Rice MYB Protein OSMYB5 Specifically Binds to the AACA Motif Conserved among Promoters of Genes for Storage Protein Glutelin

Binding analyses revealed that the AAC--A sequence in glutelingene promoters is the target site of OSMYB5 protein and thatboth the distal and proximal AACA motifs are recognized by thisprotein. These results suggest that the OSMYB5 protein functionsas a trans-acting factor for glutelin gene expression. (Received November 25, 1997; Accepted February 18, 1998)     

The Spirulina platensis Adenylate Cyclase Gene, cyaC, Encodes a Novel Signal Transduction Protein

A cyaC gene encoding an adenylate cyclase of the filamentouscyanobacterium Spirulina platensis was se-quenced. The predictedamino acid sequence of the C-ter-minal region of cyaC is similarto the catalytic domains of adenylate cyclases in other cyanobacteriaand eukaryotes. The sequences of other regions are similar tothose of proteins consisting of the bacterial two-componentsignal transduction system: the sensory kinase and the responseregulator. The predicted gene product of cyaC contains, fromthe N-terminal end, a receiver domain of the response regulatorprotein (Rl), a domain similar to the ETR1 of Arabi-dopsis thaliana,a transmitter domain of the sensory kinase protein, a receiverdomain of the response regulator protein (R2), and a catalyticdomain of adenylate cyclase. The cyaC gene was expressed asan affinity-tagged protein in Escherichia coli, and the recombinantprotein was purified. The purified protein had adenylate cyclaseactivity which was activated by Mn²+. The results of Westernblotting using an anti-CyaC antiserum and the S. platensis cellextract confirmed that cyaC gene is expressed in S. platensis (Received February 27, 1997; Accepted April 26, 1997)     

B型烟粉虱和温室白粉虱热激蛋白90基因（hsp90）的全长cDNA克隆与系统发育分析

B型烟粉虱Bemisia tabaci (Gennadius) biotype B和温室白粉虱Trialeurodes vaporariorum均为全球普遍发生的重要害虫。本研究以其他昆虫热激蛋白90基因（hsp90）保守区域设计兼并引物扩增两种粉虱hsp90中间片段, 然后利用RACE技术获得全长cDNA。温室白粉虱hsp90全长cDNA的开放性阅读框2 166 bp, 编码722个氨基酸; 烟粉虱hsp90全长cDNA的开放性阅读框2 160 bp, 编码720个氨基酸。两种粉虱HSP90的完整氨基酸序列相似性高达92.94％, 并均具有定义HSP90家族签名序列的5个氨基酸保守区域和末尾基序“MEEVD”。通过real-time PCR技术, 探测到两个基因在mRNA水平上皆能高温诱导表达。采用昆虫纲所有完整HSP90氨基酸序列进行Kimura双参数遗传距离分析并构建NJ进化树, 结果显示hsp90在昆虫纲低级阶元水平和高级阶元水平系统进化上能得到一个较理想结果。本研究结果为B型烟粉虱和温室白粉虱抗逆适应性研究提供基础, 并进一步验证保守的功能基因hsp90可以作为研究生物系统发育的手段之一     

Intergenic transcripts containing a novel human cytochrome P450 2C exon 1 spliced to sequences from the CYP2C9 gene

The cytochrome P450 2C (CYP2C) gene locus was found to includea novel exon 1 sequence with high similarity to the canonicalexon 1 of CYP2C18. Rapid amplification of cDNA ends (RACE) andPCR amplifications of human liver cDNA revealed the presenceof several intergenic species containing the CYP2C18 exon 1–likesequence spliced to different combinations of exonic and intronicsequences from the CYP2C9 gene. One splice variant was foundto have an open reading frame starting at the canonical translationinitiation codon of the CYP2C18 exon 1–like sequence.Another variant consisted of the nine typical CYP2C9 exons splicedafter the CYP2C18 exon 1–like sequence through a segmentof CYP2C9 5' flanking sequences. Moreover, analysis of bacterialartificial chromosome (BAC) clones revealed that the CYP2C18exon 1–like sequence was located in the intergenic regionbetween the CYP2C19 and CYP2C9 genes. The finding that a solitaryexon is spliced with sequences from a neighboring gene may beinterpreted as representing a general evolutionary mechanismaimed at using the full expression potential of a cell's genomicinformational content.     

