of Arabidopsis acts as a functional KTI when produced in bacteria and in planta. Expression of AtKTI1 is induced late in response to bacterial and fungal elicitors and to salicylic acid. RNAi silencing of the AtKTI1 gene results in enhanced lesion development after infiltration of leaf tissue with the PCD-eliciting fungal toxin fumonisin B1 （FB1） or the avirulent bacterial pathogen Pseudomonas syringae pv tomato DC3000 carrying avrB （Pst avrB）. Overexpression of AtKTI1 results in reduced lesion development after Pst avrB and FB1 infiltration. Interestingly, RNAi silencing of AtKTI1 leads to enhanced resistance to the virulent pathogen Erwinia carotovora subsp, carotovora SCC1, while overexpression of AtKTI1 leads to higher susceptibility towards this pathogen. Together, these data indicate that AtKTI1 is involved in modulating PCD in plant-pathogen interactions.

关键词： CASPASE-LIKE PROTEASES PATHOGEN ERWINIA-CAROTOVORA HYPERSENSITIVE RESPONSE SERINE-PROTEASE transgenic plants GENE-EXPRESSION SOYBEAN KUNITZ

RNA Interference-based Suppression of Phosphoenolpyruvate Carboxylase Results in Susceptibility of Rapeseed to Osmotic Stress

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Journal of Integrative Plant Biology 2010年第6期52卷 585-592页

作者： Mei Chen Yunlai Tang Jingmei Zhang Mingfeng Yang Yinong Xu Key Laboratory of Photobiology Instiiute of Botany Chinese Academy of Sciences Beijing 100093 China College of Life Science and Engineering Southwest University of Science and Technology Mianyang 621010 China

The diverse functions of phosphoenolpyruvate carboxylase （PEPCase; EC 4.***.1.31） in C3 plants are not as well understood as in C4 plants. To investigate the functions of PEPCase in C3 plants, rapeseed （Brassica napus L.） PEPCase gene （referred to as BNPE15） was silenced by the RNA interference （RNAi） technique. Under normal growth conditions, no significant difference in lipid content and fatty acid composition were found between wild-type （WT） and transgenic rapeseed plants. However, when these plants were subjected to osmotic stress induced by osmoticum polyethylene glycol （PEG-6000）, membrane permeability and membrane lipid peroxidization in roots and leaves of transgenic plants were higher than those of WT plants. It suggested that transgenic plants are more susceptible to osmotic stress than WT plants. Taken together, the results showed that the suppression of PEPCase by RNAi leads to susceptibility to osmotic stress in rapeseed, and PEPCase is involved in the response of C3 plants to environmental stress.

关键词： C3 plants plants Planting Rapeseed transgenic plants

Targeting of anti-microbial proteins to the hyphal surface amplifies protection of crop plants against Phytophthora pathogens

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Molecular Plant 2021年第8期14卷 1391-1403页

作者： Yang Zhou Kun Yang Qiang Yan Xiaodan Wang Ming Cheng Jierui Si Xue Xue Danyu Shen Maofeng Jing Brett M.Tyler Daolong Dou Key Laboratory of Plant Immunity College of Plant ProtectionAcademy for Advanced Interdisciplinary StudiesNanjing Agricultural UniversityNanjing 210095China College of Plant Protection China Agricultural UniversityBeijing 100091China Center for Genome Research and Biocomputing and Department of Botany and Plant Pathology Oregon State UniversityCorvallisOR 97331USA

Phytophthora pathogens are a persistent threat to the world's commercially important agricultural crops,including potato and soybean.Current strategies aim at reducing crop losses rely mostly on disease-resistance breeding and chemical pesticides,which can be frequently overcome by the rapid adaptive evolution of pathogens.transgenic crops with intrinsic disease resista nee offer a promising alternative and con tinue to be developed.Here,we explored Phytophthora-derived PI3P(phosphatidylinositol 3-phosphate)as a novel control target,using proteins that bind this lipid to direct secreted anti-microbial peptides and proteins(AMPs)to the surface of Phytophthora pathogens.In transgenic Nicotiana benthamiana,soybean,and potato plants,significantly enhanced resistance to different pathogen isolates was achieved by expression of two AMPs(GAFP1 or GAFP3 from the Chinese medicinal herb Gastrodia elata)fused with a PI3P-specific binding domain(FYVE).Using the soybean pathogen P.sojae as an example,we demonstrated that the FYVE domain could boost the activities of GAFPs in multiple independent assays,including those performed in vitro,in vivo,and in planta.Mutational analysis of P.sojae PI3K1 and PI3K2 genes of this pathogen confirmed that the enhanced activities of the targeted GAFPs were correlated with PI3P levels in the pathogen.Collectively,our study provides a new strategy that could be used to confer resistance not only to Phytophthora pathogens in many plants but also potentially to many other kinds of plant pathogens with unique targets.

