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Sequence analysis of mRNA polyadenylation signals of rice genes

Chinese Science Bulletin 2006年第9期51卷 1069-1077页

作者： LU Ying1, 2. GAO Chenxi1,2.3.& HAN Bin1,3.41. National Center for Gene Research,{2., Chinese Academy of sciences, shanghai 2.02.0}, China 2. Graduate School of the Chinese Academy of sciences, Beijing 10003., China 3. institute of plant physiology and ecology,{2., Chinese Academy of sciences, shanghai 2.003., China National Center for Gene Research Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200233 China Graduate School of the Chinese Academy of sciences Beijing 100039 China institute of plant {2. and {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China

The formation of eukaryotic mRNAs involves the cleavage and polyadenylation of pre- mRNAs. To investigate the sequence requirement of putative polyadenylation signals (PASs), poly(A) sites and downstream elements (DUEs) in 3.-end-pro- cessing in rice, we compared expressed sequences tags (ESTs) with poly(A) extremity to full-length cDNA sequences and constructed a database of 12.69 pre- mRNA sequences in ?40―+40 nt surrounding the poly(A) sites, which were from 9953.genes. The al- ternative poly(A) sites were revealed in approxi- mately 2.% of mRNAs. Nearly 80% of pre-mRNAs showed stringent requirement of the YA (CA or UA) at poly (A) sites for polyadenylation. About 7.9% had the AAUAAA signals on ?40―?1 nt upstream of the poly(A) sites. Over 60% of mRNAs probably used the one- or two-base variants of AAUAAA hexamers as their PASs in 3. fragments. The single-base variants of AAUGAA revealed the high frequency in 11.5% of 3. fragments. The DUEs were detected in 90% of pre- mRNAs, especially more than half of the pre-mRNAs with multi-base variants of AAUAAA had the DUEs surrounding the poly(A) site. The location of DUE is also important for defining the cleavage site. Al- though most of the rice pre-mRNAs did not contain AAUAAA signal, the existence of downstream ele- ments ensured the efficiency of cleavage-polyade-

关键词：多聚腺苷酸序列分析 mRNA 水稻基因工程

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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 {3. & ecology, shanghai institutes for biological sciences,the Chinese Academy of sciences, shanghai 2.003., 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 -2.9 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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Agrobacterium tumefaciens-mediated transformation of rice with the spider insecticidal gene conferring resistance to leaffolder and striped stem borer

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Cell Research 2001年第2期11卷 149-155页

作者： HUANG JIAN QIU, ZHI MING WEI, HAI LONG AN, YU XIAN ZHU National Laboratory of plant Molecular Genetics, institute of plant {3. and ecology, shanghai institutes for biological sciences, Chinese Academy of sciences, shanghai 2.003., China National La National Laboratory of plant Molecular Genetics Institute of Plant Physiology and Ecology Chinese Academy of Sciences Shanghai. jqhuang@***

Immature embryos of rice varieties 'Xiushui11' and 'Chunjiang 11' precultured for 4d were infected and transformed by Agrobacterium tumefaciens strain EHA101/pExT7 (containing the spider insecticidal gene). The resistant cant were transferred onto the differentiation medium and plants were regenerated. The transformation frequency reached 56%-72. measured as numbers of Geneticin (G418)-resistant calli produced and 3.%-60% measured as numbers of transgenic plants regenerated, respectively. PCR and Southern blot analysis of transgenic plants confirmed that the T-DNA had been integrated into the rice genome. Insect bioassays using T1 transgenic plants indicated that the mortality of the leaffolder (Cnaphalocrasis medinalis) after 7d of leaf feeding reached 3.%-61% and the corrected mortality of the striped stem borer (Chilo suppressalis) after 7d of leaf feeding reached 16%-75%. The insect bioassay results demonstrated that the transgenic plants expressing the spider insecticidal protein conferred enhanced resistance to these pests.

关键词： Rice Agrobacterium tumefaciens spider insecticidal gene transgenic plant Leaffolder striped stem borer insect bioassay.

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Regeneration of plants from callus tissues of hairy roots induced by Agrobacterium rhizogenes on Alhagi pseudoalhagi

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Cell Research 2001年第4期11卷 279-284页

作者： WANG YU MEI, JIANG BO WANG, DA LUO , JING FEN JIA National Laboratory of plant Molecular Genetics, institute of plant {3. and ecology, shanghai institutes for biological sciences, Chinese Academy of sciences, shanghai 2.003., China Department of Bi institute of plant {2. and {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences.

The legume forage Alhagi pseudoalhagi was transformed by the Agrobacterium rhizogenes strain A4 using cotyledon and hypocotyl segments as infection materials. Regenerated plants were achieved from sterile calli derived from hairy roots, which occurred at or near the infection sites. The regenerated plants from hairy root were characterized by normal leaf morphology and stem growth but a shallow and more extensive root system than normal plants. Opine synthesis, PCR and Southern blot confirmed that T- DNA had been integrated into the A. pseudoalhagi genome. Acetosyringone (AS) was found to be vital for successful transformation of A. pseudoalhagi.