东亚飞蝗中肠几丁质酶基因的克隆、序列分析及组织定位

通过RACE方法,克隆了东亚飞蝗Locusta migratoria manilensis (Meyen)几丁质酶基因 (LmChi)cDNA全序列 (GenBank 登录号：EF092841)。获得的cDNA全长1 604 bp,其中可读框1 452 bp, 编码483个氨基酸。推测其氨基酸序列与18家族昆虫几丁质酶有较高的相似性。与其他几丁质酶一样,东亚飞蝗几丁质酶序列也包含一个信号肽、一个几丁质酶活性位点、一个碳端丝氨酸富集区和一个几丁质结合域。半定量RT-PCR研究表明,LmChi基因只在东亚飞蝗不同发育阶段的中肠组织中表达,而在东亚飞蝗体壁、前肠和后肠均没有发现LmChi基因的转录。     

The Influences of Two Plant Nuclear Matrix Attachment Regions (MARs) on Gene Expression in Transgenic Plants

Nuclear matrix attachment regions (MARs) are thought to influencegene expression by anchoring active chromatin to the proteinaceousnuclear matrix. In this study, two plant DNA fragments withstrong MAR activity were selected and tested for their effectson expression of a linked reporter gene in transgenic tobacco.One MAR was isolated from the 5' flanking region of a pea vicilingene previously reported to be expressed in a copy number-dependentmanner in transgenic tobacco. A second MAR was isolated fromthe genome of Arabidopsis thaliana by preselection for autonomouslyreplicating sequence (ARS) activity in yeast. Flanking copiesof the A. thaliana MAR stimulated median reporter gene expressionin transgenic plants by five to ten fold. Neither MAR significantlyreduced the variation in transgene expression between individualtransformants, or conferred copy number-dependence in gene expression. (Received July 24, 1997; Accepted November 10, 1997)     

The Gene Encoding Flavanone 3-Hydroxylase is Expressed Normally in the Pale Yellow Flowers of the Japanese Morning Glory Carrying the speckled Mutation Which Produce Neither Flavonol nor Anthocyanin but Accumulate Chalcone, Aurone and Flavanone

The Japanese morning glory carrying the recessive mutable speckledallele with the dominant speckled-activator bears colorlessflowers with fine and round colored spots distributed over thecorolla whereas the plant without the speckled-activator producespale yellow flowers. Previous chemical analysis has indicatedthat a mutation in the gene for flavanone 3-hydroxylase (F3H)is a likely candidate for the speckled allele. However, theF3HmRNA without sequence alteration accumulates normally inthe pale yellow flowers, indicating that the speckled alleleis neither the F3H gene nor a regulatory gene acting on theF3H gene expression. (Received April 4, 1997; Accepted June 2, 1997)     

Expression of Plasma Membrane Water Channel Genes under Water Stress in Nicotiana excelsior

Deduced amino acid sequences encoded by the cDNAs related tothe MIP gene family from Nicotiana excelsior were characterized.Phylogenetic characterization of the products of correspondinggenes named NeMip1, NeMip2, and NeMip3 strongly suggested thatthey are water channel proteins localized in the plasma membrane.Organ specificity of the gene expression was examined in leaves,roots, and reproductive organs. NeMip1 was expressed in rootsand reproductive organs; however, it was hardly detectable inleaves. Two other genes, NeMip2 and NeMip3, were expressed inall of organs examined. mRNA accumulation from the genes wasinvestigated in leaves under salt- and drought-stresses. Theresults demonstrated that mRNA accumulation from all three genesincreased under salt- and drought-stresses within one day. However,they showed different accumulation patterns. In addition totheir up-reg-ulation under salt- and drought-stresses, dailychanges in NeMip2 and NeMip3 mRNA accumulation was observedunder unstressed conditions in leaves. (Received May 2, 1997; Accepted September 3, 1997)     

Hydroxypyruvate Reductase with a Carboxy-Terminal Targeting Signal to Microbodies is Expressed in Arabidopsis

Five Arabidopsis EST cDNA clones of hydroxypyruvate reductase(HPR), a photorespiratory enzyme in leaf peroxisomes, were sequenced.Deduced amino acid sequences revealed that HPR in Arabidopsiscontained the carboxy-terminal targeting signal to microbodies.Nucle-otide sequence analysis showed that the cDNA with thelongest insert contained an open reading frame of 1,158 bp whichencoded a polypeptide with 386 amino acids with a calculatedmolecular mass of 42,251 Da. A Southern blot analysis suggestedthat the Arabidopsis HPR gene, like that of the pumpkin HPRgene, exists as a single copy. Two kinds of pumpkin HPR mRNAmight be produced from a single gene by alternative splicing,but the structure of the genomic DNA indicated that the ArabidopsisHPR gene did not undergo alternative splicing. We detected apolypeptide with a molecular mass of 42 kDa in green leavesof Arabidopsis using an HPR-specific antibody. Immunoelectronmicroscopy revealed that Arabidopsis HPR protein was exclusivelylocalized in leaf peroxisomes in green leaves. These resultsindicate that HPR is expressed in a form with a carboxy-terminaltargeting signal to microbodies and is localized in microbodiesin Arabidopsis, suggesting that the differences in the genestructure and the regulation of gene expression of HPR are probablydue to species-specific differences in plants. (Received November 11, 1996; Accepted February 1, 1997)     

