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Transiently Expressed Short Hairpin RNA Targeting 126 kDa Protein of Tobacco Mosaic Virus Interferes with Virus Infection

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Acta Biochimica et Biophysica Sinica 2006年第1期38卷 22-28页

作者： Ming-Min ZHAO~(1,2),De-Rong AN~(1*),Jian ZHAO~2,Guang-Hua HUANG~3,Zu-Hua HE~4,and Jiang-Ye CHEN~3 ~1 College of plant Protection,Northwest Science and Technology University of Agriculture and Forestry,Yangling 712100,china ~2 College of Agriculture,Yangtze University,Jingzhou 434025,china ~3 state key laboratory of molecular Biology,institute of Biochemistry and Cell Biology,shanghai institutes for biological sciences, chinese academy of sciences,shanghai 200031,china ~4 state key laboratory of plant molecular genetics,institute of plant physiology and ecology,shanghai institutes for biological sciences, chinese academy of sciences,shanghai 200032,china College of plant Protection Northwest Science and Technology University of Agriculture and Forestry Yang ling 712100 China College of Agriculture Yangtze University Jingzhou 434025 China state key laboratory of molecular Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China state key laboratory of plant molecular genetics Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China

RNA-interference (RNAi) silences gene expression by'guiding mRNA degradation in asequence-specific *** interfering RNA (siRNA),an intermediate of the RNAi pathway,has beenshown to be very effective in inhibiting virus infection in mammalian cells and cultured plant ***,wereport that Agrobacterium tumefaciens-mediated transient expression of short hairpin RNA (shRNA) couldinhibit tobacco mosaic virus (TMV) RNA accumulation by targeting the gene encoding the replication-asso-ciated 126 kDa protein in intact plant *** results indicate that transiently expressed shRNA efficientlyinterfered with TMV *** interference observed is sequence-specific,and time-and *** expressed shRNA corresponding to the TMV 126 kDa protein gene did not inhibit cucumbermosaic virus (CMV),an unrelated *** order to interfere with TMV accumulation in tobaccoleaves,it is essential for the shRNA constructs to be infiltrated into the same leaves as TMV *** support the view that RNAi opens the door for novel therapeutic procedures against virus *** propose that a combination of the RNAi technique and Agrobacterium-mediated transient expressioncould be employed as a potent antiviral treatment in plants.

关键词： tobacco mosaic virus 126 kDa protein RNA interference short hairpin RNA

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Expressional Analysis of an EREBP Transcription Factor Gene OsEBP-89 in Rice

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Acta Biochimica et Biophysica Sinica 2004年第1期36卷 21-26页

作者： Hui SHEN and Zong-Yang WANG( The state key laboratory of plant molecular genetics, institute of plant physiology & ecology, shanghai institutes for biological sciences,the chinese academy of sciences, shanghai 200032, china ) The state key laboratory of plant molecular genetics Institute of Plant Physiology & Ecology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences Shanghai 200032 China

OsEBP-89 gene encodes an ethylene responsive element binding protein (EREBP) transcription factor from rice (Oryza sativa). Northern blot analysis revealed that OsEBP-89 was expressed in root, stem, seeds, flowers and leaves. Histochemical assay showed that β-glucuronidase (GUS) was expressed mainly in phloem of vascular tissues of the root and stem transition region (RST), basal part of sheath roots, stem node and basal part of adventitious roots, also in endosperm of seeds in transgenic rice harboring OsEBP-89/GUS construct (pNSG). A sequence region from -279 to -97 was found to play an important role for OsEBP-89 gene expression through promoter deletion assay. The possible function of OsEBP-89 gene was discussed.

关键词： OsEBP-89 gene rice EREBP subfamily transcription factor tissue expression

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Transcriptional, post-transcriptional and post-translational regulations of gene expression during leaf polarity formation

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Cell Research 2007年第6期17卷 512-519页

作者： Lin Xu Li YangHai Huang National laboratory of plant molecular genetics, shanghai institute of plant physiology and ecology, shanghai institutes for biological sciences, chinese academy of sciences, 300 Fenglin Road, shanghai 200032, china National laboratory of plant molecular genetics Shanghai Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China Present address: IBMP CNRS Universite Louis Pasteur 67084 Strasbourg France Present address: Department of Biology University of Pennsylvania Philadelphia PA 19104 USA