关键词： Alhagi pseudoalhagi Agrobacterium rhizogenes hairy root regeneration.

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Tissue Distribution and Purification of Prophenoloxidase in Larvae of Asian Corn Borer, Ostrinia furnacalis Guenée (Lepidoptera: Pyralidae)

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Acta Biochimica et Biophysica Sinica 2004年第5期36卷 360-364页

作者： Cong-Jing FENG1,2.and Wen-Jun FU1* 1institute of plant {2. and {3., shanghai institute for biological sciences, the Chinese Academy of sciences, shanghai 2.002., China 2.he GraduateSchool of the Chinese Academy of sciences, Beijing 10003., China institute of plant {2. and {3. Shanghai Institute for Biological Sciences the Chinese Academy of Sciences The Graduate School of the Chinese Academy of sciences

Using ammonium sulphate precipitation, Blue-Sepharose CL-6B, Phenyl-Sepharose CL-4B, prophenoloxidase (PPO) was isolated and purified from hemolymph of Ostrinia furnacalis larvae. This zy- mogen was a heterodimer, and composed of two subunits with the relative molecular mass ranging from 66.2. kD to 97.4 kD determined by SDS-PAGE. Western blotting and indirect immunofluorescence test showed that PPO was present in integument, hemolymph plasma and cell membrane of granular hemocytes and oenocytoids of O. furnacalis larvae.

关键词： Ostrinia furnacalis prophenoloxidase tissue distribution purification

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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 {2. & {3..shanghai institutes for biological sciences,Chinese Academy of sciences,shanghai 2.003.,China Laboratory of Proteomics,institute of Biochemistry and Cell Biology,shanghai institutes for biological sciences,Chinese Academy of sciences,shanghai 2.003.,China College of sciences,Zhejiang University.Hangzhou 3.002.,China Graduate School of the Chinese Academy of sciences,shanghai 2.003.,China institute of plant {2. & {3. Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200032 China Graduate School of the Chinese Academy of sciencesshanghai 2.00{2. 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 2.0}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 2.0}kDa ***,it was found that the negative charges of carboxylgroups of this protein did not participate in binding with the extrinsic 2.0}kDa protein associated with PSⅡmembranes.

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

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Auxin distribution and transport during embryogenesis and seed germi-nation of Arabidopsis

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Cell Research 2001年第4期11卷 273-278页

作者： NI DI AN, LING JIAN WANG, CHUN HONG DING, ZHI HONG XU, institute of plant {2. and {3., shanghai institutes for biological sciences, Chinese Academy of sciences, shanghai 2.003., China College of Bioscience, East China Normal University, Shangha 1institute of plant {2. and {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China 1institute of plant {2. and {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China 2.ollege of Bioscience East China Normal University Shanghai 200062 China 3.ollege of Life sciences Peking University Beijing 100871 China

Auxin distribution during embryogenesis and seed germination were studied with transgenic Arabidopsis plants expressing GUS gene driven by a synthetic DR5 promoter,an auxin responsive promoter. The results showed that GUS activity is higher in ends of hypophysis and cotyledon primordia of heart-, torpedo- and cotyledon-stage embryos, leaf tip area, lateral root primordia, root apex and cotyledon of young seedlings. And GUS accumulated in root apex of the seedlings grown on auxin transport inhibitor containing media. All these suggested that above-mentioned part of the organs and tissues have a higher level of auxin, and auxin polar transport inhibitor could cause the accumulation of auxin in root apex. And auxin transport inhibitor also resulted in aberration of Arabidopsis leaf pattern formation, root gravitropism and elongation.

关键词： Auxin auxin distribution auxin polar transport auxin-responsive promoter transgenic Ara- bidopsis.

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plant fertility defects induced by the enhanced expression of microRNA167

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Cell Research 2006年第5期16卷 457-465页

作者： Peng Ru~1 Lin Xu~1 Hong Ma~(2.3. Hai Huang~(1,2. ~1National Laboratory of plant Molecular Genetics,{2. institute of plant physiology and ecology,{2. institute for biological sciences,3.0 Fenglin Road,{2. 2.003.,China ~2.IBS-SJTU-PSU Joint Center for Life sciences,{2. institute of plant physiology and Ecologgy,{2. institute for biological sciences,3.0 Fenglin Road,{2. 2.003.,China ~3.epartment of Biology and the Huck institute for the Life sciences,The Pennsylvania State University,University Park,PA 16802.USA National Laboratory of plant Molecular Genetics Shanghai Institute of Plant Physiology and Ecology Shanghai Institute for Biological Sciences Shanghai China SIBS-SJTU-PSU Joint Center for Life sciences Shanghai Institute of Plant Physiology and Ecology Shanghai Institute for Biological Sciences Shanghai China Department of Biology and the Huck institute for the Life sciences The Pennsylvania State University University Park USA