Molecular Cloning of Plant Spermidine Synthases

Four cDNAs for spermidine synthase (SPDS), which converts thediamine putrescine to the higher polyamine spermidine usingdecarboxylated S-adenosylmethionine as the co-factor, were isolatedfrom Nicotiana sylvestris, Hyoscyamus niger, and Arabidopsisthaliana. When the N. sylvestris SPDS cDNA was expressed ina SPDS-deficient E. coli mutant, the recombinant protein showedhigh SPDS activity, but did not have any spermine synthase activity.The plant SPDSs have molecular masses of about 34 kDa, possessthe co-factor binding motifs which have been proposed for S-adenosylmethionine,and are more homologous in amino acid sequence to tobacco putrescineN-methyltransferase (PMT) than to SPDSs from mammals and E.coli. The SPDS gene is expressed in root, stem, and leaf inN. sylvestris, whereas the PMT gene is expressed only in root.The potential evolution of plant SPDS and PMT, and their evolutionaryrelationships with animal SPDS are discussed. (Received September 3, 1997; Accepted November 5, 1997)     

Mapping of ribosomal DNA and (TTAGGG)n telomeric sequence by FISH in the bivalve Patinopecten yessoensis (Jay, 1857)

The chromosome set of Patinopecten yessoensis (Jay, 1857) wascharacterized using Giemsa staining, DAPI staining and fluorescencein situ hybridization (FISH) with three repetitive DNA probes[18S–28S rDNA, 5S rDNA and telomeric (TTAGGG)_n]. DAPIstaining showed that AT-rich regions were located on the centromereof almost all chromosomes and interstitial banding was not observed.FISH showed that 18S–28S rDNA spread over the short armsof two subtelocentric chromosome pairs and 5S rDNA was locatedon the long arm of one subtelocentric chromosome pair. SequentialFISH demonstrated that 18S–28S and 5S rDNA were locatedon different chromosomes. FISH also showed that the vertebratetelomeric sequence (TTAGGG)_n was located on both ends of eachchromosome and no interstitial signals were detected. Sequential18S–28S rDNA and (TTAGGG)_n FISH indicated that repeatedunits of the two multicopy families were closely associatedon the same chromosome pair. (Received 4 January 2007; accepted 1 September 2007)     

Structure and expression of a cDNA encoding zeaxanthin epoxidase, isolated from a wilt-related tomato (Lycopersicon esculentum Mill.) library

Zeaxanthin epoxldase (ZE) catalyses two early steps in the abscisicacid (ABA) biosynthetic pathway. The sequence of a cDNA cloneencoding ZE from Nicotiana plumbaginifolia was reported In 1996and represented the first DNA sequence data on an ABA biosyntheticenzyme. The N. plumbaginifolia cDNA has been used to providea heterologous probe to isolate a ZE cDNA from tomato (Lycopersiconesculentum Mill.). DNA and amino acid sequence differences areconsidered in relation to putative functional domans withinthe enzyme. The results of northern analysis in tomato are discussedin relation to the effects of water stress on ZE mRNA levels. Key words: ABA biosynthesis, zeaxanthin epoxidase, tomato     

Cloning and Characterization of High-CO2-Specific cDNAs from a Marine Microalga, Chlorococcum littorale, and Effect of CO2 Concentration and Iron Deficiency on the Gene Expression

Two cDNA clones exclusively induced under an extremely high-CO₂concentration (20%) were isolated from Chlorococcum littoraleby differential screening and named HCR (high-CO₂ response)1 and 2, respectively. The amino acid sequence of the proteinencoded by HCR2 exhibited homology to the gp91-phox protein,a critical component of a human phagocyte oxidoreductase, andto the yeast ferric reductases, Saccharomyces cerevisiae FRE1and FRE2 and Schizosaccharomyces pombe Frpl. The induction ofboth HCR mRNAs required extremely high-CO₂ conditions and irondeficiency, being suppressed under air conditions and by ironsufficiency, suggesting that the expression of these two HCRgenes required extremely high-CO₂ conditions and iron deficiencyin combination. The HCR2 protein was detected in the membranefractions of cells grown under conditions which would favorthe induction of HCR2-mRNA and the protein level was loweredwhen the cells were transferred from iron deficient to 10 µMFeSO₄ conditions (with 20% CO²). (Received September 10, 1997; Accepted November 14, 1997)     