Leaf morphogenesis requires the establishment of adaxial-abaxial polarity after primordium initiation from the shootapical meristem (SAM). Several families of transcription factors are known to play critical roles in promoting adaxial orabaxial leaf fate. Recently, post-transcriptional gene silencing pathways have been shown to regulate the establishmentof leaf polarity, providing novel and exciting insights into leaf development. For example, microRNAs (miR165/166)and a trans-acting siRNA (TAS3-derived tasiR-ARF) have been shown to repress the expression of several key transcrip-tion factor genes. In addition, yet another level of regulation, post-translational regulation, has been revealed recently bystudies on the role of the 26S proteasome in leaf polarity. Although our understanding regarding the molecular mecha-nisms underlying establishment of adaxial-abaxial polarity has greatly improved, there is still much that remains *** review aims to discuss recent progress, as well as the remaining questions, regarding the molecular mechanismsunderlying leaf polarity formation.

关键词： Arabidopsis leaf development polarity formation

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nifH Promoter Activity Is Regulated by DNA Supercoiling in Sinorhizobium meliloti

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Acta Biochimica et Biophysica Sinica 2005年第4期37卷 221-226页

作者： Yan-Jie LIU Biao HU Jia-Bi ZHU Shan-Jiong SHEN Guan-Qiao YU National laboratory of plant molecular genetics,institute of plant physiology and ecology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200032,china National laboratory of plant molecular genetics Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China

In prokaryotes,DNA supercoiling regulates the expression of many genes;for example,theexpression of Klebsiella pneumoniae nifLA operon depends on DNA negative supercoiling in anaerobicallygrown cells,which indicates that DNA supercoiling might play a role in gene regulation of the *** the expression of the nifH promoter in Sinorhizobium meliloti is not repressed by oxygen,itis proposed that the status of DNA supercoiling may not affect the expression of the nifH *** this hypothesis by analyzing nifH promoter activity in wild-type and gyr Escherichia coli in thepresence and absence of DNA gyrase *** results show that gene expression driven by the *** nifH promoter requires the presence of active DNA *** DNA gyrase increases thenumber of negative superhelical turns in DNA in the presence of ATP,our data indicate that negativesupercoiling is also important for nifH promoter *** study also shows that the DNA supercoiling-dependent *** nifH promoter activity is related to the trans-acting factors NtrC and NifA that *** supercoiling appeared to have a stronger effect on NtrC-activated nifH promoter activity than onNifA-activated promoter ***,these results from the *** nifH promoter model systemseem to indicate that,in addition to regulating gene expression during anaerobic signaling,DNA supercoilingmay also provide a favorable topology for trans-acting factor binding and promoter activation regardless ofoxygen status.

关键词： nifH DNA supercoiling gyrase Sinorhizobium meliloti gene expression

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Substitutions of the Conserved Gly47 Affect the CF1 Inhibitor and Proton Gate Functions of the Chloroplast ATP Synthase ε Subunit

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Acta Biochimica et Biophysica Sinica 2005年第7期37卷 453-462页

作者： Hui DONG Zhang-Lin NI Jia-Mian WEI institute of plant physiology & ecology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200032,china institute of plant physiology & ecology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200032 China

The conserved residue Gly47 of the chloroplast ATP synthase ε subunit was substituted withLeu,Arg,Ala and Glu by site-directed *** process generated the mutants εG47L,εG47R,εG47A and εG47E,*** the ε variants showed lower inhibitory effects on the soluble CF1（-ε）Ca2+-ATPase compared with wild-type ε.In reduced conditions,εG47E and εG47R had a lower inhibitoryeffect on the oxidized CF1（-ε）Ca2+-ATPase compared with wild-type ε.In contrast,εG47L and εG47Aincreased the Ca2+-ATPase activity of soluble oxidized CF1（-ε）.The replacement of Gly47 significantlyimpaired the interaction between the subunit ε and γ in an in vitro binding *** study showed thatall ε variants were more effective in blocking proton leakage from the thylakoid *** enhancedATP synthesis of the chloroplast and restored ATP synthesis activity of the reconstituted membranes to alevel that was more efficient than that achieved by wild-type ε.These results indicate that the conservedGly47 residue of the ε subunit is very important for maintaining the structure and function of the ε subunitand may affect the interaction between the ε subunit,β subunit of CF1and subunit Ⅲ of CF_o,therebyregulating the ATP hydrolysis and synthesis,as well as the proton translocation role of the subunit Ⅲ of CF_o.