关键词： Phytophthora PI3P AMPs transgenic plants disease resistance

Senescence-Inducible Expression of Isopentenyl Transferase Extends Leaf Life,Increases Drought Stress Resistance and Alters Cytokinin Metabolism in Cassava

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Journal of Integrative Plant Biology 2010年第7期52卷 653-669页

作者： Peng Zhang Wen-Quan Wang Gen-Liang Zhang Miroslav Kaminek Petre Dobrev Jia Xu Wilhelm Gruissem Shanghai Center for Cassava Biotechnology National Laboratory of Plant Molecular Genetics Institute of Plant Physiology & Ecology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences Shanghai 200032 China Department of Biology Plant Biotechnology ETH Zurich Universitatstrasse 2 8092 Zurich Switzerland Institute of Tropical Biosciences and Biotechnology Chinese Academy of Tropical Agricultural Science Haikou 571101 China Institute of Experimental Botany Academy of Sciences of the Czech Republic Rozvojova 263 CZ-165 02 Prague 6 Czech Republic Key Laboratory of Synthetic Biology Institute of Plant Physiology & Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China

Cassava （Manihot esculenta Crantz） sheds its leaves during growth, especially within the tropical dry season. With the production of SAG12-1PT transgenic cassava we want to test the level of leaf retention and altered cytokinin metabolism of transgenic plants via the autoregulatory senescence inhibition system. After confirmation of transgene expression by molecular analysis and phenotype examination in greenhouse plants, two transgenic plant lines, 529-28 and 529-48, were chosen for further investigation. Detached mature leaves of 529-28 plants retained high levels of chlorophyll compared with wild-type leaves after dark-induced senescence treatment. Line 529-28 showed significant drought tolerance as indicated by stay-green capacity after drought stress treatment. Field experiments proved that leaf senescence syndrome was significantly delayed in 529-28 plants in comparison with wild-type and 529-48 plants. Physiological and agronomical characterizations of these plants also revealed that the induced expression of IPT had effects on photosynthesis, sugar allocation and nitrogen partitioning. Importantly, the 529-28 plants accumulated a high level of trans-zeatin-type cytokinins particularly of corresponding storage O-glucosides to maintain cytokinin homeostasis. Our study proves the feasibility of prolonging the leaf life of woody cassava and also sheds light on the control of cytokinin homeostasis in cassava leaves.

关键词： Sheds transgenic plants Cassava Leaves Cytokinins transgenics Homeostasis Drought Senescence plants

Recombinase-mediated Gene Stacking as a Transformation Operating System

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Journal of Integrative Plant Biology 2011年第7期53卷 512-519页

作者： David W.Ow South China Botanical Garden

The current method for combining transgenes into a genome is through the assortment of independent loci, a classical operating system compatible with transgenic traits created by different developers, at different times and/or through different transformation techniques. However, as the number of transgenic loci increases over time, increasingly larger populations are needed to find the rare individual with the desired assortment of transgenic loci along with the non-transgenic elite traits. Introducing a transgene directly into a field cultivar would bypass the need to introgress the engineered trait. However, this necessitates separate transformations into numerous field cultivars, along with the characterization and regulatory approval of each independent transformation event. Reducing the number of segregating transgenic loci could be achieved if multiple traits are introduced at the same time, a preferred option if each of the many traits is new or requires re-engineering. If reengineering of previously introduced traits is not needed, then appending a new trait to an existing locus would be a rational strategy. The insertion of new DNA at a known locus can be accomplished by site- specific integration, through a host-dependent homology-based process, or a heterologous site-specific recombination system. Here, we discuss gene stacking through the use of site-specific recombinases.

关键词： site-specific recombination transgenic plants DNA integration marker removal Cre-lox

Improvement of plant abiotic stress tolerance through modulation of the polyamine pathway

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Journal of Integrative Plant Biology 2014年第2期56卷 114-121页

作者： Haitao Shi Zhulong Chan Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture Wuhan Botanical Gardenthe Chinese Academy of Sciences

Polyamines （mainly putrescine （Put）, spermidine （Spd）, and spermine （Spin）） have been widely found in a range of physiological processes and in almost all diverse environ- mental stresses. In various plant species, abiotic stresses modulated the accumulation of polyamines and related gene expression. Studies using loss-of-function mutants and transgenic overexpression plants modulating polyamine metabolic pathways confirmed protective roles of polyamines during plant abiotic stress responses, and indicated the possibility to improve plant tolerance through genetic manipulation of the polyamine pathway. Additionally, puta- tive mechanisms of polyamines involved in plant abiotic stress tolerance were thoroughly discussed and crosstalks among polyamine, abscisic acid, and nitric oxide in plant responses to abiotic stress were emphasized. Special attention was paid to the interaction between polyamine and reactive oxygen species, ion channels, amino acid and carbon metabolism, and other adaptive responses. Further studies are needed to elucidate the polyamine signaling pathway, especially polyamine-regulated downstream tar- gets and the connections between polyamines and other stress responsive molecules.

关键词： Abiotic stress crosstalk polyamine polyamine metabolic pathway transgenic plants