Genes Encoding the Group I Intron-containing tRNALeu and Subunit L of NADH Dehydrogenase from the Cyanobacterium Synechococcus PCC 6301

A part of the tRNA^Leu (UAA) gene containing a 240-nucleotidegroup I intron was amplified by PCR from cyanobacterium SynechococcusPCC 6301 genomic DNA. The pre-tRNA synthesized from the clonedPCR product was efficiently self-spliced in vitro under physiologicalconditions. The gene encoding the tRNA^Leu (UAA), trnL-UAA, wasisolated from a Synechococcus PCC 6301 genomic library and thenucleotide sequence of a 2,167-bp portion was determined. ThetrnL-UAA consists of a 34-bp 5' exon, a 240-bp group I intronand a 50-bp 3' exon. In addition, three open reading frames(ORF1, ORF2 and ORF3) were found in the 5' and 3' flanking regionsof trnL-UAA. The predicted protein sequence of ORF3, which islocated 74-bp upstream from trnL-UAA on the opposite strand,shows 66.2% amino acid identity to that of the SynechocystisPCC 6803 gene encoding subunit L of NADH dehydrogenase (ndhL).     

Water deficits and heat shock effects on photosynthesis of a transgenic Arabidopsis thaliana constitutively expressing ABP9, a bZIP transcription factor

The effects of water deficits (WD), heat shock (HS), and both(HSWD) on photosynthetic carbon- and light-use efficienciestogether with leaf ABA content, pigment composition and expressionsof stress- and light harvesting-responsive genes were investigatedin ABP9 [ABA-responsive-element (ABRE) binding protein 9] transgenicArabidopsis (5P2). WD, HS, and HSWD significantly decreasedphotosynthetic rate (A) and stomatal conductance (g_s) in wild-typeplants (WT). A and g_s of 5P2 transgenic plants were slightlyreduced by a single stress and were hardly modified by HSWD.Although A and electron transport rate (ETR) in 5P2 plants weredepressed under optimal growth conditions (control) in relationto WT, they were enhanced under HS and HSWD. These results indicatethat ABP9 transgenic plants are less susceptible to stress thanthe WT. In addition, the increased ABA contents in both WT and5P2 plants in response to WD and/or HS stresses suggest thatdeclines in A and g_s might have been due to ABA-induced stomatalclosure. Moreover, compared with WT, 5P2 plants exhibited higherABA content, instantaneous water use efficiency (IWUE), Chla/b, NPQ, and lower Chl/carotenoid ratios. Finally, alteredexpression of stress-regulated or light harvesting-responsivegenes was observed. Collectively, our results indicate thatconstitutive expression of ABP9 improves the photosyntheticcapacity of plants under stress by adjusting photosyntheticpigment composition, dissipating excess light energy, and elevatingcarbon-use efficiency as well as increasing ABA content, IWUE,and expression of stress-defensive genes, suggesting an importantrole of ABP9 in the regulation of plant photosynthesis understress. Key words: ABP9, ABA, heat shock, photosynthesis, stress tolerance, water deficits Received 1 September 2007; Revised 19 December 2007 Accepted 21 December 2007     

Isolation, Properties and a Possible Function of a Water-Soluble Chlorophyll a/b-Protein from Brussels Sprouts

A water-soluble Chl a/b-protein (CP673) was isolated and purifiedfrom Brussels sprouts (Brassica oleracea L. var. gemmifera DC).The protein had a molecular mass of 78 kDa and an isoelectricpoint of 4.7, consisted of three or four subunits of 22 kDaand was extremely heat-stable. Although CP673 contained aboutone Chl a per protein, the blue and red absorption bands ofChl a that consisted of three or four Chl a forms with differentabsorption maxima suggested that there are several differentmodes or sites of binding for Chl a. Chl a/b ratio of largerthan 10 also indicated that Chl b is present only in a smallfraction of CP673. The heterogeneity of CP673 in terms of compositionand binding of Chl suggests that Chl is not an intrinsic componentof the Chl-protein. Homology search showed that the N-terminalamino acid sequence of CP673 is highly homologous with thatof a 22 kDa protein that accumulates in water-stressed leavesof two Brassicaceae plants, rapeseed and radish, but not withthose of the light-harvesting Chl a/b-proteins of photosynthesis.A possible function of the water-soluble Chl-protein was discussed. (Received September 17, 1996; Accepted November 18, 1996)