关键词： chloroplast ATP synthase ε subunit GST pull-down assay site-directed mutagenesis

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Are small RNAs a big help to plants?

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Science china(Life sciences) 2009年第3期52卷 212-223页

作者： MAO YingBo,XUE XueYi & CHEN XiaoYa National key laboratory of plant molecular genetics,institute of plant physiology and ecology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200032,china National key laboratory of plant molecular genetics Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China

The discovery of RNA interference(RNAi) has augmented our knowledge of gene regulation and presents a fascinating technology that has a great potential for application in genetic analysis,disease therapy,plant protection,and many other *** this review,we will focus on the biological functions of RNAi and its application in agriculture with a brief introduction to the history of its discovery and molecular mechanism.

关键词： RNA interference gene regulation plant protection

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A Viral Protein Suppresses siRNA-directed Interference in Tobacco Mosaic Virus Infection

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Acta Biochimica et Biophysica Sinica 2005年第4期37卷 248-253页

作者： Ming-Min ZHAO De-Rong AN Guang-Hua HUANG~1 Zu-Hua HE~2 Jiang-Ye CHEN~1 College of plant Protection,North West Science and Technology University of Agriculture and Forestry,Shaanxi 712100,china ~1state key laboratory of molecular Biology,institute of Biochemistry and Cell Biology,shanghai institutes for biological sciences. chinese academy of sciences,shanghai 200031,china ~2state key laboratory of plant molecular genetics.institute of plant physiology and ecology,shanghai institutes for biological sciences.chinese academy of sciences,shanghai 200032.china College of plant Protection North West Science and Technology University of Agriculture and ForestryShaanxi 712100 China state key laboratory of molecular Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China state key laboratory of plant molecular genetics Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200032 China

plant viruses encode suppressors of post-transcriptional gene silencing (PTGS), an adaptivedefense response that limits virus replication and its spread in *** helper component proteinase (HC-Pro) of the potato virus A (PVA,genus Potyvirus) suppresses PTGS of silenced ***,the effectof HC-Pro on siRNA-directed interference in the tobacco mosaic virus (TMV) was examined by using atransient Agrobacterium tumefaciens-based delivery system in intact *** was shown that the interferenceeffect was completely blocked by co-infiltration with HC-Pro plus siRNA constructs in both systemic andhypersensitive *** the system host,all plants agro-infiltrated with HC-Pro plus siRNA constructsdisplayed the same symptoms as the negative ***,TMV RNA accumulation was found to beabundant in the upper leaves using reverse transcriptase-PCR (RT-PCR) and Northern blot *** thecontrary,plants agro-infiltrated with the siRNA construct alone were free of ***,ourstudy suggests that the transient expression of HC-Pro inhibited the siRNA-directed host defenses againstTMV infection.

关键词： siRNA HC-Pro tobacco mosaic virus suppressor

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Positive Charges on Lysine Residues of the Extrinsic 18 kDa Protein Are Important to Its Electrostatic Interaction with Spinach Photosystem Ⅱ Membranes

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Acta Biochimica et Biophysica Sinica 2005年第11期37卷 737-742页

作者： Jin-Peng GAO Zhen-Hua YONG Feng ZHANG Kang-Cheng RUAN Chun-He XU Gen-Yun CHEN institute of plant physiology & ecology.shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200032,china laboratory of Proteomics,institute of Biochemistry and Cell Biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200031,china College of sciences,Zhejiang University.Hangzhou 310029,china Graduate School of the chinese academy of sciences,shanghai 200032,china institute of plant physiology & ecology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200032 China Graduate School of the chinese academy of sciencesshanghai 200032 China College of sciences Zhejiang UniversityHangzhou 310029 China laboratory of Proteomics Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China

To determine the contribution of charged amino acids to binding with the photosystem Ⅱcomplex(PSⅡ),the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N-succinimidyl propionate(NSP)or glycine methyl ester(GME)in the presence of a water-soluble carbodiimide,*** on isoelectric point shift,4-10 and 10-14 amino groups were modified in the presence of2 and 4 mM NSP,***,3-4 carboxyl groups were modified by reaction with *** of negatively charged carboxyl groups with GME did not alter the binding activity of theextrinsic 18 kDa ***,the NSP-modified 18 kDa protein,in which the positively charged aminogroups had been modified to uncharged methyl esters,failed to bind with the PSⅡ membrane in the presenceof the extrinsic 23 kDa *** defect can not be attributed to structural or conformational alterationsimposed by chemical modification,as the fluorescence and circular dichroism spectra among native,GME-and NSP-modified extrinsic 18 kDa proteins were ***,we have concluded that the positive chargesof lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSⅡ membranes in thepresence of the extrinsic 23 kDa ***,it was found that the negative charges of carboxylgroups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSⅡmembranes.