The plant hormone auxin plays a critical role in regulating plant growth and *** advances havebeen made in the understanding of auxin response pathways,primarily by the characterization of auxin response mu-tants in *** addition,microRNAs (miRNAs) have been shown to be critical regulators of genes importantfor normal plant development and ***,little is known about possible interactions between miRNAsand hormonal signaling during norlnal *** we show that an Arabidopsis microRNA,miR167,whichhas a complementary sequence to a portion of the A UXIN RESPONSE FACTOR6 (ARF6) and ARF8 mRNAs,can causetranscript degradation for ARFS,but not for *** report phenotypic characterizations of3.S::MIR167b transgeniclines,and show that severe 3.S::MIR167b transgenic lines had phenotypes similar to those of an arf6 arf8 double *** transgenic phenotypes suggest that miR167 may repress ARF6 at the level of *** demonstrate that thetransgenic plants are defective in all four whorls of floral *** the transgenic flowers,filaments were abnormallyshort,anthers could not properly release pollen,and pollen grains did not *** results provide an importantlink between the miRNA-mediated regulatory pathway of gene expression and the auxin signaling network promotingplant reproductive development.

关键词： Arabidopsis auxin response factor microRNA167 floral organ pollen germination

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Overview of Rim2.Hipa transposon superfamily:Structure, distribution, transposition and utilization

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Progress in Natural Science:Materials International 2008年第4期18卷 375-379页

作者： Pingfang Tian a, Zuhua He b a College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 10002., China b shanghai institute of {2. {3. and ecology, shanghai institute for biological sciences, Chinese Academy of sciences, shanghai 2.003., China College of Life Science and Technology Beijing University of Chemical Technology Beijing 100029 China shanghai institute of {2. {3. and ecology Shanghai Institute for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China

The rice Rim2.Hipa is a unique transposon superfamily in Oryza genomes, which can be transcriptionally activated by the infection of the rice blast pathogen Magnaporthe grisea and its elicitors. Rim2.Hipa elements belong to En/Spm family, with perfect terminal inverted repeats in both ends, direct and inverted sub-terminal repeats, and 3.bp target site duplication upon insertion. Intriguingly, the typical ’CACTG’ end sequences in the terminal inverted repeats of most Rim2.Hipa elements distinguish them from previously reported CACTA elements, such as En/Spm, Tam1, and Tgm1, which harbor ’CACTA’ sequence in their terminal inverted repeats. In particular, the plentiful Rim2.Hipa elements unevenly distribute over all 12.chromosomes and make up at least 1% of the entire genome. Southern hybridization revealed the existence of Rim2.Hipa elements in all rice varieties examined and the Rim2.Hipa core sequence is unique to rice genomes. Evidence derived from sequence analysis indicated the occurrence of transposition events during the long evolution. The abundance and insertion polymorphisms suggested their applications as novel molecular marker in rice fingerprinting and fine chromo- some mapping. This paper aims at outlining the Rim2.Hipa elements with respect to their structure, distribution, transposition and utilization.

关键词： Rim2.Hipa elements Structure Distribution Transposition Utilization

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Alpha-picolinic acid,a fungal toxin and mammal apoptosis-inducing agent,elicits hypersensitive-like response and enhances disease resistance in rice

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Cell Research 2004年第1期14卷 27-33页

作者： Hai Kuo ZHANG~1, Xin ZHANG~1, Bi Zeng MAO~(2.) Qun LI~1, Zu Hua HE~(1,2.) ~1SHARF and National Laboratory of plant Molecular Genetics, shanghai {2. of plant physiology and ecology, shanghai institutes for biological sciences, Chinese Academy of sciences, shanghai 2.003., China. ~2.iotechnology {2., Zhejiang University, Hangzhou 3.002., China. 1SHARF and National Laboratory of plant Molecular Genetics Shanghai Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China. 1SHARF and National Laboratory of plant Molecular Genetics Shanghai Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China. 2.iotechnology institute Zhejiang University Hangzhou 310029 China. 1SHARF and National Laboratory of plant Molecular Genetics Shanghai Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China. 2.iotechnology institute Zhejiang University Hangzhou 310029 China.

Alpha-picolinic acid (PA), a metabolite of tryptophan and an inducer of apoptosis in the animal cell, has been reported to be a toxin produced by some of plant fungal pathogens and used in screening for disease resistant mutants. Here, we report that PA is an efficient apoptosis agent triggering cell death of hypersensitive-like response in planta. Confirmed by Fluorescence Activated Cell Sorter (FACS), rice suspension cells and leaves exhibited programmed cell death induced by PA. The PA-induced cell death was associated with the accumulation of reactive oxygen species that could be blocked by diphenylene iodonium chloride, indicating that the generation of reactive oxygen species was NADPH-oxidase dependent. We also demonstrated the induction of rice defense-related genes and subsequent resistant enhancement by PA against the rice blast fungus Magnaporthe grisea. Hence, it was concluded that the PA-stimulated defense response likely involves the onset of the hypersensitive response in rice, which also

关键词： picolinic acid cell death hypersensitive responses Magraporche grisea rice.

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