关键词： extrinsic 18 kDa protein circular dichroism electrostatic interaction chemical modification

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Sequencing and Analysis of a Genomic Fragment Provide an Insight into the Dunaliella viridis Genomic Sequence

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Acta Biochimica et Biophysica Sinica 2006年第11期38卷 812-820页

作者： Xiao-Ming SUN~(1#) Yuan-Ping TANG~(1#) Xiang-Zong MENG~1 Wen-Wen ZHANG~1 Shan LI~1 Zhi-Rui DENG~1 Zheng-Kai XU~(1,2) Ren-Tao SONG~1* 1 shanghai key laboratory of Bio-energy Crops,School of Life sciences,shanghai University,shanghai 200444,china 2 institute of plant physiology & ecology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200032,china shanghai key laboratory of Bio-energy Crops School of Life Sciences Shanghai UniversityShanghai 200444 China institute of plant physiology & ecology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200032 China

Dunaliella is a genus of wall-less unicellular eukaryotic green *** exceptional resistancesto salt and various other stresses have made it an ideal model for stress tolerance ***,very littleis known about its genome and genomic *** this study,we sequenced and analyzed a 29,268 bpgenomic fragment from DunalieIla *** fragment showed low sequence homology to the *** the nucleotide level,only a segment with significant sequence homology to 18S rRNA *** fragment contained six putative genes,but only one gene showed significant homology at theprotein level to GenBank *** average GC content of this sequence was 51.1%,which was muchlower than that of close related green algae Chlamydomonas （65.7%）.Significant segmental duplicationswere found within this *** duplicated sequences accounted for about 35.7% of the *** amounts of simple sequence repeats （microsatellites） were found,with strong bias towards（AC）_n type （76%）.Analysis of other Dunaliella genomic sequences in the GenBank database （total 25,749bp） was in agreement with these *** sequence features made it difficult to sequence Dunaliellagenomic *** investigation should be made to reveal the biological significance of these uniquesequence features.

关键词： Dunaliella viridis genomic sequence sequence feature sequence duplication simple sequence repeat

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Isolation and functional characterization of the C-terminus of rice phos-phatidylinositol 4-kinase in vitro

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Cell Research 2003年第2期13卷 131-139页

作者： XIANG FENG KONG, ZHI HONG XU, HONG WEI XUE, National laboratory of plant molecular genetics, institute of plant physiology and ecology, shanghai institutes for biological Science (SIBS), The chinese academy of sciences Partner Group of Max-Planck-institute of molecular plant physiology (MPI-MP) on "plant molecular physiology and Signal Transduction", 300 Fenglin Road, 200032 shanghai, china National laboratory of plant molecular genetics Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Science (SIBS) The Chinese Academy of Sciences Partner Group el Max-Planck-institute of molecular plant physiology (MPI-MP) on “plant molecular physiology and Signal Transduction” Shanghai China

A partial rice (Oryza sativa L.) cDNA clone. OsPMK1c, was isolated through screening of a cDNA library constructed from tillering materials. OsPI4Klc encoded a peptide of 608 amino acids with a calculated molecular mass of 68.4 kDa. The OsPI4Klc peptide shared high homology and possessed the highly conserved domains present in most isolated cloned PI4-kinases, i.e. a lipid kinase unique (LKU) domain and a catalytic (CAT) domain. A region with similarity to pleckstrin homology (PH) domain was present in OsPI4K1c as well. Further comparison with genomic sequences in databases revealed that OsPI4K1c is located at the 3'-end of a putative rice PI 4-kinase coding gene OsPI4K1, and its coding region corresponded to the C-terminal half of OsPI4Kl protein. Twelve exons (49-562 bp in size) and 11 introns (77-974 bp in size) were identified in OsPI4K1c. The recombinant protein expressed in Escherichia coli phosphorylates phosphatidylinositol at the D4 position of the inositol ring. OsPI4K1 transcript levels were

关键词： Oryza sativa phosphatidylinositol 4-kinase.

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